Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACOIUNETIC-BASED CULTURE SYSTEM WITH BIOLOGICAL
BARRIERS
Related AMlications and Claim of Priority
[0001] This application is a continuation-in-part of U.S. Patent Application
No.
10/133,977 filed April 25, 2002, titled "DEVICES AND METHODS FOR
PHARMACOKINETIC-BASED CELL CULTURE SYSTEM," which claims the benefit of
U.S. Provisional Patent Application No. 60/286,493 filed Apri125, 2001; and
this application
also claims the benefit of U.S. Provisional Pateiit Application No. 60/682,131
filed May 18,
2005, titled "MICROSCALE, IN VITRO, CELL CULTURE DEVICE WITH A
MICROPOROUS SURFACE THAT MIMICS PHYSIOLOGICAL PARAMETERS"; and all
of the foregoing applications are hereby incorporated by reference herein in
their entirety.
STATEMENT REGARDING GOVERNMENT RIGHTS
[0002] At least some portion of the disclosure herein was supported at least
in
part under grant number NAG8-1372 from the National Aeronautics and Space
Administration. The U.S. Government may have certain rights.
Back ound
Field
[0003] The present disclosure relates to cell culture technology, and more
particularly, to systems. and method for facilitating interactions between
fluidic systems at
microscale level for pharmacokinetic studies.
Description of the Related Art
[0004] Pharmacolcinetics is the study of the fate of pharmaceuticals and other
biologically active compounds from the time they are introduced into the body
until they are
eliminated. For example, the sequence of events for an oral drug can include
absorption
through the various mucosal surfaces, distribution via the blood stream to
various tissues,
biotransformation in the liver and other tissues, action at the target site,
and elimination of
drug or metabolites in urine or bile. Pharmacokinetics provides a rational
means of
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approaching the metabolism of a conlpound in a biological system. For reviews
of
phaimacokinetic equations and models, see, for example; Poulin and Theil
(2000) J Pharm
Sci. 89(l):16-35; Slob et al. (1997) Crit Rev Toxicol. 27(3):261-72; Haddad et
al. (1996)
Toxicol Lett. 85(2):113-26; Hoang (1995) Toxicol Lett. 79(1-3):99-106; Knaak
et al. (1995)
Toxicol Lett. 79(1-3):87-98; and Ball and Scliwartz (1994) Coiiiput Biol Med.
24(4):269-76.
[0005] One of the fundamental _ challenges researchers face in drug,
environmental, nutritional, consunier product safety, and toxicology studies
is the
extrapolation of metabolic data and risk assessnlent from in vitro cell
culture assays to
animals. Although some conclusions can be drawn with the application of
appropriate
pharmacokinetic principles, there are still substantial limitations. One
concern is that current
screening assays utilize cells under conditions that do not replicate their
function in their
natural setting. The circulatory flow, interaction with other tissues, and
other parameters
associated with a physiological response are not found in standard tissue
culture formats. For
example, in a macroscale cell culture analog (CCA) system, cells are grown at
the bottom of
chambers. These systems have non-physiological high liquid-to-cell ratios, and
have an
unrealistic ratio of cell types (e.g., ratio of liver to lung cells). In a
variant form of the
macroscale CCA system the cells are grown on microcarrier beads. These systems
more
closely resemble pllysiological conditions, but are still deficient because
they do not mimic
physiological conditions accurately enough for predictive studies. Therefore,
the resulting
assay data is not based on the pattern of drug or toxin exposure that would be
found in an
animal.
[0006] Within living beings, concentration, time and metabolism interact to
influence the intensity and duration of a pharmacologic or toxic response. For
example, in
vivo the presence of liver function strongly affects drug metabolism and
bioavailability.
Elimination of an active drug by the liver occurs by biotransformation and
excretion.
Biotransformation reactions include reactions catalyzed by the cytochrome P450
enzymes,
which transform many chemically diverse drugs. A second biotransformation
phase can add a
liydrophilic group, such as glutathione, glucuronic acid or sulfate, to
increase water solubility
and speed elimination tlirough the kidneys.
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[0007] While biotransfoimatioii can be beneficial, it may also have
undesirable
consequences. Toxicity results from a complex interaction between a compound
and the
organism. During the process of biotransforination, the resulting metabolite
can be more
toxic than the parent compound. The single-cell assays used by many for
toxicity screening
miss these complex inter-cellular and inter-tissue effects.
[0008] Consequently, accurate prediction of human responsiveness to potential
phaniiaceuticals is difficult, often unreliable, and invariably expensive.
Traditional methods
of predicting human response utilize surrogates--typically eitlier static,
homogeneous in vitro
cell culture assays or in vivo animal studies. In vitro cell culture assays
are of limited value
because they do not accurately mimic the complex environment a drug candidate
is subjected
to within a human and tlius cannot accurately predict human risk. Siniilarly,
while in vivo
animal testing can account for these complex inter-cellular and inter-tissue
effects not
observable from in vitro cell-based assays, in vivo animal studies are
extremely expensive,
labor-intensive, time consuming, and often the results are of doubtful
relevance when
coiTelating human risk.
[0009] U.S. Pat. No. 5,612,188 issued to Shuler et al. describes a
multicompartmental cell culture system. This culture system uses large
components, such as
culture chambers, sensors, and pumps, which require the use of large
quantities of culture
media, cells and test compounds. This system is very expensive to operate and
requires a
large amount of space in which to operate. Because this system is on such a
large scale, the
physiological parameters vary considerably from those found in an in vivo
situation. It is
impossible to accurately generate physiologically realistic conditions at such
a large scale.
[0010] The development of microscale screening assays and devices that can
provide better, faster and more efficient prediction of in vivo -toxicity and
clinical drug
performance is of great interest in a number of fields, and is addressed in
the present
invention. Such a microscale device would accurately produce physiologically
realistic
parameters and would more closely model the desired in vivo system being
tested.
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Sunlmary
[0011] Devices, in vitro cell cultures, and methods are provided for a
microscale
cell culture analog (CCA) device. The devices of the inveiition permit cells
to be maintained
in vitro, under conditions with pharmacokinetic parameter values similar to
those found in
vivo. Pharmacokinetic parameters of interest include interactions between
cells, liquid
residence time, liquid to cell ratios, relative size of organs, metabolism by
cells, shear stress,
and the like. By providing a phamlacokinetic-based culture system that mimics
the natural
state of cells, the predictive value and iii vivo relevance of screening and
toxicity assays is
enhanced.
[0012] The microscale culture device comprises a fluidic network of channels
segregated into discrete but interconnected chambers. The specific chamber
geometry is
designed to provide cellular interactions, liquid flow, and liquid residence
parameters that
correlate with those found for the corresponding cells, tissues, or organs in
vivo. The fluidics
are designed to accurately represent primaiy elements of the circulatory or
lymphatic systems.
In one embodiment, these components are integrated into a chip format. The
design and
validation of these _geometries is based on a pliysiological-based
pharmacokinetic (PBPK)
model; a mathematical model that represents the body as interconnected
conipartinents
representing different tissues.
[0013] The device can be seeded with the appropriate cells for each culture
chamber. For example, a chamber designed to provide liver pharmacolcinetic
parameters is
seeded with hepatocytes, and may be in fluid connection with adipocytes seeded
in a chamber
designed to provide fat tissue pharmacokinetics. The result is a
pharmacokinetic-based cell
culture system that accurately represents, for example, the tissue size ratio,
tissue to blood
volume ratio, drug residence time of the animal it is modeling.
[0014] In one embodiment, a system includes a first microscale culture device
and
a control instrument. The first microscale culture device has a number of
microscale
chambers with geometries that siniulate a plurality of in vivo interactions
with a culture
medium, wherein each chamber includes an inlet and an outlet for flow of the
culture
medium, and a microfluidic channel interconnecting the chambers. The control
instrument is
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coupled to the first microscale culture device, and includes a computer to
acqtiire data from,
and control pharmacokinetic parameters of, the first microscale culture
device.
[0015] In another embodiment, a conlputer includes a niicroprocessor, a
general
memory, a non-volatile storage element, ari input/output interface tllat
includes an interface to
a microscale culture device having one or more sensors, and coinputer
software. The
computer software is executable on the microprocessor. to analyze data from
the sensors to
measure physiological events in a number of chambers of the microscale culture
device,
regulate fluid flow rates of a culture medium in the chambers of the
microscale culture
device, detect biological or toxicological reactions in the chambers of the
microscale culture
device, and upon detection, change one or more pharmacolcinetic parameters of
the
microscale culture device.
[0016] As used herein the singular forms "a" and "tlie" include plural
referents
unless the context clearly dictates otherwise. For example, "a compound"
refers to one or
more of such compounds, while "the cell" includes a particular cell as well as
other family
members and equivalents thereof as known to those slcilled in the art.
[0017] One embodiment of the present disclosure relates to an apparatus that
includes at least one feature dimensioned to maiutain biological material
under conditions
that provide a value of at least one pharmacokinetic parameter in vitro that
is comparable to
the value of at least one pharmacokinetic parameter found in vivo. The
apparatus furtller
includes a permeable material.
[0018] -In one embodiment, the feature is a microscale feature. In one
embodiment, the permeable material is selected from at least one of the group
consisting of a
membrane, a porous membrane, microporous silicon, a semi-permeable , membrane,
a
microporous material, a microporous polyiner, alginate, collagen, MATRIGEL,
cells, cellular
material, tissue, and pieces of tissue.
[0019] In one embodiment, the permeable material further includes organic or
inorganic material in, on or near a microporous surface.
[0020] In one embodinlent, the permeable material is configured to simulate at
least one of a biological barrier, passage of substances in or through a
biological barrier, or
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absoiption of substaiices in, through or by a biological barrier. hl oiie
embodiment, the
biological barrier is selected from at least one of the group consisting of a
gastrointestinal
barrier, a blood-brain barrier, a pulmonary barrier, a placental barrier, axi
epidermal barrier,
ocular barrier, olfactory barrier, a gastroesophageal barrier, a mucous
membrane, a blood-
urinary barrier, air-tissue barrier, a blood-biliary barrier, oral barrier,
anal rectal barrier,
vaginal barrier, and urethral barrier.
[0021] Iil one embodiment, the at least one pharniacokinetic paranieter is
selected
from at least one of the group consisting of tissue size, tissue size ratio,"
tissue to blood
volume ratio, drug residence time, interactions between cells, liquid
residence time, liquid to
cell ratios, metabolisin by cells, shear stress, flow rate, geometry,
circulatory transit time,
liquid distribution, interactions between tissues and/or organs, and molecular
transport by
cells.
[0022] In one embodiment, the device determines absoiption, metabolism, or
distribution of a substance in, through or by the pei-meable material. In one
enibodiment, the
feature is configured to represent at least one of the group consisting of at
least portions of
central nervous, circulatory, digestive,- biliary, pulmonary, urinary, ocular,
olfactory,
epidermal, and lymphatic systems. In one embodiment, the peimeable material is
located in
or external to the device.
[0023] In one embodiment, the apparatus further includes at least one
microfluidic channel connected to the permeable material.
[0024] In one embodiment, the flow of fluid in, througll, or in proximity to
the
permeable material provides the at least one phamiacokinetic parameter. In one
embodiment,
the characteristics of the fluid flow through the device are based on a
matheinatical model. In
one enzbodiment, the mathematical model is a pliysiologically-based
pharmacokinetic
("PBPK") model.
[0025] In one enlbodiment, the feature or the permeable material is integrated
into
a chip format.
[0026] In one embodiment, the permeable material includes a layer of
gastrointestinal enterocytes cultured on a niicroporous material. In one
enibodiment, at least
a portion of the layer of gastrointestinal enterocytes is positioned in the
device such that fluid
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may flow along eitller side of but not through the layer. In one embodiment,
at least a first
microscale feature located on a first side of the layer of gastrointestinal
enterocytes represents
the gastrointestinal tract, and at least a second microscale feature located
on a second side of
the monolayer represents a circulatory system. In one einbodiment, the
apparatus further
includes a tliird nlicroscale feature that is configured to contain the same
or a different type of
biological material.
[0027] In one embodiment, the pemieable material includes a microporous
material coated at least in part with an organic material.
[0028] In one embodiment, the apparatus further includes cells located in, on
or
near both sides of the permeable material. hi one embodiment, the device
provides
absorption characteristics, metabolic enzyme activity and/or expression
levels. In one
embodiment, the cells on either side of the permeable material are of the same
type or of
different types.
[0029] In one embodiment, the apparatus further includes hepatocytes in, on or
near a microporous surface of the permeable material. In one einbodiment, at
least a portion
of the microporous surface includes proteins that polarize the hepatocytes.
[0030] In one embodiment, the permeable material includes a cell line capable
of
foiming a confluent monolayer.
[0031] In one embodiment, the apparatus further includes a binder that binds
hepatocytes to the permeable material. In one embodiment, the binder polarizes
the
hepatocytes. In one embodiment, the binder includes at least one selected from
the group
consisting of a protein, connexin 32, a tight junction protein, occludin,
claudin-1, ZO- 1, ZO-
2, an adherens junction protein, E-cadherin, beta-catenin, a cell adhesion
molecule, and
uvomoiulin.
[0032] hi one embodiment, the apparatus further includes a second type of
biological material in, on or near the permeable material.
[0033] In one embodiment, the apparatus further includes fibroblasts in, on or
near the permeable material.
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[0034] In one enlbodiment, the apparatus further includes a blood suiTogate
flow
in proximity to a first side of the permeable material. In one embodiment, the
apparatus
further includes a bile surrogate flow in proxiniity to "a second side of the
pernieable material.
[0035] One enzbodiment of tlie present -disclosure relates to a method that
includes maintaining biological material under conditions that provide a value
of at least one
phannacokinetic parameter in vitro that is coinparable to the value of at
least one
phai7nacokinetic parameter found in vivo. The method furtlier includes passing
a substance
through at least a portion of a permeable materiaL
[0036] In one embodiment, the method further includes maintaining the
biological
material within or in proximity to a microscale feature.
[0037] In one embodiment, the permeable material is selected from at least one
of
the group consisting of a membrane, a porous membrane, microporous silicon, a
semi-
pemieable membrane, a microporous material, a microporous polymer, alginate,
collagen,
MATRIGEL, cells, cellular material, tissue, and pieces of tissue.
[0038] In one embodiment, the permeable material further includes organic or
inorganic material in, on- or near a microporous surface.
[0039] hi one embodiment, the pernieable material is configured to simulate at
least one of a biological barrier, passage of substances in or through a
biological barrier, or
absorption of substances in, through or by a biological barrier. In one
embodiment, the
biological barrier is selected from at least one of the group consisting of a
gastrointestinal
barrier, a blood-brain barrier, a blood-biliaiy ban-ier, a pulmonary barrier,
a placental barrier,
an epidermal barrier, ocular barrier, olfactory barrier, a gastroesophageal
barrier, a mucous
membrane, a blood-urinary barrier, an air-tissue barrier, oral barrier, anal
rectal barrier,
vaginal barrier, -and urethral barrier.
[0040] In one embodiment, the at least one phannacokinetic parameter is
selected
fiom at least one of the group consisting of tissue size, tissue size ratio,
tissue to blood
volume ratio, drug residence time, interactions between cells, liquid
residence time, liquid to
cell ratios, metabolism by cells, shear stress, flow rate, geometry,
circulatory transit time,
liquid distribution, interactions between tissues and/or organs, and,
molecular transport by
cells.
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[0041] In one embodiment, the method fiu-ther includes deteimining absoiption,
metabolism, or distribution of the substance in, througli or by the pei7neable
material. In one
embodiment, the feature is configtued to represent at least one of the group
consisting of at
least portions of central neivous, circulatory, digestive; biliary, pulmonary,
urinary, ocular,
olfactory, epidernial, and lymphatic systems.
[0042] In one embodiment, the method further includes locating the permeable
material in or external to a microscale device.
[0043] In one embodiment, the metliod further includes flowing fluid througll
at
least one microfluidic channel connected to the permeable material.
[0044] In one embodiment, the flow of fluid in, througli, or in proximity to
the
permeable material provides the at least one pharmacokinetic parameter. In
oneembodiment,
the characteristics of the fluid flow through the device are based on a
mathematical model. In
one embodiment, the mathematical model is a pliysiologically-based
pharmacolcinetic
("PBPK") model.
[0045] In one embodiment, the metllod further includes integrating the
microscale
feature or the peimeable material into a chip format.
[0046] In one embodinient, the permeable material includes a layer of
gastrointestinal enterocytes cultured on a microporous material. In one
embodiment, the
method further includes positioning at least a portion of the layer of
gastrointestinal
enterocytes such that fluid may flow along eitlier side of but not through the
layer. In one
embodiment, at least a first microscale feature located on a first side of the
layer of
gastrointestinal enterocytes represents -the gastrointestinal tract and at
least a second
microscale feature located on a second side of the monolayer represents a
circulatory system.
In one embodiment, a third microscale feature is configured to contain the
same or a different
type of biological material.
[0047] In one embodiment, the permeable material includes a microporous
material coated at least in part with an organic material.
[0048] In one embodiment, the method fiirther includes locating cells in, on
or
iiear both sides of the permeable material. In one embodiment, the method
further includes
providing absorption characteristics, metabolic enzyme activity and/or
expression levels. In
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one enzbodiment, the cells on eitlier side of the permeable material are of
the same type or of
different types.
[0049] In one embodiment, the method further includes locating hepatocytes in,
on or near a microporous surface of the perineable material. In one
embodiment, at least a
portion of the microporous surface includes proteins that polarize the
hepatocytes.
[0050] In one einbodiment, the permeable material includes a cell line capable
of
forming a confluent monolayer and polarizing.
[0051] In one embodiment, the method further includes binding hepatocytes to
the
permeable material. In one embodiment, the method fiu-ther includes polarizing
the
hepatocytes. In one einbodiment, the binding includes a binder that is at
least one selected
from the group consisting of a protein, connexin 32, a tight junction protein,
occludin,
claudin-l, ZO-1, ZO-2, an adherens juinction protein, E-cadherin, beta-
catenin, a cell
adhesion molecule, and uvomorulin.
[0052] In one embodiment, the method further includes locating a second type
of
biological material in, on or near the permeable material.
[0053] In one embodiment, the metliod further includes locatiiig fibroblasts
in, on
or near the peimeable material.
[0054] In one embodiment, the method further includes flowing a blood
surrogate
in proximity to a first side of the permeable material. In one embodiment, the
method further
includes flowing a bile surrogate in proximity to a second side of the
permeable material.
[0055] One embodiment of the present disclosure relates to a method of forming
a
device. The method includes forming a feature that is configured to niaintain
biological
material under conditions that provide a value of at least one
pharmacolcinetic parameter in
vitro that is comparable to the value of at least oue pharmacokinetic
parameter found in vivo.
The method furtlier includes adding, forming, or providing for a permeable
material. The
peimeable material is configured such that a substance passes through at least
a por tion of the
permeable material.
[0056] One embodiment of the present disclosure relates to a device having
means for maintaining biological material under conditions that provide a
value of at least
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one pharnlacokinetic parameter in vitro that is comparable to the value of at
least one
pharmacokinetic parameter found in vivo, and ineans for providing a perineable
barrier.
[0057] One embodiment of the present disclosure relates to a device that
includes
microscale peimeable material, and at least one binder configured to polarize
a substance,
wliere the substance manifests at least one characteristic of liver function.
[00581 In one embodiment, the substance is one or more hepatocytes. hi one
embodiment, the substance is a genetically engineered biological material. In
one
embodiment, the binder binds and polarizes hepatocytes to the microscale
permeable
material.
[0059] In one embodiment, the device further includes a second substance type.
In one embodiment, the device further includes one or more fibroblasts located
near at least
one surface of the microscale permeable material.
[0060] In one embodiment, the microscale peimeable material is selected from
at
least one of the group consisting of organic material, inorganic material, a
membrane, a
porous membrane, microporous silicon, a semi-permeable membrane, a microporous
material, a microporous polymer, alginate, collagen, MATRIGEL, cells, cellular
material,
tissue, and pieces of tissue. In one embodiment, the microscale permeable
material is in, on
or near a microporous surface. In one embodiment, the niicroscale pemieable
material is
configured to simulate at least one of a biological barrier, passage of
substances in or through
a biological barrier, or absorption of substances in, through or by a
biological barrier.
[0061] In one embodiment, the device processes the substance in by or through
the microscale permeable material. In one einbodiment, the processing further
includes at
least one of the group consisting of absorption, extraction, excretion,
metabolism, and
distribution of molecules.
[0062] In one embodiment, the microscale permeable material is located in or
external to the device.
[0063] In one enzbodiment, the device furtller includes at least one
microfluidic
channel connected to the microscale permeable material.
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[0064] hi one enlbodiment, the characteristics of fluid flow through the
device are
based on a mathematical model. In one enlbodiment, the matliematical model is
a
physiologically-based pharmacokinetic ("PBPK") model.
[0065] In one enibodiment, the feature or the microscale perineable material
is
integrated into a chip format. In one embodiment, the device provides
absorption
characteristics, metabolic enzyine activity and/or expression levels.
[0066] In one embodiment, the device further includes biological material
located
in, on or near both sides of the microscale pemieable material. In one
embodiment, the
biological material on eitlier side of the microscale permeable material are
of the same type
or of different types.
[0067] hi one embodiment, the microscale peimeable material includes a cell
line
capable of forming a confluent monolayer. In one embodimeiit, the binder
includes at least
one selected from the group consisting of a protein, connexin 32, a tight
junction protein,
occludin, claudin-1, ZO-1, ZO-2, an adherens junction protein, E-cadherin,
beta-catenin, a
cell adhesion molecule, and uvomorulin.
[0068] In one embodiment, the device further includes a blood surrogate flow
in
proximity to a first side of the microscale peimeable material. In one
embodiment, the device
further includes a bile surrogate flow in proximity to a second side of the
microscale
permeable material.
[0069] One embodiment of the present disclosure relates to a method that
includes binding a substance that manifests at least one characteristic of
liver function to a
microscale permeable material in a manner that polarizes the substance.
[0070] In one embodiment, the substance is one or more hepatocytes. In one
embodiment, the substance is a genetically engineered biological material.
[0071] In one embodiment, the method furtlier includes providing a second
substance type. In one embodiment, the metllod further includes locating one
or more
fibroblasts located near at least one surface of the microscale permeable
material.
[0072] In one embodiment, the microscale penneable material is selected from
at
least one of the group consisting of organic material, inorganic material, a
membrane, a
porous membrane, microporous silicon, a semi-permeable membrane, a microporous
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material, a microporous polymer, alginate, collagen, MATRIGEL, cells, cellular
material,
tissue, and pieces of tissue.
[00731 In one embodiment, the method furtller includes locating the microscale
pemieable material in, on or near a microporous sttrface.
[00741 In one embodiment, the microscale peimeable material simulates at least
one of a biological barrier, passage of substances in or tlirough a biological
barrier, or
absorption of substances in, through or by a biological barrier.
[0075] In one embodiment,the method further includes processing the substance
in, through or by the microscale peimeable material. In one embodiment, the
processing
further includes at least one of the group consisting of absorption,
extraction, excretion,
metabolism, and distribution of molecules.
[0076] In one embodiment, the method further includes locating the microscale
permeable material in or external to a device.
[0077] hi one embodiment, method furtlier includes providing at least one
microfluidic ehannel connected to the microscale permeable material.
[0078J In one _embodiment, the characteristics of fluid flow associated with
the at
least one characteristic of liver function are based on a mathematical model.
In one
embodiment, the mathematical model is a physiologically-based pharnlacokinetic
("PBPIC")
model.
[0079] In one embodiment, the metllod furtlier includes integrating the
microscale
permeable material into a chip fomiat.
[0080] hi one embodiment, the method further includes providing absorption
characteristics, metabolic enzyme activity and/or expression levels.
[0081J In one embodiment, the metliod further includes locating biological
material in, on or near both sides of the microscale peimeable material. In
one embodiment,
the biological material is on either side of the microscale permeable material
is of the same
type or of different types.
[00821 In one embodiment, the microscale permeable material includes a cell
line
capable of forming a confluent monolayer. In one embodiment, the binding
includes
providing a binder selected from at least one of the group consisting of a
protein, connexin
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32, a tight junction protein, occludin, claudin-1, ZO-1, ZO-2, an adherens
junction proteiil, E-
cadlierin, beta-catenin, a cell adhesion molecule, and uvomonilin.
[0083] In one embodiment, the nsethod- further includes providing a blood
surrogate flow in proximity to a first side of the microscale perineable
material. In one
einbodiment, the method further includes providing a bile suiTogate flow in
proximity to a
second side of the microscale permeable material.
[0084] One embodiment of the present disclosure relates to a method of forming
a
device. The metliod includes forming a microscale permeable material that is
configured to
bind to and polarize a substance that manifests at least one characteristic of
liver fiinction.
[0085] One embodiment of the present disclosure relates to a microscale
apparatus having means for binding a substance that manifests at least one
characteristic of
liver function to a microscale permeable material in a manner that polarizes
the substance.
[0086] One embodiment of the present disclosure relates to a device that
includes
a microscale peimeable material, and at least one substance configured to
manifest at least
one characteristic of liver function, where molecules processed by the
substance are directed
to pass through at least a portion of the microscale pemieable material.
[0087] One embodiment of the present disclosure relates to a method that
includes directing molecules processed by a substance through at least a
portion of a
microscale permeable material, where the substance is configured to manifest
at least one
characteristic of liver function.
[0088] One embodiment of the present disclosure relates to a method of forming
a
device. The method includes fom7ing a microscale perrrieable material that is
configured to
direct molecules processed by a substance through at least a portion of the
microscale
permeable material, where the substance is configured to manifest at least one
characteristic
of liver function.
[0089] One embodiment of the present disclosure relates to a device having
means for directing molecules processed by a substance througli at least a
portion of a
microscale permeable material, where the substance is configured to manifest
at least one
characteristic of liver function.
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Brief Descri-ption of the Drawings
[0090] FIG. 1 is a block diagram of a system in accordance with the present
invention.
[0091] FIG. 2 is a siniplified perspective view of one embodimeiit of the
exterior
of the system of the present invention.
[0092] FIG. 3 is a detailed schematic view of another enzbodiment of the
system
of the present invention.
[0093] FIG. 4 is a schematic view of yet another embodiment of the system of
the
present invention.
[0094] FIGS. 5A through 5G show steps used to fabricate a chip from plastic.
FIG. 5A shows coating a silicon wafer with a positive photoresist material.
FIG. 5B shows
exposing resist-coated silicon wafer to UV light through a photomaterial. FIG.
5C shows
developing the photoresist material. FIG. 5D shows etching silicon.FIG. 5E
shows striping
the photoresist material and evaporating gold. FIG. 5F shows electroplating
nickel. FIG. 5G
shows removing silicon and embossing polymer.
[0095] FIG. 6 is a schematic view of still anotlier embodiment of the system
of
the present invention.
[0096] FIG. 7 is a scliematic detailing a computer associated with the chips.
[0097] FIG. 8 is a schematic showing more than one chip located within a
housing.
[0098] FIG. 9 is a schematic of a system that includes sets of chips from
different
housings.
[0099] FIG. 10 is a schematic of yet another embodiment of a chip.
[0100] FIG. 11 is an isometric partially exploded view of a system.
[0101] FIG. 12 is an isometric view of the steps for fabricating the chin
associated
with the system shown in FIG. 11.
[0102] FIG. 13 is an isometric view of a single trough elastomeric portion of
a
pump associated with the system shown in FIG. 11.
[0103] FIG. 14 is an isometric view of a lnultiple trough elastomeric portion
of a
pun7p.
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[0104] FIG. 15 is a scliematic diagram of the four-conlpartiizent chip.
[0105] FIG. 16 Tegafur dose response. Chips were seeded with HepG2-C3A cells
in the liver conzpartnzent and HCT-1-16 colou cancer cells in the target
tissues compart7nent.
The chips were treated with indicated concentrations of tegafur for 24 hours.
The first graph
(FIG. 16A) is a plot of percentage dead cells vs. tegafur or 5-FU
concentration after 24 hours
of re-circulationon the chip. The second graph (FIG. 16B) is a similar dose
response using a
traditional in vitro cell culture assay with HCT 116 cells using a 48 hour
exposure. HCT-116
cells were seeded on poly-lysine treated glass coverslips and exposed to
eitller tegafur or 5-
FU at the indicated concentrations. After a 48 hr incubation, coverslips were
treated as
described above and the percentage of cell deatli was determined.
[0106] FIG. 17A depicts a "first generation" tliree compartment device. FIG.
17B
shows a cross-sectional view of the device.
[0107] FIG. 18A depicts a "second generation" device. FIG. 18B depicts 5 m
tall
ridges in a chamber, and FIG. 18C depicts 20 m tall pillars in a chamber.
[0108] FIG. 19 depicts a "third generation" device.
[0109] FIG. 20 is a flow diagram for a five compartment PBPK model CCA.
[0110] FIG. 21 depicts a human biochip prototype that contains compartments
for
lung, target tissues, and other tissues. The dimensions of the compartments
and channels are
as follows:
[0111] Inlet: 1 mm by 1 mni
[0112] Liver: 3.2 mm wide by 4 mm long
[0113] Target Tissues: 2 mm wide by 2 mm long
[0114] Otlier Tissues: 340 m wide by 110 min long
[0115] Outlet: 1 nmi by 1 mnl
[0116] Channel Connecting Liver to Y connection: 440 in wide
[0117] Channel from Y connectioii to Target Tissue: 100 m wide
[0118] FIG. 22 depicts a schematic drawing of the microscale chip system.
[0119] FIG. 23 depicts basal CYP expression levels for Hep G2, HepG2/C3A,
and human liver. Std. error from 3 separate deteiminations.
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[0120] FIG. 24A depicts HepG2/C3A growth cuives in EMEM, DMEM,
McCoy's and RPMI. FIG. 24B depicts HCT1 16 growth curves in EMEM, DMEM,
McCoy's
and RPMI. Standard error from 3 separate determinations.
[0121] FIG. 25 depicts RT-PCR determination of CYP isofoims expression in
HepG2/C3A under different growth media conditions.
[0122] FIG. 26 depicts RT-PCR determination of CYP isoforms expression in
HepG2/C3A grown on different substrates.
[0123] FIG. 27 depicts a human bio-chip prototype.
[0124] FIG. 28A is a block-diagrain view illustrating a system for controlling
a
microscale culture device, according to one embodiment of the present
invention. FIG. 28B is
a block-diagram view illustrating a system for controlling a microscale
culture device,
according to another embodiment of the present invention.
[0125] FIG. 29 is a flow-diagram view illustrating a computerized method for
dynamically controlling a microscale culture device, according to one
embodiment of the
present invention.
[0126] FIG. 30 is a block-diagram view illustrating a computer for controlling
a
nlicroscale culture device, according to one embodiment of the present
invention.
[0127] Figure 31 shows that in one embodiment, interaction between first and
second fluidic systems can be provided and maintained in vitro under
conditions with
physiological parameter values similar to those found in vivo;
[0128] Figure 32 shows a block diagrani of some example fluidic systems anlong
wliich various inter-system interactions can be simulated in vitro;
[0129] Figure 33A shows an example interaction between two fluidic systems;
[0130] Figure 33B shows that in one embodiment, a given fluidic system can
interact with more than one fluidic system;
[0131] Figure 33C shows that in one embodiment, a given fluidic system can
interact witli more than two fluidic systems;
[0132] Figure 33D shows that in one embodiment, fluidic system interactions
can
provide recirculation ftinctionality;
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[0133] Figure 34A shows a partially exploded view of an exanlple embodiment of
a two-fluidic-systenz configuration, where inter-system interaction can be
facilitated by a
permeable material;
[0134] Figure 34B shows an assenibled view of the two-fluidic-system of Figure
34A;
[0135] Figure 34C shows a top view of the two-fluidic-system of Figure 34A;
[0136] Figure 34D shows one enibodiment of a variation of the system of Figure
34A;
[0137] Figure 35A shows a partially exploded view of an example embodiment of
a three-fluidic-system configuration, where two inter-system interactions can
be facilitated by
one or more types of permeable materials;
[0138] Figure 35B shows an asserribled view of the three-fluidic-systeni of
Figure
35A;
[0139] Figure 36 shows a block diagram of an example three-fluidic-system
where an organ system is depicted as interacting with a drug delivery system
such as
gastrointestinal (GI) system and witli a target system such as brain system;
[0140] Figure 37 shows a block diagram of an example configuration involving
various inter-system interactions involving a l'iver, where such interactions
can be part of a
recirculating process such as enterohepatic circulation;
[0141] Figure 38 shows a block diagram depicting the enterohepatic circulation
of
Figure 37;
[0142] Figure 39- shows one embodinient of a microscale permeable device
having a permeable material that can facilitate one or more interactions
between two fluidic
systems;
[0143] Figure 40A shows one embodiment of the microscale permeable device
configured to facilitate interaction between blood and bile systems;
[0144] Figure 40B shows one embodiment of the microscale permeable device
configured to facilitate interaction between GI and blood systems;
[0145] Figures 41A and 41B show partially exploded and assenibled views of one
embodinient of an enterohepatic circulation simulation device;
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[0146] Figure 41C shows another partially exploded view of Figure 41A, where
one embodiment of the microscale peinzeable device is shown in greater detail;
[0147] Figure 42 shows an example schematic depiction showiiig various fluid
flows that can be implemented in the example enterohepatic circulation
simulation device of
Figures 41 A and 41 B;
[0148] Figures 43A to 43E show various stages of fabricatioi-i of one
embodiment
of the microscale permeable device of Figure 39;
[0149] Figure 44 shows one emVodiment of a process for fabricating the
microscale peimeable device of Figures 43A to 43D;
[0150] Figure 45 shows non-limiting examples of inter-system interactions that
can be facilitated by the microscale permeable device;
[0151] Figure 46 shows a generalized depiction of the inter-system interaction
between first and second systems facilitated by the microscale permeable
device; and
[0152] Figure 47 shows that in one embodiment, a niicroscale permeable device
can be configured so as to facilitate inter-system interaction between two
compartments
formed on a same layer, where the two compartments are parts of two different
systems.
[0153] These and other aspects, advantages, and novel features of the present
teachings will become apparent upon reading the following detailed description
and upon
reference to the accompanying drawings. In the drawings, similar elements may
have similar
reference numerals.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0154] The present inventors have developed a microscale cell culture analog
(CCA) system. Such a microscale CCA system has many advantages over the
earlier
macroscale systems. The microscale systems use smaller quantities of reagents,
fewer cells
(which allow the use of autlientic primary cells rather than cultured cells),
are more
pllysiologically realistic (e.g., residence times, organ ratios, shear
stresses), have a lower
device cost, and are smaller in size (nlultiple tests and statistical analysis
available).
Moreover, multiple biosensors can be incorporated on the same chip.
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[0155] In simplest terms, the chip of the present invention provides an
accurate in
vitro surrogate of an wliole animal or human. To accomplish this, an initial
design was
produced using a physiological-based phariiiacokinetic (PBPK) model--a
matllematical
model that represents the body as interconnected compartments specific for a
particular
organ. From the PBPK model and published empirical data, a lengthy and
extensive
development prograni resulted in a microscale device that accurately mimics
the lcnown
tissue size ratio, tissue to blood volume ratio, drug residence time, and
other important
pliysiological parameters of a whole animal or human. In essence, the chip
technology of the
preseiit invention is a microscale model of a whole animal or human (-
1/100,000t" for
human).
[0156] In operation, the device replicates a re-circulating multi-organ system
by
segregating living cells into discrete, interconnected "organ" compartments
(see e.g., FIG.
15). The fluidics are designed such that the primary elenlents of the
circulatory system and
the interactions of the organ systems are accurately mimicked. Each organ
compartment
contains a particular cell type carefully selected or engineered to mimic the
primary
function(s) of the corresponding whole organ (e.g. xenobiotic metabolism by
the liver). The
cell type may be adherent or non-adherent and derived from standard cell
culture lines or
primary tissue. Human cells are used for human surrogates or cells from other
species as
appropriate.
[0157] The organ compartments are connected by a re-circulating culture medium
that acts as a "blood surrogate." Test agents in the medium are distributed
and interact with
the cells in the organ compartments much as they would in the human body or
wliole animal.
The effects of these compounds and/or their metabolites on the various cell
types are detected
by measuring or monitoring key physiological events such as cell death, cell
proliferation,
differentiation, immune response, or perturbations in metabolism or signal
transduction
patliways. In addition, pharmacolcinetic data can be determined by collecting
and analyzing
aliquots of the culture medium for drug metabolites.
[0158] The microscale chip device of the present invention offers both the
cost
and tlirougl-iput advantages of traditional cell culture assays and also the
high informational
content of whole animal models. Unlike whole animal tests however, the chip is
inexpensive
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and largely disposable. The low fluid volunie (-5 l) of the device provides
the high
sensitivity and througliput characteristic of microfluidic devices. Moreover,
the readout of the
device is highly flexible and assay independent--almost any cell type or assay
can be used
without modification. Numerous biological assays based on optical
interrogation and readout
(e.g., fluorescence, luininescence) are available, tllus making real-tiine
monitoring feasible.
Alternatively, standard pathology, biochemical, genomic or proteomic assays
can be utilized
directly as the system can be designed to be fully conipatible witli the
traditional coverslip
(22 nlm x 22 mm) or 96 well foi-niat. Further, genetically engineered cells
can be used for
specialized end-user applications. In addition, "3D" chips can be used to
encompass
additional compartments and modules to analyze gastrointestinal tract or blood-
brain barrier
absorption.
[0159] Unlike traditional in vitro assays, the chip of the present invention
more
closely mimics the complex multi-tissue (liver, lung, adipose, circulatory
system, etc.)
biology of the whole organism. Drug candidates are exposed to a more realistic
animal or
human physiological environment thus providing higher and more accurate
informational
content .(e.g., absorption, distribution, bioaccumulation, metabolism,
excretion, efficacy and
toxicity) than typical in vitro assays. These benefits directly affect the
safety and efficacy
predictions of drug leads and particularly, their prioritization before
entering into expensive
and time-consuming non-clinical or clinical trials. This prioritization
increases drug
development throughput, reduces the number of animals needed for toxicological
screening,
decreases the costs of non-clinical studies, and increases the efficiency of
clinical trials by
allowing rapid and direct assessment of potential toxicity or lack of efficacy
prior to entering
these trials.
[0160] These demonstrate some of the advantages of the chip technology of the
present invention. In summary, acquisition of data is rapid when compared to
traditional in
vitro cell culture assays, animal studies, or clinical trials. The data is
also robust, providing
highly predictive content not available from traditional in vitro assays. The
chip platfoi-ln is
designed such that it is fully compatible with existing assays--either in the
standard coverslip
or 96 well format. The device itself is configurable for any animal species or
combination of
multiple organ compartments. Individual chips are priced cost-effectively as
disposables.
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Moreover, the low volume of the device further reduces reagent costs in
screening potential
compounds.
[0161] Unlilce currently available technologies, the present chip system
greatly
increases the success rates not only at the clinical phase, but also in
reducing the number of
conipounds that need to undergo pre-clinical testing. Consequently, a
pharinaceutical
company can (1) determine which drug candidates have the potential to be toxic
to humans
early in the development process; (2) better select the animal species that
best predict human
response; and (3) determiize which drug candidate has the poteiitial to be
efflcacious. Thus,
the chip of the present invention greatly increases the success rates and
decrease the
development time of marlcetable drugs.
Pharmokinetic-based Microscale Culture Device
[0162] Devices, in vitro cell cultures, and methods are provided for a CCA
device. The subject methods and devices provide a means whereby cells are
maintained in
vitro in a pliysiologically representative environment, thereby improving the
predictive value
and in vivo relevance of screening and toxicity assays. A microscale
pharniacolcinetic culture
device of the present invention is seeded with the appropriate cells for each
culture chamber,
which culture system can then be used for compound screening, toxicity assays,
models for
development of cells of interest, models of infection kinetics, and the lilce.
An input variable,
which may be, for example, a compound, sample, genetic sequence, pathogen,
cell (such as a
stem or progenitor cell), is added to an established culture system. Various
cellular outputs
may be assessed to determine the response of the cells to the input variable,
including pH of
the niedium, concentration of 02 and CO2 in the medium, expression of proteins
and other
cellular marlcers, cell viability, or release of cellular products into the
culture medium.
[0163] The design and geometry of the culture substrate, or device, provides
for
the unique growtli conditions of the invention. Each device comprises one or
more chambers,
which are interconnected by fluidic channels. Each chamber may have a
geometric
configuration distinct from other chamber(s) present on the device. For
example, one
embodiment of the device consists of chainbers representing lung, liver, and
other tissues
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(FIG. 18A). The lung chamber in this embodiment contains 5 n1 tall ridges iii
order to
achieve realistic cell to liquid volume ratio and liquid residence time (FIG.
18B). The liver
chamber in this embodiment contains 20 m tall pillars to achieve realistic
cell to liquid
volume ratio and liquid residence time (FIG. 18C). The device also comprises
inlet and outlet
ports so that the culture medium can be circulated.
[0164] In one embodiment, the culture device is in a chip format, i.e., the
chambers and fluidic channels are fabricated or molded from a fabricated
master, such that
the device is formed either as a single unit or as a modular system with one
or more chambers
on separate units. Generally the chip format is provided in a small scale,
usually not more
than about 10 cm on a side, or even not more tlian about 5 cm on a side. It
may even be only
about 2 cm on a side or smaller. In anotlier example, the chip may be housed
in a 96 well
format in which the individual chips are less than 0.9 cm x 0.9 cm. The
chambers and fluidic
channels are correspondingly micro-scale in size.
[0165] In another embodiment, the culture device is in the form of an
integrated
device consisting of a table-top instrument housing multiple microscale chips
fabricated as
disposable plastic polymer-based components. The instrument may consist of a
base with
depressions to accommodate individual cell chips or alternatively, a single
"chip" in a
standard 96 well format (i.e., 96 individual chips in a 8 x 12 format). The
instrument top,
when closed seals the chips and provide fluid interconnects. The instrument
contains low
volume pumps to re-circulate fluid to the chips and small 3-way valves with
injection loops
to provide introduction of test compounds, or alternatively draws compounds
directly from a
96- or 384-well plate. Multiple compounds can be evaluated simultaneously for
efficacy,
toxicity, and/or metabolite production using this instrument. The instrument
may also
integrate on-chip fluorescence detection for real-time pllysiology monitoring
using well-
characterized biomarlcers.
[0166] The device may include a mechanism for obtaining signals fiom the cells
and culture medium. The signals from different chambers and channels can be
monitored in
real time. For example, biosensors can be integrated or external to the
device, which permit
real-time readout of the physiological status of the cells in the system.
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[0167] The present invention provides an ideal system for high-througliput
screening to identify positive or negative response to a range of substances
such as, for
exanlple, phamlaceutical conipositions, vaccine preparations, cytotoxic
chemicals, mutagens,
cytokines, chemokines, growth factors, hormones, inhibitory compounds,
chemotherapeutic
agents, and a host of other compounds or factors. The substance to be tested
can be either
naturally-occurring or synthetic, and can be organic or inorganic.
[0168] For example, the activity of a cytotoxic compound can be measured by
its
ability to damage or kill cells in culture. This may readily be assessed by
vital staining
tecliniques. The effect of growth/regulatory factors may be assessed by
analyzing the cellular
content of the matrix, e.g., by total cell counts, and differential cell
counts. This may be
accomplished using standard cytological and/or histological techniques
including the use of
immunocytochemical techniques employing antibodies that define type-specific
cellular
antigens. The effect of various drugs on normal cells cultured in the device
may be assessed.
For example, drugs that increase red blood cell formation can be tested on
bone marrow
cultures. Drugs that affect cholesterol metabolism, e.g., by lowering
cholesterol production,
can be tested on a liver system. Cultures of tumor cells may be used as model
systems to test, -
for example, the efficacy of anti-tumor agents.
[0169] The device of the invention may be used as model systems for the study
of
physiologic or patllologic conditions. For example, in a specific embodiment
of the
invention, a device can be used as a model for the blood-brain barrier; such a
model system
can be used to study the penetration of substances through the blood-brain
barrier. In an
additional embodiment, and not by way of limitation, a device containing
mucosal epithelium-
rnay be used as a model system to study herpesvii-us or papillomavirus
infection; such a
model system can be used to test the efficacy of anti-viral medications.
[0170] The device of the present invention may also be used to aid in the
diagnosis and treatment of malignancies and diseases. For example, biopsies of
any tissue
(e.g., bone marrow, skin; liver) may be talcen from a patient suspected of
having a
malignancy. The patient's culture can be used in vitro to screen cytotoxic
and/or
pharmaceutical compounds in order to identify those that are most efficacious;
i.e., those that
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kill the inalignant or diseased cells, yet spare the nornial cells. These
agents can then be used
to therapeutically treat the patient.
[0171] In yet another embodiment of the invention, the device can be used in
vitro
to produce biological products in high yield. For example, a cell that
nattirally produces large
quantities of a particular biological product (e.g., a growth factor,
regulatoiy factor, peptide
honnone, antibody), or a host cell genetically engineered to produce a foreign
gene product,
can be clonally expanded using the in vitro device. If a transformed cell
excretes the gene
product into the nutrient medium, the product may be readily isolated fiom the
spent or
conditioned medium using standard separation techniques (e.g., HPLC, column
chromatography, electrophoretic techniques, to name but a few). A"bioreactor"
can be
devised that would talce advantage of the continuous flow method for feeding
cultures in
vitro. Essentially, as fresh media is passed through the cultures in the
device, the gene
product will be washed out of the culture along with the cells released, from
the culture. The
gene product can be isolated (e.g., by HPLC column chromatography,
electrophoresis) from
the outflow of spent or conditioned media.
- [0172] The present invention also provides a system for screening or
measuring
the effects of various environmental conditions or compounds on a biological
system. For
example air or water conditions could be mimicked or varied in the device. The
inipact of
different known or suspected toxic substances could be tested. The present
invention further
provides a system for screening consumer products, such as cosmetics,
cleansers, or lotions.
It also provides a system for determining the safety and/or efficacy of
nutriceuticals,
nu.tritional supplements, or food additives. The present invention could also
be used as a
miniature bioreactor or cellular production platform to produce cellular
products in quantity.
[0173] Typical efficacy or toxicity experiments using the cliip format
microscale
culture device of the present invention are conipleted within 24 to 48 hours
or less depending
on experimental design. Extended experiments, however, can be performed in
order to test
for the effects of chronic exposure (e.g., genotoxicity, carcinogenicity, or
latent diseases.
[0174] The present invention provides novel devices, systems and metllods as
set
forth within this specification. In general, all technical and scientific
teizns used herein have
the same meaning as commonly understood to one of ordinaiy skill in the art to
which this
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invention belongs, unless clearly indicated otllerwise. For clarification,
listed below are
definitions for certain terins used herein to describe the present invention.
These definitions
apply to the terms as they are used tllrougllout this specification, unless
otlierwise clearly
indicated.
Definition of Terins
[0175) Pliarmacolcinetic-based culture system: An in vitro cell culture
system,
wherein the cells are maintained under conditions providing pharmacokinetic
parameter
values that model those found in vivo. A pharmacokinetic culture device
coniprises a fluidic
network of channels segregated into discrete butintercomiected chambers, where
the specific
chaniber geometry is designed to provide cellular interacti ons, liquid flow,
and liquid
residence parameters that correlate with those found for the corresponding
cells, tissue, or
organ system in vivo. The device is seeded with cells that are appropriate for
conditions being
modeled, e.g., liver cells in a liver-based culture chamber, lung cells in a
lung-based culture
chamber, and the like, to provide the culture system.
[0176] The culture systems of the invention provide for at least one
pharmacokinetic parameter value that is comparable to values obtained for the
cell, tissue, or
organ system of interest in vivo, preferably at least two parameter values,
and may provide
for three or more comparable parameter values. Pharmacolcinetic parameters of
interest
include, for example, interactions between cells, liquid residence time,
liquid to cell ratios,
metabolism by cells, or shear stress.
[0177] By comparable values, it is meant that the actual values do not deviate
more than 25% from the theoretical values. For example, the calculated or
theoretical value
for the liquid residence time in the lung compartment for a rat is 2 seconds
and the actual
value measured in the lung cell culttire chamber of a rat CCA device was 2.5+/-
0.7 seconds.
[0178] The pharmacokinetic parameter value is obtained by using the equations
of
a PBPK model. Such equations have been described in the art, for example see
Poulin and
Theil (2000) J Phartn Sci. 89(l):16-35; Slob et al. (1997) Crit Rev Toxicol.
27(3):261-72;
Haddad et al. (1996) Toxicol Lett. 85(2): 113-26; Hoang (1995) Toxicol Lett.
79(1-3):99-
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106; Kliaak et al. (1995) Toxicol Lett. 79(1-3):87-98; and Ball and Schwartz
(1994) Comput
Biol Med. 24(4):269-76, herein incoiporated by reference. Pharmacokinetic
paranieters can
also be obtained from the published literature, for exaniple see Buclcpitt et
al., (1984) J.
Pharmacol. Exp. Ther. 231:291-300; DelRaso (1993) Toxicol. Lett. 68:91-99;
Haies et al.,
(1981) Ain. Rev. Respir. Dis. 123:533-541.
[0179] Specific physiologic parameters of interest include tissue or organ
liquid
residence time, tissue or organ mass, liquid-to-cell volume ratio, cell shear
stress, etc.
Physiologically relevant parameter values can be obtained empirically
according to
conventional methods, or can be obtained from values known in the art and
publicly
available. Pharmacokinetic parameter values of interest are obtained for an
animal, usually a
mammal, although otlier animal models can also find use, e.g., insects, fish,
reptiles, or
avians. Mamnials include laboratory animals, e.g., mouse, rat, rabbit, or
guinea pig mammals
of economic value, e.g., equine, ovine, caprine, bovine, canine, or feline;
primates, including
monkeys, apes, or humans; and the lilce. Different values may be obtained and
used for
animals of different ages, e.g., fetal, neonatal, infant, child, adult, or
elderly; and for different
physiological states, e.g., diseased, after contact with a pharmaceutically
active agent, after
infection, or under conditions of altered atmospheric pressure.
[0180] Information relevant to the pharmacokinetic parameter values, as well
as
mass balance equations applicable to various substances to be modeled in the
system, is
optionally provided in a data processing component of the culture system,
e.g., look-up tables
,in general purpose inemory set aside for storage, and the like. These
equations represent
physiologically-based phamiacokinetic models for various biological/chemical
substances in
systems.
[0181] Pharmacokinetic culture device: The culture device of the invention
provides a substrate for cell growth. Each device coniprises at least one
chamber, usually at
least two chambers, and may comprise three or more chainbers, where the
chambers are
interconnected by fluidic channels. The chambers can be on a single substrate
or on different
substrates. Preferably each chamber has a geometric configuration distinct
from other
chamber(s) present on the device. The device contains a cover to seal the
chambers and
channels and coinprises at least one inlet and one outlet port that allow for
recirculation of the
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culture medium. The device contains a mechanism to pump the culture medium
tllrough the
system. The culture medium is designed to maintain viability of the cultured
cells. The device
contains a mechanism by wliich test conipounds can be introdticed to the
system.
[0182] In one embodinient of the invention, the device is fabricated on a
microscale as a single unit of not more than about 2.5 cm in a side,
preferably comprising at
least two interconnected chanlbers. The two organ conlpartments are connected
by a channel
of from about 50-150 m wide and 15-25 m deep. For example, one chamber may
represent
the lung, coinprising an interconnected airay of parallel channels, usually at
least about 10
channels, preferably at least about 20 channels. Such channel may have typical
microfluidic
dimensions, e.g., about 30-50 m wide, 5-15 m deep and 3-7 mm long. Another
coinpartnlent may represent the liver, comprising two or more parallel
channels, usually from
about 50-150 m wide, 15-25 m deep and 5-15 cm long in a seipentine shape.
[0183] The device will usually include a mechanism for obtaining signals from
the cells and culture medium. The signals from different chambers and channels
can be
monitored in real time. For example, biosensors can be integrated or external
to the device,
which pennit real-time readout of the physiological status of the cells in the
system.
[0184] The pharmacokinetic culture device of the present invention may be
provided as a chip or substrate. In addition to enhancing the fluid dynamics,
such
microsystems save on space, particularly wllen used in highly parallel
systems, and can be
produced inexpensively. The culture device can be formed from a polymer such
as but not
liniited to polystyrene, and disposed of after one use, eliminating the need
for sterilization. As
a result, the in vitro subsystem can be produced inexpensively and widely
used. In addition,
the cells may be grown in a three-dimensional manner, e.g., to form a tube,
which more
closely replicates the iv vivo environment.
[0185] To model the metabolic response of an animal for any particular agent,
a
bank of parallel or multiplex arrays comprising a plurality (i.e., at least
two) of the cell
culture systems, where each system can be identical, or can be varied with
predetermined
paranleter values or uiput agents and concentrations. The array may comprise
at least about
10, or may even be as many as 100 or more systems. Advantageously, the cell
culture systems
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on microchips can be housed within a siiigle chamber so that all the cell
culture systems
under are exposed to the same conditions during an assay. .
[0186] Alternatively, multiple chips may be interconnected to form a single
device, e.g., to mimic gastrointestinal barriers or the blood brain barrier.
[0187] Cells: Cells for use in the assays of the invention can be an organism,
a
single cell type derived from an organism, and can be a mixture of cell types,
as is typical of
in vivo situations. The culture conditions may include, for example,
temperature, pH,
presence of factors, presence of other cell types, and the like. A variety of
aninial cells can be
used, including any of the animals for wliich pham7acokinetic paraineter
values can be
obtained, as previously described.
[0188] The invention is suitable for use witll any cell type, including
primary
cells, stem cells, progenitor cells, iiormal, genetically-modified,
genetically altered,
iminortalized, and transformed cell lines. The present invention is suitable
for use with single
cell types or cell lines, or with combinations of different cell types.
Preferably the cultured
cells maintain the ability to respond to stiniuli that elicit a response in
their naturally
occurring counteiparts. These may be _ derived from all sources such as
eukaiyotic or
prokaryotic cells. The eukaryotic cells can be plant, or animal in nature,
such as human,
simian, or rodent. They may be of any tissue type (e.g., heart, stomach,
kidney, intestine,
lung, liver, fat, bone, cartilage, skeletal muscle, smooth muscle, cardiac
muscle, bone
marrow, muscle, brain, pancreas), and cell type (e.g., epithelial,
endothelial, mesenchymal,
adipocyte, hematopoietic). Further, a cross-section of tissue or an organ can
be used. For
example, a cross-section of an artery, vein, gastrointestinal tract,
esophagus, or colon could
be used.
[0189] In addition, cells that have been genetically altered or modified so as
to
contain a non-native "recombinant" (also called "exogenous") nucleic acid
sequence, or
modified by antisense technology to provide a gain or loss of genetic function
may be utilized
with the invention. Methods for generating genetically modified cells are
known in the art,
see for example "Current Protocols in Molecular Biology," Ausubel et al., eds,
John Wiley &
Sons, New Yorlc, N.Y., 2000. The cells could be temlinally differentiated or
undifferentiated,
such as a stem cell. The cells of the present invention could be cultured
cells from a variety of
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genetically diverse individuals wlio may respond differently to biologic and
pharmacologic
agents. Genetic diversity can have indirect and direct effects on disease
susceptibility. hi a
direct case, even a single nucleotide change, resulting in a single nucleotide
polymorphism
(SNP), can alter the amino acid sequence of a protein and directly contribute
to disease or
disease susceptibility. For example, certain APO-lipoprotein E genotypes have
been
associated with onset and progression of Alzheimer's disease in some
individuals.
[0190) When certain polymoiphisms are associated with a particular disease
phenotype, cells from individuals identified as carriers of the polymorphism
can be studied
for developmental anomalies, using cells from non-carriers as a control. The
present
invention provide an experimental system for studying developmental anomalies
associated
with particular genetic disease presentations since several different cell
types can be studied
simultaneously, and linked to related cells. For example, neuronal precursors,
glial cells, or
other cells of neural origin, can be used in a device to characterize the
cellular effects of a
compound on the nervous system. Also, systems can be set up so that cells can
be studied to
identify genetic elements that affect drug sensitivity, chemokine and cytokine
response,
response to growth factors, hormones, and inhibitors, as well as responses to
changes in
receptor expression and/or function. This information can be iuivaluable in
designing
treatment methodologies for diseases of genetic origin or for which there is a
genetic
predisposition.
[0191) In one embodiment of the invention, the cells are involved in the
detoxification and metabolism of pharmaceutically active compounds, e.g.,
liver cells,
including hepatocytes; kidney cells including tubule cells; fat cells
including adipocytes that
can retain organic compounds for long periods of time. These cells may be
combined in a
culture system with cells such as lung cells, which are involved in
respiration and
oxygenation processes. These cells may also be combined with cells that are
particularly
sensitive to dainage from an agent of interest, e.g., gut epithelial cells,
bone marrow cells, and
other normally rapidly dividing cells for agents that affect cell division.
Neural cells may be
present to monitor for the effect of an agent for neurotoxicity, and the like.
[0192] The growth characteristics of tumors, and the response of surrounding
tissues and the immune system to tunlor growth are also of interest.
Degenerative diseases,
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including affected tissues and surrounding areas may be exploited to determine
botll the
response of the affected tissue, and the interactions with other parts of the
body.
[0193] The terin "environment" or "culture condition" encompasses cells,
media,
factors, time and teinperature. Environments may also include drugs and other
compounds,
particular atmospheric conditions, pH, salt composition, minerals, etc. Cell
culturing is
typically performed in a sterile environment mimicking physiological
conditions, for
example, at 37 C. in an incubator containing a humidified 92-95% air/5-8% CO2
atmosphere. Cell culturing may be cairied out in nutrient mixtures containing
undeflned
biological fluids such a fetal calf serum, or media that is fully defined and
serum fiee. A
variety of culture media are known in the art and are commercially available.
[0194] The term "physiological conditions" as used herein is defined to mean
that
the cell culturing conditions are very specifically monitored to mimic as
closely as possible
the natural tissue conditions for a particular type of cell in vivo. These
conditions include
such parameters as liquid residence time (i.e., the time that a liquid stays
in an organ); cell to
blood volume ratio, sheer stress on the cells, size of compartment comparable
to natural
organ.
[0195] Screening Assays: Drugs, toxins, cells, pathogens, samples, etc.,
herein
refeiTed to generically as "input variables" are screened for biological
activity by adding to
the pharmacokinetic-based culture system, and then assessing the cultured
cells for changes
in output variables of interest, e.g., consumption of 02, production of CO2i
cell viability, or
expression of proteins of interest. The input variables are typically added in
solution, or
readily soluble fonn, to the medium of cells in culture. The input variables
may be added
using a flow through system, or alternatively, adding a bolus to an otherwise
static solution.
In a flow-through system, two fluids are used, where one is a physiologically
neutral solution,
and the other is the same solution with the test compound added. The first
fluid is passed
over the cells, followed by the second. In a single solution method, a bolus
of the test input
variables is added to the volume of medium surrounding the cells. The overall
composition of
the culture medium should not change significantly with the additioii of the
bolus, or between
the two solutions in a flow througli method.
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[0196] Preferred input variables foi7nulations do not include additional
components, such as preservatives, that have a significant effect on the
overall formulation.
Thus, preferred formulations include a biologically active agent and a
physiologically
acceptable carrier, e.g., water, ethanol, or DMSO. However, if aii agent is
liquid without an
excipient, the formulation may be only the compound itself.
[0197] Preferred input variables include, but are not limited to, viruses,
viral
particles, liposomes, nanoparticles, biodegradable polyiilers, radiolabeled
particles,
radiolabeled biomolecules, toxin-conjugated particles, toxin-conjugated
biomolecules, and
particles or biomolecules conjugated witli stabilizing agents. A "stabilizing
agent" is an agent
used to stabilize drugs and provide a controlled release. Such agents include
albumin,
polyethyleneglycol, poly(ethylene-co-vinyl acetate), and poly(lactide-co-
glycolide).
[0198] A plurality of assays inay be iun in parallel with different input
variable
concentrations to obtain a differential response to the various
concentrations. As la-iown in
the art, determining the effective concentration of an agent typically uses a
range of
concentrations resulting from 1:10, or other log scale, dilutions. The
concentrations may be
further refined with a second series of dilutions, if necessaiy. Typically,
one of these
concentrations serves as a negative control, i.e., at zero concentration or
below the level of
detection.
[0199] Input variables of interest enconzpass numerous chemical classes,
thouglz
frequently they are organic molecules. A preferred embodiment is the use of
the methods of
the invention to screen samples for toxicity, e.g., environmental samples or
drug. Candidate
agents may coinprise functional groups necessary for structural interaction
with proteins,
particularly hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or
carboxyl group, preferably at least two of the functional chemical groups. The
candidate
agents often comprise cyclical carbon or heterocyclic stnictures and/or
aromatic or
polyaromatic structures substituted with one or more of the above functional
groups.
Candidate agents are also found among biomolecules including peptides,
saccliarides, fatty
acids, steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0200] Included are pharmacologically active drugs and genetically active
molecules. Compounds of interest include chemotherapeutic agents, anti-
inflammatory
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agents, hoi-liiones or hoi7ilone antagonists, ion channel modifiers, and
neuroactive agents.
Exemplary of pharmaceutical agents suitable for this invention are those
described in "The
Pharmacological Basis of Therapeutics," Goodnlaii and Gilnlan, McGraw-Hill,
New Yorlc,
N.Y., (1996), Nintli edition, under the sections: Drugs Acting at Synaptic and
Neuroeffector
Junctional Sites; Diugs Acting on the Central Nervous System; Autacoids: Drug
Therapy of
Inflanimation; Water, Salts and Ions; Drugs Affecting Renal Function and
Electrolyte
Metabolisnl; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function;
Drugs
Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy
of
Microbial Diseases; Chemotlierapy of Neoplastic Diseases; Drugs Used for
Immunosuppression; Drugs Acting on Blood-Fol-lning Organs; Hormones and
Hormone
Antagonists; Vitamins, Dermatology; and-Toxicology, all incoiporated herein by
reference.
Also included are toxins, and biological and chemical warfare agents, for
example see
Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New Yorlc,
1992).
[0201] Test compounds include all of the classes of molecules described above,
and may f-urther conlprise samples of unknown content. While many samples will
comprise
compounds in solution, solid samples that can be dissolved in a suitable
solvent may also be
assayed. Samples of interest include environmental samples, e.g., ground
water, sea water, or
mining waste; biological samples, e.g., lysates prepared from crops or tissue
samples;
manufacturing samples, e.g., time course during preparation of
pharmaceuticals; as well as
libraries of compounds prepared for analysis; and the like. Samples of
interest include
conlpounds being assessed for potential therapeutic value, e.g., drug
candidates from plant or
fungal cells.
[0202] The term "samples" also includes the fluids described above to which
additional components have been added, for exaniple, components that affect
the ionic
strength, pH, or total protein concentration. hi addition, the samples may be
treated to achieve
at least partial fractionation or concentration. Biological samples may be
stored if care is
talcen to reduce degradation of the compound, e.g., under nitrogen, frozen, or
a combination
thereof. The volume of sample used is sufficient to allow for measurable
detection, usually
from about 0.1 l to 1 ml of a biological sample is sufficient.
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[0203] Conipounds and candidate agents are obtained from a wide variety of
sources including libraries of syiithetic or natural coinpounds. For exanlple,
numerous means
are available for random and directed syntliesis of a widevariety of organic
compounds and
biomolecules, including expression of randomized oligonucleotides and
oligopeptides.
Altei7iatively, libraries of natural compounds in the form of bacterial,
fungal, plant and
animal extracts are available or readily produced. Additionally, naturally or
syntlietically
produced libraries and compounds are readily modified through conventional
chemical,
physical and biochemical means, and n7ay be used to produce combinatorial
libraries. Known
phaimacological agents may be subjected to directed or random chemical
modifications, such
as acylation, allcylation, esterification, amidification to produce structural
analogs.
[0204] Output variables: Output variables are quantifiable elements of cells,
particularly elements that can be accurately measured in a high throughput
system. An output
can be any cell component or cell product including, e.g., viability,
respiration, metabolism,
cell surface determinant, receptor, protein or conformational or
posttranslational modification
thereof, lipid, carbohydrate, organic or inorganic molecule, mRNA, DNA, or a
portion
derived from. such a cell component. While most outputs will provide a
quantitative readout,
in some instances a semi-quantitative or qualitative result will be obtained.
Readouts may
include a single. detemlined value, or may include mean, median value or the
variance.
Characteristically a range of readout values will be obtained for each output.
Variability is
expected and a range of values for a set of test outputs can be established
using standard
statistical methods.
[0205] Various methods can be utilized for quantifying the presence of the
selected marlcers. For measuring the amount of a molecule that is present, a
convenient
metliod is to label the molecule with a detectable moiety, which may be
fluorescent,
luminescent, radioactive, or enzymatically active. Fluorescent and luminescent
moieties are
readily available for labeling virtually any biomolecule, stiucture, or cell
type.
Immunofluorescent moieties can be directed to bind not only to specific
proteins but also
specific conformations, cleavage products, or site modifications like
phosphorylation.
Individual peptides and proteins can be engineered to autofluoresce, e.g., by
expressing them
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as green fluorescent protein chimeras inside cells (for a review, see Jones et
al. (1999) Trends
Biotechnol. 17(12):477-81).
[0206] Output variables may be measured by immunoassay tecliniques such as,
inununohistochemistry, radioimmunoassay (RIA) or enzynie linlced
inimunosorbance assay
(ELISA) and related non-enzyniatic techniques. These techniques utilize
specific antibodies
as reporter molecules that are particularly useful due to their higli degree
of specificity for
attaching to a single molecular target. Cell based ELISA or related non-
enzymatic or
fluorescence-based methods enable measuremerit of cell surface parameters.
Readouts from
such assays may be the mean fluorescence associated with individual
fluorescent antibody-
detected cell surface molecules or cytolcines, or the average fluorescence
intensity, the
niedian fluorescence intensity, the variance in fluorescence intensity, or
some relationship
among these.
[0207] Data analysis: The results of screening assays may be compared to
results
obtained from reference compounds, concentration curves, controls, etc. The
comparison of
results is accomplished by the use of suitable deduction protocols, Al
systems, statistical
comparisons, etc.
[0208] A database of reference output data can be compiled. These databases
may
include results from known agents or combinations of agents, as well as
references from the
analysis of cells treated under environmental conditions in wliich single or
multiple
environmental conditions or parameters are removed or specifically altered. A
data matrix
may be generated, where each point of the data matrix corresponds to a readout
from a output
variable, where data for each output may come from replicate determinations,
e.g., multiple
individual cells of the same type.
[0209] The readout may be a mean, average, median or the variance or other
statistically or mathematically derived value associated witli the
measurement. The output
readout information may be further refined by direct comparison with the
corresponding
reference readout. The absolute values obtained for each output under
identical conditions
will display a variability that is inherent in live biological systems and
also reflects individual
cellular variability as well as the variability inherent between individuals.
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Cell Culttues and Cell Culture Devices
[0210] The culture devices of the inventioii comprise a microfluidic networlc
of
channels segregated into one or more discrete but interconnected chambers,
preferably
integrated into a chip format. The specific chamber geometry is designed to
provide cellular
interactions, liquid flow, and liquid residence parameters that correlate with
those found for
the corresponding cells, tissue, or organ systems in vivo.
[0211] Optimized chamber geometries can be developed by repeating the
procedure of testing parameter values in response to fluid flows aiid chaiiges
in dimensions,
until the selected values are obtained. Optimization of the substrate includes
selecting the
number of chambers, choosing a chamber geometiy that provides the proper cell
to volume
ratio, selecting a chamber size that provides the proper tissue or organ size
ratio, choosing the
optimal fluid flow rates that provides for the coiTect liquid residence time,
then calculating
the cell shear stress based on these values. If the cell shear stress is over
the maximum
allowable value, new parameter values are selected and the process is
repeated. Another
embodiment of the CCA device includes where the cells are grown within hollow
tubes
rather than on the bottom and sides of channels or chambers. It has been
demonstrated that
cells growing in such a three-dimensional tissue constiuct are more authentic
with respect to
certain in vivo tissues (Griffith (1998) PhARMA Biol. Biotech. Conf.,
Coronado, Calif.,
March 15-18).
[0212] Three primary design parameters are considered in creating the 3-D
culture
device. The first is the residence time that the fluid is in contact with a
particular tissue or
within a well. The residence times are chosen to reflect the amount of time
blood stays in
contact with organ tissue, represented by a well, in one pass of the
circulatory system. The
second is the radius of the tubes the cells are grown in. For example, the
radius of the tubes
for replicating liver are witliin a range of 200-400 m. It should be noted
that if the radius of
the tubes gets too large, the cells will essentially see a flat surface and
will form a monolayer
on the tube.
[0213] The third parameter is the proportion of flow that arrives at each
module.
Adjusting the geometry of the flow channels partitions the flow fiom the
chambers. The
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channels or tubes to each module or chamber are typically of different lengths
to equilibrate
the pressure drops and balance the flow. After the fluid leaves the other
tissues, it can be re-
circulated by a pump. The flow rate through the tubes was calculated from the
tube
dimensions and the resideiice time. Given a flow rate, the shear stress on the
cells was
calculated to ensure that the value did not exceed the cells' stress limit.
The very short
residence time required in the lung tissue malces it impossible to use a well
and tube approach
for this organ. The shear stress is too high and tlierefore, the lung tissue
section remains flow-
over with a lung tissue monolayer.
[0214] Since the system of the present invention is interactive (i.e., the
computer
not only senses but also controls the conditions within the test), corrections
can be
dynamically instituted into the system and appropriately noted and documented
for apprising
researchers of the dynainics of the test being run.
[0215] Data gathering by the computer consists of the collection of data
required
for continuous in-line monitoring of test chemical effluent from each
compartment. Sensors,
preferably of the flow-tllrough type, are disposed in-line with the outflow
from each
compartment, to thus detect, analyze and provide quantitative data regarding
the test chemical
effluent from each coinpartment.
[0216] Microprocessors can also serve to conlpute a physiologically-based
pharmacokinetic (PBPI,',-) model for a particular test chemical. These
calculations may sei-ve
as the basis for setting the flow rates among compartments and excretion rates
for the test
cheinical from the system. However, they may also serve as a theoretical
estimate for the test
chemical. At the conclusion of the experiment, predictions concerriing the
concentrations of
test chemicals and metabolites made by the PBPK determination can be compared
to the
sensor data. Hard copy output compares the PBPK model witli experimental
results.
[0217] Several prototype CCA systeins have been constructed and tested. FIG.
17A depicts a "first generation" tliree compartinent device. The dimensions
were as follows:
wafer was 2 em x 2 em; lung chaniber had 20 channels (5 mnl long) 40 m x 20
m (w x d);
liver cliamber had 2 channels (100 mm long) 100 m x 20 m (w x d). The first
step in using
this device is to inject the fluid using a syringe pump until all the channels
filled up. Second,
a peristaltic pump is used to recirculate the fluid. FIG. 17B shows a cross-
sectional view of
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the device, demonstrating the fluidics of the system. It was found that 400
ni thick elastomer
gave a better seal, and that plexiglass and gel-loading tips are much less
fragile than otlier
materials. This device had problems with a high pressure drop and lealcs
occurred at 90
bends.
[0218] Cell attachment studies were perfomied using this "flrst generation"
device. L2 cells were placed in the lung chamber and H4IIE cells were placed
in the liver
chamber. Poly-D-lysine was adsorbed to the surface of the chambers to promote
attachment
of the cells within the channels. Unfortunately, cells attached outside the
trenches, so
different substrates were tested and surfaces were modified.
[0219] FIG. 18A depicts a "second generation" device. The dimensions were as
follows: chip was 2 cm x 2 cm; etcliing is 20 m deep; lung chainber was 2 mm
x 2 mm (w x
1); liver chamber was 7.5 mm x 10 mm (w x 1). The lung chamber contained 5 m
tall ridges
to increase cell attachment (FIG. 18B), and the liver chamber contained 20 m
tall pillars to
simulate percolation (FIG. 18C).
[0220] FIG. 19 depicts a "third generation" device. The dimensions were as
follows: chip was 2 cm x 2 cm; lung chamber was 2 mm x 2 mm (w x 1); liver
chamber was
3.7 mm x 3.8 mm (w x 1); and the "other tissue" chaniber was 7 mm x 7 mm (w x
1). Fluid
was split from the lmlg chamber, with 20% going to the liver and 80% to the
other tissue
chamber. Portions of the chambers (dashed) are 100 m deep to reduce pressure
drops, and
other portions (solid) are 20 m deep to give realistic liquid-cell ratios.
[0221] FIG. 20 is a flow diagram for a five compartment PBPK model CCA. This
device adds chambers for fat cells, a chamber for slowly perfused fluid and
for rapidly
perfused fluid. Such a device can be used for bioaccumulation studies,
cytotoxicity studies
and metabolic - activities. Other devices can be developed with various
permutations. For
example, a diaphragm pump with gas exchange can be added, or an online
biosensor, or a
microelectromechanical (MEM) pump, or a biosensor and electronic interface. A
device can
be developed to mimic oral delivery of a pharmaceutical. Alternatively, a
device can be
developed to mimic the blood-brain barrier.
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Fabrication
[0222] The cell culture device typically comprises an aggregation of separate
elements, e.g., chambers, channels, inlet, or outlets, wlzich when
appropriately mated or,
joined together, foi7n the culture device of the invention. Preferably the
elements are
provided in an integrated, "chip-based" fornlat.
[0223] The fluidics of a device are appropriately scaled for the size of the
device.
hi a chip-based format, the fluidic connections are "microfluidic," such a
system contains a
fluidic element, such as a passage, chanzber or conduit that has at least one
internal cross-
sectional dimension, e.g., depth or width, of between about 0.1 m and 500 m.
In the
devices of the present invention, the channels between chambers typically
include at least one
microscale channel.
[0224] Typically, microfluidic devices comprise a top portion, a bottom
portion,
and an interior portion, wlierein the interior portion substantially defines
the channels and
chambers of the - device. In preferred aspects, the bottom portion will
coniprise a solid
substrate that is substantially planar in structure, and which has at least
one substantially flat
upper surface. A variety of substrate materials may be employed as the bottom
portion.
Typically, because the devices are microfabricated, substrate materials will
generally be
selected based upon their compatibility with known microfabrication
techniques, e.g.,
photolithography, thin-film deposition, wet chemical etching, reactive ion
etching,
inductively coupled plasma deep silicon etching, laser ablation, air abrasion
techniques,
injection molding, embossing, and other techniques.
[0225] The substrate materials of the present invention comprise polyineric
materials, e.g., plastics, such as polystyrene, polymethylmethacrylate (PMMA),
polycarbonate, polytetrafluoroethylene (TEFLONTM), polyvinylchloride (PVC),
polydimethylsiloxane (PDMS), polysulfone, and the like. Such substrates are
readily
manufactured from microfabricated masters, using well known molding
techniques, such as
injection molding, einbossing or stamping, or by polymerizing the polymeric
precursor
material within the mold. Such polynleric substrate materials are preferred
for their ease of
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inanufacttue, low cost and disposability, as well as their general inerhiess
to most extreme
reaction conditions. These polymeric materials may include treated surfaces,
e.g., derivatized
or coated surfaces, to enhance their utility in the system, e.g., provide
enhanced fluid
direction, cellular attachment or cellular segregatioil.
[0226] The channels and/or chambers of the microfluidic devices are typically
fabricated into the upper surface of the substrate, or bottom portion, using
the above
described microfabrication techniques, as microscale grooves or indentations.
The lower
surface of the top portion of the microfluidic device, wliich top portion
typically comprises a
second planar substrate, is then overlaid upon and bonded to the surface of
the bottom
substrate, sealing the channels and/or chambers (the interior portion) of the
device at the
interface of these two components. Bonding of the top portion to the bottom
portion may be -
carried out using a variety of known methods, depending upon the nature of the
substrate
material. For example, in the case of glass substrates, thermal bonding
techniques may be
used that employ elevated temperatures and pressure to bond the top portion of
the device to
the bottom portion. Polymeric substrates may be bonded using similar
techniques, except that
the teinperatures used are generally lower to prevent excessive melting of the
substrate
material. Alternative methods may also be used to bond polymeric parts of the
device
together, including acoustic welding techniques, or the use of adhesives,
e.g., UV curable
adhesives, and the like.
[0227] The device will generally comprise a punip, such as a low flow rate
peristaltic pump. A small bore flexible tubing would be attached to the outlet
of the device,
passing through the peristaltic pump and attached to the inlet of the device,
tlius forming a
closed loop system. The pump generally operates at flow rates on the order of
1 gL/min. The
pump system can be any fluid pump device, such as a diaphragm, and can be
either integral to
the CCA device (chip-based system) or a separate component as described above.
[0228] The device can be connected to or interfaced with a processor, which
stores and/or analyzes the signal from each the biosensors. The processor in
tuni forwards the
data to computer memory (either hard disk or RAM) from where it can be used by
a software
program to further analyze, print and/or display the results.
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Description of Exemplaiy Embodiments
[0229] In the following detailed description of specific enibodiments,
reference is
made to the acconzpanying drawings, which fornz a part hereof, and' in whicli
are shown by
way of illustration specific embodiments in which the invention may be
practiced. It is to be
understood that other embodinients may be utilized and structural chauges may
be made
witllout departing from the scope of the present invention.
[0230]- FIG. 1 is-a block diagram of an in vitro systeni in accordance with
the
present invention. Lung cell sinzulating chamber 102 receives oxygenated
culture medium
fiom gas exchange device 103. Such oxygenated medium is obtained by contacting
culture
medium with oxygen-containing gas so that the culture medium absorbs - oxygen-
containing
gas and desorbs carbon dioxide-containing gas. The culture medium exiting lung
cell
sin7ulating chainber 102 is analogous to arterial blood 106 in mammals. The
oxygen-
containing culture medium constituting arterial blood 106 is then supplied to
liver simulating
chamber 108, other tissue simulating chanzber 110, fat simulating chamber 112,
and kidney
simulating chamber 114. The culture mediunl departing from liver simulating
chamber 108,
other tissue simulating chamber 110, fat simulating chamber 112, and kidney
simulating
chamber 114 is analogous to venous blood 104 in manlmals. As shown in FIG. 1,
the culture
medium corresponding to venous blood 104 is returned to luiig cell simulating
chamber 102.
The system of the present invention also includes gut simulating chamber 116
and peritoneal
cavity simulating chamber 118, both of which constitute sites for introduction
of test
compounds. As in mammals, waste liquid 115 is withdrawn from kidney simulating
chamber
114.
[0231] FIG. 2 is a simplified schematic view of one embodinient of the system
200 of the present invention. The system 200 includes a lung cell culture
chamber 210, a liver
cell culture chamber 212, a fat cell culture chamber 213, an otlier tissues
chamber 214, and a
gas exchange chamber 250. The chambers 210, 212, 213, 214, and 250 are fonned
on a
substrate of silicon that is commonly refeiTed to as a chip 230. It should be
noted that more
than four cell culture chambers may be housed or formed on a single chip 230.
A fluid path
240 connects the chambers 210, 212, 213, 214, and 250.
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[0232] The chambers have an inlet 211 and an outlet 215. The inlet 211 is
located
at one end of the gas exchange chamber 250. The outlet 215 is located at one
end of the liver
cell culture chamber 212. The chambers 210, 212, 213, 214, and 250 and the
fluid path 240
are located substantially between the inlet 211 and the outlet 215. The system
includes a
pump 260 for circulating the fluid in the system 200. A microtube 270 connects
between the
outlet 215 and the inlet side of the pump 260. A microtube 271 connects the
outlet side of the
pump 260 to the inlet 211. The cell culture chambers 210, 212, 213, 214 the
gas exchange
chamber 250, the fluid path 240, and the pump 260 foml the system 200. The
system may
include additional cell culture chambers. One common cell culture chamber
added is one
sinlulating kidney.
[0233] FIG. 3 is a schematic of another enlbodiment of the invention. In FIG.
3 a
first signal path 310, a second signal path 320, and a third signal path 330
are provided on the
chip 230. Signals for monitoring various aspects of each cell culture system
200 can be taken
from the chip 230 and at specific locations on the chip 230 and moved to
outputs off the chip
230. One example, the signal paths 310, 320, 330 on the chip 230 are
integrated buried
waveguides. The chip 230, in such an embodiment, could be made of silicon,
glass or a
polyiner. The waveguide 310, 320, 330 would caily light to the edge of the
chip where a
transducer 312, 322, 332 would be located to transform the light signal to an
electrical signal.
The cells within the system 200 could then be monitored for fluorescence,
luminescence, or
absorption or all these properties to interrogate and monitor the cells within
the system 200.
Checking fluorescence requires a light source. The light source is used to
interrogate the
molecule and the signal carrier, such as a waveguide 310, 320, 330 or a fiber
optic captures
the signal and sends it off the chip 230. The signal carrier, 310, 320, 330
would direct light to
a photodetector near the end of the signal calTying portion of the chip 310,
320, 330.
[0234] FIG. 4 is a schematic view of another embodin-ient of the system 200 of
the present invention. In this embodiment, biosensors 410, 420, 430, 440, 450,
and 460 are
positioned on the chip tipstream and downstream of each of the cell culture
chambers of the
chip 230. The biosensors 410, 420, 430, 440, 450, 460 monitor the oxygen,
carbon dioxide,
and/or pH of the medium. These sensors allow monitoring of the system 200 and
adjustment
of gas levels as needed to maintain a healthy environment: In addition, if
positioned just
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upstream and downstreain of each cell conipartinent, biosensors provide useful
infomiation
on cellular metabolism and viability.
[0235] FIGS. 5A tlirough 5G show steps used to fabricate a polymer-based
disposable chip 230. A silicon wafer 20 is spin coated with a thiil layer of
photoresist 21
(FIG. 5A). The photoresist 21 is exposed to UV liglit 22 through a photomask
23 containing
the desired features (FIG. 5B). The UV exposed photoresist 21 is developed
away in an
appropriate solvent tllus exposing the silicon 20 (FIG. 5C). The silicon 20 is
etched to a
desired depth using an inductively coupled plasma etching system (FIG. 5D).
The remaining
photoresist is removed with an appropriate solvent (FIG. 5E). A veiy thin gold
(or Ti) plating
base 24 is deposited on the silicon substrate 20 creating a template for the
electroplating
process, as shown in FIG. 5E. The sample is immersed in a nickel sulfamate
type plating bath
and nickel 25 is electroplated onto the silicon teniplate 20 until the niclcel
thiclcness is
sufficient, with the gold acting as a conducting layer. The nickel master
grows off the gold
layer, and the gold becomes a part of the nickel master. This forms Ni
features 25, shown in
FIG. 5F. The plating rate, which is a function of plating current, teniplate
diameter and
teniplate thiclcness, is calibrated for about 45 nm/min. After fabrication,
the features 25 are
examined using a microscope to verify the feature dimensions. The resulting
nickel features
25 must be uniform and have the desired shape. The nickel master 25 and the
polymer
substrate 26 are heated to just above the glass transition temperature of the
polymer. The
nickel master 25 and polymer 26 are brought into contact and the features of
the nickel
master 25 are embossed into the polymer substrate 26. The nickel master 25 is
removed thus
producing a polymer 26 containing the identical features of the original
silicon wafer 20
(FIG. 5G).
[0236] FIG. 6 is a schematic view of a third embodiinent of the system 200 of
the
present invention. In this embodiment, biosensors 600, 602, 604 are positioned
about the
periphery of the chip 230. The biosensors 600, 602, 604 are used to fiu-ther
monitor the status
of the cells of the system 200 created on the cliip 230. Advantageously, by
positioning the
biosensors 600, 602, 604 about the periphery of the chip 230, the chip 230
could be made to
be disposable with the least amount of cost. hi other words, the biosensors
600, 602, 604
would not have to be thrown away with the chip 230. It should be noted that
biosensors 600,
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602, 604 may also be provided on- board -the disposable chip 230. This
particular option
would not be as cost effective since the biosensors 600, 602, 604 disposing
the chip 230 also
results in tlirowing away the liiosensors 600, 602, 604. It is more cost
effective when the
biosensors 600, 602, 604 are positioned off the chip 230 since the biosensors
600, 602, 604
are reused rather than disposed of after each use. Each of the biosensors 600,
602, 604 is
connected tothe inputs of a computer 620.
[0237] FIG. 7 is a schematic further detailing the computer 620. The computer
620 monitors and regulates operations of the system 200 of each chip 230.
Computer 620
includes a microprocessor provided with input/output interface 700 and
internal
register/cache memory 702. As shown, microprocessor 798 interfaces to keyboard
704
through connection 716, to non-volatile storage memoiy 706, general purpose
memory 708,
and look-up tables 710 througli connector 718, and to printer/plotter recorder
712 and display
714 through connector 720.
[0238] Non-volatile storage memoiy 706 may be in the form of a CD writeable
memory, a magnetic tape memory, disk drive, or the like. Look-up tables 710
may physically
comprise a portion of general purpose memory 708 that is set aside for storage
of a set of
mass balance equations applicable to various substances to be modeled in the
system. These
equatioizs represent physiologically-based pharmacokinetic models for various
biological/chemical substances in systems. hiternal register/cache memory 702_
and general
purpose memoiy 708 contain a system program in the form of a plurality of
program
instructions and special data for automatically controlling virtually every
function in the
system 200 of each chip 230. The computer can also control and regulate the
pump 260
associated with the system 200.
[0239] Fluid flow may also be provided as inputs to microprocessor 798
tlirough
input/output interface 700 from flow meters. This permits precise control over
fluid flow
rates within the system by adjustment of program conimands that are
transmitted to pumps
260 tlirough pump control lines, respectively. For example, the flow rates may
be set to 9.5
L/min. in conduit 58, 2.5 L/min. through flow meter 66, 7 L/min. through
flow meter 78,
and 2.5 L/min. in conduit 70. The temperature of culture medium in reservoir
50 may also
be regulated by microprocessor 798, which receives, through input/output
interface 700 and
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temperature indicator line 728, tempcrature measurements from temperature
probe 792. In
response to these signals, heater coi1790 is tunied on and off by
microprocessor 798 througli
input/output interface 700 and heater coil control line 730.
[0240] Biological and toxicological reactions/changes in cell culture
chanzbers
210 and 212 are detected by sensors 600, 602 and 604, respectively, and
communicated to
microprocessor 798 througli control lines as well as input/output interface
700. The sensors
can be designed to represent test results in terins of specific values or
ranges of wavelengths
to represent test results.
[0241] Microprocessor 798 is also quite easily adaptable to include a program
to
provide the researcher with interactive control via keyboard 704. This
permits, for example,
directing the coniputer to specifically check on the conditions of any of the
culture
compartments at any given time.
[0242] A further option provided by the present invention is the ability to
recall
previously stored test results for siniilar experinients by recalling
infomlation from the
CD/tape memory 706. Thus, memory 706 may be preprogrammed to hold historical
data
talcen from published information, data gathered fiom previously run tests
conducted with the
system of the present invention or data derived from tlieoretical
calculations. The provision
of the CD/tape memory also permits the system to be used as an inforination
researching tool.
It can, for example, obtain the research data pertaining to a particular test
chemical, or to a
particular culture line, based on selection infoimation inputted into
microprocessor 798 via
keyboard 704. By including or developing a large library of information in
manlory 706,
researchers will be able to configure and plan test iuns more intelligently.
[0243] FIG. 8 is a schematic showing that more tlian one chip 230 can be
housed
within a single housing 800. The housing 800 can be an environmental chainber
that
maintains the same conditions for each of the chips 230 within the housing.
The housing 800
inch.tdes a plurality of chip locations 810, 812, 814, 816. The outputs from
each chip 230 or
chip location 810, 812, 814, 816 is input to a coniputer 620. The computer 620
is then able to
monitor the systems 200 from multiple chips 230 in real time.
[0244] FIG. 9 is a schematic showing that a test may include sets of chips 230
in
different housings 800, 900. The outputs of each of the chips 230 can be
monitored for
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changes in the environment, such as when temperature-is slightly elevated, or
the like. It is
further contemplated that each of the chips in one housing may have the same
cell culture
tliereon or that the chips 230 in the housing 800 may have chips
interconnected to one
another to forin different portions of a mainmal or interdependent organs
witliin a housing.
[0245] The chips 230 discussed with respect to FIGS. 2-4 and 6-9 use two
dimensional cell culture chambers 210, 212, 213, 214. Since tliree dimensional
tissue culture
constructs may be more authentic in their metabolism, yet another of the chip
1000 addresses
the inclusion of three dimensional constructs. The following describes the
creation of a
microscale cell culture analogous device ("CCA"), wliich incorporates three
dimensional
tissues in a modular format. The CCA device or chip 1000 incorporates a flow
over approach
for lung cell chambers and a flow-through approach for other organs. The flow-
through
approach to CCA design is further discussed below.
[0246] FIG. 10 shows a schematic and flow regime for a chip 1000. The chip.
1000 includes four wells or tissue modules. The chip 1000 includes a lung well
1010, a liver
well 1020, a fat well 1030, and a slowly perfused well 1040, and a rapidly
perfused well
1050. Tubes are used to circulate a fluid through the chip 1000. A pump 1060
moves the
fluid through the tubes. The lung well 1010 initially receives all of the
flow. After the lung
1010, the fluid will partition into the four tissue modules. The liver module
will get 25% of
the flow, the fat module 9%, the slowly perfused module 15% and the rapidly
perfused
section 51%. Adjusting the geometry of the flow chamiels will partition the
flow from the
lung well 1010. The channels to each module will be of different lengths to
equilibrate the
pressure drops and balance the flow. After the fluid leaves the other tissues,
it will be re-
circulated back into the lung compartment via the pump 1060. Each of the wells
or tissue
modules 1020, 1030, 1040, 1050 holds tissue. The tissue is held in microscale
tubes 1022,
1032, 1042, 1052 witliin the wells 1020, 1030, 1040, 1050. As shown in FIG.
10, there is
only one microscale tube 1022, 1032, 1042, 1052 per well 1020, 1030, 1040,
1050. It should
be noted that a plurality of microtubes may be placed in a well. -
[0247] In operation, there are two methods that allow three dimensional tissue
to
be incorporated into a CCA device or chip 1000. Both methods involve the flow
of
inoculated medium through microscale tubes of polystyrene or glass. The cells
under test
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adhere to the inside of the tubes and aggregate into three dimensional tissue.
The tubes are
collected, bundled and placed into wells on a chip 1000. Each well becomes an
organ module
that the aqueous dilig will flow tllrough to contact the tissue.
[0248] The first metliod to allow incoiporatioii of three dimensional tissue
involves a flow-through reactor strategy. Openings are formed in a silicon
wafer and
channeled medium-is then passed through the openings. The silicon on the
inside surface of
the openings provided a scaffold for the cells and they aggregated into three
dimensional
tissue. To apply this technique to a polymer CCA 1000, the polymer tubes can
either be
treated with an adhesion protein or the cells can be cultured in serum-added
medium. Botli
serum and an adhesion protein allow the cells to stick to the inside surface
of the tube.
[0249] The secoiid method involves culturing the cells in a HARV microgravity
reactor. By scaffolding the tubes in the center of the rotating reactor, or by
introducing free-
floating tubes into the culture medium, the cells form three dimensional
aggregates in some
of the tubes. Due to the heightened activity of cells grown in microgravity,
these tissue
constricts have superior function compared to two dimensional tissue or the
tissue formed in
the metliod above. The tubes with tissue inside of them can be separated
according to weight
or density and placed on the device.
[0250] FIG. 11 is a partially exploded isometric view of a cell culture analog
device 1100 that incoiporates chip 1000. The chip 1000 includes a lung cell
culture area 1010
and a plurality of wells that are connected to the lung cell culture area
1010. The wells
include a liver tissue well 1020, a fat tissue well 1030, a slowly perfused
well 1040, and a
rapidly perfused well 1050. Microscale tubes containing the various tissues
fit within the well
1020, 1030, 1040, and 1050. Each well includes an output-to an elastomeric
bottom 1110 that
is attached to the chip 1000. The elastomer 1110 is part of a pump. An
actuator 1120 presses
against the elastomer to produce a pumping action to move the fluid of the
system 1100 or to
circulate the fluid of the system 1100 from the wells back to the lung tissue
module 1010 via
a return line 1130. A glass layer is placed over the top of the chip to cover
the lung tissue
module 1010 and the various wells 1020, 1030, 1040, and 1050. It should be
noted that the
channels 1021, 1031, 1041, and 1051 are dimensioned to produce certain flow
rates through
the various wells 1020, 1030, 1040, and 1050. Rather than adjust the length
and width of the
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various channels 1021, 1031, 1041, 1051 it is contemplated that otlier flow
restrictors can be
placed along the channel in order to provide for variability within the flow
rates to the
various wells 1020, 1030, 1040, and 1050. The glass top 1140 can be replaced
with a
niembrane that flexes and plunger ball-type valves can be added so that the
flows in the
channels 1021, 1031, 1041, and 1051 can be regulated by other than the
dimensions of the
channel.
[0251] The chip 1100 can be made out of silicon but is more cost effective to
nialce the chip 1000 out of polystyrene or some other suitable plastic. Each
chip is first
formed in silicon by conventional means. A nickel master is then formed from
the silicon. In
otlier words, the chip 1000 is manufactured by replica molding polystyrene and
silicone
elastomer on silicon and nickel masters. Of course, the first step in the
manufacture of a
polynzer chip is to produce the chip on a silicon wafer. Initially, a layer of
photoresist 1210 is
placed_ on a silicon wafer 1200. A mask is placed over the photoresist 1210.
The mask
contains the pattern of a lung tissue culture area 1010. The mask allows UV
light to pass to
the photoresist to expose just the portion corresponding to the lung area
1010. The
photoresist is then developed to produce an opening 1211, which coiresponds to
the lung
tissue culture area 1010. The silicon wafer with the photoresist is then
etched to produce the
lung opening 1010 within the silicon wafer 1200. The photoresist 1210 is then
removed from
the silicon wafer 1200 leaving the silicon wafer with the lung well 1010.
Another layer of
photoresist 1220 is then placed onto the wafer 1200. A mask is placed over the
wafer. The
mask allows for exposure of the various wells or fluid channels including
1021, 1031, 1041,
and 1051, which are used to connect the lung well 1010 with the various wells
1020, 1030,
1040, and 1050. The mask exposes the photoresist in the area of the fluid
channel. The
photoresist is then developed to remove the exposed photoresist corresponding
to the fluid
flow channels. The exposed area is then etched to a desired depth. Afterwards,
the remaining
photoresist 1220 is removed leaving a silicon wafer 1200 with a lung well 1010
and other
wells 1020, 1030, 1040, and 1050. The next step is to apply yet a third layer
of photoresist
1230. A mask is placed over the photoresist and the mask has openings
corresponding to the
various wells 1020, 1030, 1040, and 1050. The photoresist is masked and
exposed to UV
light to produce openings corresponding to the various wells. The photoresist
is developed
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leaving the exposed silicon areas for wells 1020,- 1030, 1040, and- 1050. The
chip and the
photoresist 1230 are then etched to produce the wells 1020, 1030, 1040, and
1050. The
openings coiTesponding to the tissue modules 1020, 1030, 1040, 1050 is etclied
with plasma
to a depth of approximately 750 micrometers. The opeiiings are then wet etched
another 250
micrometers with KOH to foim a tapered end. The KOH will etch silicon along
its
ciystallographic plane at an angle of 54.7 degrees. The photoresist is then
removed and a
silicon wafer has been forined from which the nickel master can be made.
[0252] Nickel is electroplated onto the silicon chip to create a niclcel
master 1250.
The nickel master is then used to cast or emboss the polymer substrate 1000.
For replica
molding, the polynler is melted or solubilized in an appropriate solvent and
poured onto the
nickel master 1250 and solidifies in the same shape as the initial silicon
chip For embossing,
refer to FIG. 5. The polymer chip 1000 is then mounted on a silicone elastomer
trough 1110.
The polynier and silicone are self-sealing so the layers will form a single
unit. A pneumatic
actuator 1120 is put below the chip to punip fluid collected from the various
tissue modules
1020, 1030, 1040, 1050. Every second, the trough will fill up with 0.032
microliters of fluid.
The actuator will then push up on the silicone and cause the fluid to escape
through the
microtubes back to the lung compartment 1010. The elastomeric trough 1110 and
the actuator
1120 fonn the pump 260 (shown in FIG. 12). The elastomer-coated
polymethylmethacrylate
(PLEXIGLASTM) 1140 is then sealed to the top of the wafer or chip 1000.
[0253] To balance the pressure pull created as the silicone fills up with
liquid, the
polymetliylmethacrylate (PLEXIGLASTM) over the lung cell compartment 1010 is
removed
and replaced with a silicone membrane. This membrane rises and falls in
response to the
action of the silicone pump and keeps the pressure in the device balanced. The
various
microscale tubes are "placed into the wells prior to placing the elastomer-
coated
polynlethylmethacrylate (PLEXIGLASTM) over the chip 1000. A machine for
handling the
microtubes includes an adllesive arm that lowers and collects a specific
number of tissue-
laden tubes. The machine transports the tubes to the device and tightly packs
the tubes into
the respective module wells 1020, 1030, 1040, 1050. The tight packing allows
the force of
friction to keep the tubes in place regardless of any agitation to the cell
culture analog device.
This minimizes leakage of fluid flow around the tubes in the respective wells
1020, 1030,
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1040, 1050. Even with a tight fit, approximately 5-10% of the fluid flow
circumvents the
tubes and flows directly to the silicone base or elastomer trough 1110.
[0254] FIG. 13 shows the elastomer trough. The elastomer trough is a piece of
silicone elastomer with an essentially rectangular opening therein. The
rectangular opening
acts as a fluid reseivoir for the fluids coming from the wells 1020, 1030,
1040, and 1050. The
elastomer trough 1110 has an opening in one side designated by reference
ntimeral 1300. The
return line 1130 has one end that attaches to the opening 1300 in the
elastomer trougli 1110
and another end that attaches tothe luiig well 1010 of the chip 1000.
-[0255] In yet another embodiment, the elastomer trough 1110 is replaced with
a
silicone elastomer pump 1400, which is shown in FIG.14. The silicone elastomer
pump 1400
is designed to more accurately reproduce the circulatoiy system flow on the
chip 1000 and
throughout the system depicted by reference numeral 1100. The pump 1400
includes a first
pulmonaiy chamber 1410 and a secondsystem chamber 1412, which are actuated by
separate
actuators 1420 and 1422. With the multiple chambers 1410 and 1412 a more
physiologically
realistic puniping pattern is created with the multi-trough elastomeric base
on the bottom of
the chip 1000. By creating the nlultiple chambers 1410 and 1412 in the
silicone elastomer
trough 1400 by having actuators that push up on the section of the base at
specific time
intervals, the puinping action of a heart is replicated.
[0256] FIG. 28A is a block-diagram view illustrating a system for controlling
a
microscale culture device, according to one embodiment of the present
invention. In this
embodiment, the system 2800 includes a first microscale culture device 2806
coupled to a
control instrument 2802. The first microscale culture device 2806 includes a
number of
microscale chambers (2808, 2810, 2812, and 2814) with geometries that simulate
a number
of in vivo interactions with a culture medium, wherein each chamber includes
an inlet and an
outlet for flow of the culture medium, and a microfluidic channel
interconnecting the
chambers. The control instrument 2802 includes a computer 2804 to acquire data
from, and
control pharmacokinetic parameters of, the first microscale culture device
2806.
[0257] In anotlier embodiment, the first microscale culture device 2806 is
formed
on a computerized chip. The first microscale culture device 2806 further
includes one or
more sensors coupled to the control instrument 2802 for measuring
physiological events in
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the chanlbers. The sensors include one or more biosensors that monitor the
oxygen, carbon
dioxide, or pH of the culture medium. The control instrument 2802 holds the
first microscale
culture device 2806, and seals a top of the first microscale culture device
2806 to establish
the microfluidic channel. The control instrument 2802 provides the microfluid
iilterconnects,
so that microfluid flows into and out of the device. In anotlier
inzplementation, the computer
2804 controls a pharinacokinetic parameter selected from a group consisting of
group punlp
speed, temperature, length of experiment, and- frequency of data acquisition
of the first
microscale culture device 2806. In one implementation, the computer 2804
provides a set-up
screen so that an operator may also manually specify pump speed, device
temperature, length
of experiment, and frequency of data acquisition (e.g., every fifteen
minutes). In another
implementation, the computer 2804 controls a pharmacokinetic parameter
selected from a
group consisting of flow rate, chamber geometry, and number of cells in the
first microscale
culture device 2806. In this implementation, the system 2800 provides more
rapid and more
sensitive responses as compared to whole animal studies and traditional tissue
culture studies.
By controlling parameters, the system 2800 is no longer physiologically-based.
In another
implementation, tlie computer 2804 further controls one or more pumps in the
first
microscale culture device 2806 to create culture medium residence times in the
chambers
(2808, 2810, 2812, and 2814) comparable to those encountered in the living
body. In another
implementation, the computer 2804 further controls one or more valves
distributed along the
microfluidic channel in a manner that is consistent with a pharmacokinetic
parameter value
associated with a simulated part of a living body.
[0258] In another embodiment, the system 2800 further includes a second
microscale culture device having a number of nzicroscale chambers with
geometries that
simulate a number of in vivo interactions with a culture medium, wherein each
chamber
includes an inlet and an outlet for flow of the culture medium, and a
microfluidic channel
intercoimecting the chambers. The control instrument 2802 is coupled to the
second-
microscale culture device.
[0259] FIG. 28B is a block-diagram view illustrating anotlier embodiment of a
systeni for controlling a microscale culttue device. In this embodiment, the
system 2816
includes the first microscale culture device 2806 coupled to a control
instrument 2818. The
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control instrument 2818 includes the coinputer 2804, a puinp 2820 to control
circulation of
microfluid in the microfluidic channel of the first microscale culture device
2806, a heating
element 2822 to control the temperature of the first microscale culture device
2806, a light
sotirce 2824, and a photodetector 2826 to detect fluorescent emissions fiom
cell
compartnients within the first microscale culttue device 2806. In one
implementation, the
coniputer 2804 records data for fluorescent intensity using a measuring
instrunient of a type
that is selected from a group consisting of colorimetric, fluorometric,
luminescent, and
radiometric. In another implenzentation, the heating elenient 2822 maintains
the first
microscale culture device 2806 at a temperature of thirty-seven degrees
Celsius.
[0260] FIG. 29 is a flow-diagram view illustrating a computerized metllod for
dynamically controlling a microscale culture device, according to one
embodiment of the
present invention. In this enzbodiment, the computerized method 2900 includes
blocks 2902,
2904, 2906, and 2908. Block 2902 includes analyzing data from 'a number of
sensors to
measure plrysiological events in a number of chambers of the microscale
culture device.
Block 2904 includes regulating fluid flow rates of a culture medium in the
chambers of the
microscale culture device. Block 2906 includes detecting biological or
toxicological reactions
in the chambers of the microscale culture device. Upon such detection, bloclc
2908 includes
changing one or more pharmacokinetic parameters of the microscale culture
device.
[0261] In one embodiment, block 2906 (i.e., the detecting) iiicludes detecting
a
change in dimension of a cell compartment of the microscale culture device. In
one
implementation, block 2908 (i.e., the cllanging) includes changing a
pharmacokinetic
parameter selected from a group consisting of interactions between cells,
liquid residence
time, liquid to cell ratios, metabolism by cells, and shear stress in the
microscale culture
device. In another implementation, block 2908 includes changing a
pharmacokinetic
parameter selected from a group consisting of flow rate, chamber geometry, and
nunlber of
cells in the microscale culture device.
[0262] In another embodiment, the conlputerized method 2900 further includes
optimizing chamber geometry within the microscale culture device, wherein the
optimizing
includes selecting a quantity of chambers, choosing a chaniber geometry that
provides a
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proper tissue or organ size ratio, choosing an optimal fluid flow rate that
provides a proper
liquid residence time, and calculating a cell shear stress.
[0263] In another enibodiment, the computerized nzethod 2900 further includes
regulating a temperature of the culture mediunl. In yet anotller embodiment,
the coniputerized
method 2900 fut-tlier includes detecting fluorescent emissions fiom a cell
compartment of the
microscale culture device.
[0264] In another embodiment, a computer-readable medium includes coinputer-
executable instructioiis stored tliereon to perform the various embodiments of
the
computerized method described above. In one implementation, the computer-
readable
medium includes a memory or a storage device. In another implementation, the
computer-
readable medium includes a coniputer data signal embodied in a carrier wave.
[0265] FIG. 30 is a block-diagram view illustrating a computer for controlling
a
microscale culture device, according to one embodiment of the present
invention. In this
embodiment, the computer 3000 includes a microprocessor 3002, a general memory
3004, a
non-volatile storage element 3006, an input/output interface 3008 that
includes an interface to
a microscale culture device having one or more sensors, and computer software.
The
computer software is executable on the microprocessor 3002 to regulate fluid
flow rates of a
culture medium in a nuinber of chambers in the microscale culture device,
detect biological
or toxicological reactions -in the chambers of the microscale culture device,
and upon
detection, change one or more pharmacokinetic parameters of the,microscale
culture device.
[0266] In one embodiment, the non-volatile storage element 3006 includes
historical data talcen from published infoianation, data gathered from
previously run tests, or
data derived from theoretical calculations. The computer software regulates
the fluid flow
rates by transmitting commands to one or more pumps of the microscale culture
device
through pump control lines. In one implementation, the computer software is
further
executable on the microprocessor 3002 to regulate a temperature of the culture
medium. The
computer software regulates the temperature by transmitting commands to a
heater coil of the
microscale culture device through heater coil control lines.
[0267] In another embodiment, the computer 3000 furtlier includes a look-up
table memory coupled to the general memoiy 3004 for storing a set of mass
balance
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equations that represent physiologically-based pharinacokinetic models for
various biological
or chemical substan.ces in the system, and a cache memory coupled to the
microprocessor
3002 for storing the computer software.
[0268] hZ another embodiment, the input/output interface 3008 fiirther
includes a
keyboard interface, a display interface, and a printer/plotter recorder
interface. In one
implementation, the computer 3000 uses these input/output interfaces to
connect to keyboard,
display, and printer/plotter recorder peripheral devices.
Experimental
[0269] The following examples are put forth so as to provide those of ordinary
skill in the art with a complete disclosure and description of how to malce
and use the subject
invention, and are not intended to limit the scope of what is regarded as the
iiivention.
[0270] Efforts have been made to insure accuracy with respect to the numbers
used (e.g., amounts, temperature, concentrations) but some experimental errors
and
deviations arise. Unless otherwise indicated, parts are parts by weight;
molecular weight is
weight average molecular weight, temperature is in degrees centigrade; and
pressure is at or
near atinospheric.
[0271] Methods
[0272] The following methods were used in the experimental process:
[0273] Cell culture. Cells were obtained from American Type Culture
[0274] Collection (Manassas, Va.) and propagated in the recomnzended complete
growth medium in a tissue culture incubator (95% 02/5%CO2). For HepG2 and
HepG2/C3A
cells, the recommended media is Eagle's Miniiilunl Essential medium (with
Earle's balanced
salts solution, 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential-
amino
aids, 1.5 g/L sodium bicarbonate, and 10% fetal bovine seruni) (EMEM). McCoy's
5a
medium with 1.5 mM L-glutamine, 1.5 g/L sodium bicarbonate and 10% fetal
bovine serum
is recommended for the HCT1 16.
[0275] Growth curves. Growtll cuives were determined by plating the cells at
an
initial low density in 35 mm dishes. Each day, cells were detached witli
tiypsin-EDTA and
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cell nuinber was determined by visually counting the cells using a
hemacytometer.
Determinations were done in triplicate.
[0276] Reverse transcriptase-polymerase chain reaction (RT-PCR). Cells were
culttued on glass coverslips treated with collagen, 1VIATRIGELTM, or poly-
lysine as
appropriate. HepG2/C3A grown to a-90% confluent monolayer were detached with
trypsin-
EDTA and pelleted at -500 g for 5 nzin. RNA was isolated and purified with
RNEASYTM kit
(Qiagen) according to manufacturer's protocol. Adult human liver total RNA was
purcllased
from Anzbion. The quantity and purity (260/280 nm ratio) of isolated RNA was
measured on
a BIOPHOTOMETERTM spectrophotometer (Eppendorf). The isolated RNA was then
incubated at 37 C. for 25 min with 2 U of DNase I and subsequently
inactivated with DNase
Inactivation Reagent (Ambion).
[0277] The RT reaction was performed using a mixture of 5 g RNA, 10 M
oligo dT primers heated to 72 C. for 2 minutes followed by 2 minute on ice.
Next, 5 mM
DTT, 600 M dNTP mix, 40 U rRNasin, 200 U SUPERSCRIPT TITM in reverse
transcriptase
buffer were combined and incubated at 42 C. for 1 hour.
[0278] 2.0 l of first strand cDNA was used in 50 l PCR reactions using
cytochrome P450 isoform specific primers (Rodriguez-Antona, C., Jover, R.,
Gomez-Lechon,
M. -J., and Castell, J. V. (2000). Quantitative RT-PCR measurement of huinan
cytochrome
P-450s: application to drug induction studies. Arch. Biochem. Biophys.,
376:109-116). PCR
conditions were: 94 C. for 4 minutes followed by 28 cycles of 40 seconds at
94 C., 45
seconds at 60 C., 50 seconds at 72 C., and a fmal 4 minutes extension at 72
C.
[0279] PCR products were separated by electrophoresis on a 1.2% agarose gel
and
visualized by staining with SYBR Gold and compared to appropriate molecular
weight
standards for authenticity. To quantify the amplified cDNA, 15 l of each PCR
reaction was
diluted with 0. lx Tris-EDTA buffer and stained with PICOGREENTM (Molecular
Probes) at
a final concentration of 1:400. Fluorescence was measured at 480 nm excitation
and 520 nm
emission. Results were standardized against (3-actin and done in triplicate
from at least two
separate experiments.
[0280] Cell viability, deatli and apoptosis assays. Cell viability and cell
death
were deteimined using tiypan blue exclusion or LIVE/DEAD stain (Molecular
Probes).
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Trypan blue (GIBCO), norinally excluded fiom the cytoplasm, identifies cells
witll
compromised menlbranes by visibly staining dead or dying cells blue. A 1:1
dilution of a
0.4% (w/v) solution of trypan blue is added to the re-circulating culture
nzedium of the chip
device at the conclusion of the experiment. This solution was pumped through
the chip to
waste for 30 minutes at room temperature. The housing was removed from the
puinp and
visualized under a reflecting microscope (Micromaster, Fisher).
[0281] LIVE/DEAD stain is a two-component stain consisting of calcein AM and
ethidium homodinler. Living cells actively hydrolyze the acetoxymethyl ester
(AM) moiety of
calceiii AM to produce bright green fluorescence of calcein. In contrast,
cells that have
compromised membrane integrity allow the normally membrane impermeant
etliidiuni
homodimer to stain the nucleus of dead or dying cells fluorescent red. The
cell permeant
nuclear stain, Hoechst 33342 acts as a general stain for all cells. Together
with the
appropriate filter sets, living cells fluoresce green, dying or dead cells
red, and all cells are
quantified by a blue nuclear fluorescence. For experiments described herein,
trypan blue was
used at 0.2% (w/v), calcein AM at 1:20,000, propidium iodide at 1:5,000, and
Hoechst 33342
at 10 g/ml. Cells were visualized with a M2Bio stereofluorescence microscope
(Zeiss). All
experiments were repeated at least three times and measurements done in
triplicate.
[0282] Apoptosis, or programmed cell death, can be monitored using a number of
methods (Smyth, P. G., Berman, S. A., and Bursztajn, S. (2000). Marlcers of
apoptosis:
methods for elucidating the mechanism of apoptotic cell death from the nervous
system.
Biotechniques, 32:648-665). To distinguish apoptosis from necrosis, at least
two separate
indicators of apoptosis are required-(Wronski, R., Golob, N., and Gryger, E.,
(2002). Two-
color, fluorescence-based microplate assay for apoptosis detection.
Biotechniques, 32:666-
668. One metllod, annexin V-FITC binding, relies on the observation that
annexin V binds
tiglitly to phosphatidylserine in the presence of divalent calcium
(Williamson, P., Eijnde,
S.v.d., and Schlegel, R. A. (2001). Phosphatidylserine exposure and
phagocytosis of
apoptotic cells. In Apoptosis, L. M. Scliwartz, and J. D. Ashwell, eds. (San
Diego, Academic
Press), pp. 339-364). Normally, phosphatidylserine is present on the inner
leaflet of cell
membranes, but translocates to the cell membrane early in apoptosis. Apoptotic
cells exposed
to fluorophore-labeled annexin exhibit distinct membrane staining. With the
microscale chip,
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annexin V-FITC labeling was visualized directly on-chin by first flushing the
system with
PBS, then recirculating annexin V-FITC (10 g/ml in annexin V binding buffer,
Clontech)
for 30 min. Cells were then visualized directly using a FITC filter set.
[0283] In contrast to annexin V labeling, the APOPTAGTM kit (Intergen Co., MA)
uses terminal deoxynucleotidyl transferase to label free 3'-OH DNA terniini
exposed during
apoptotic DNA degradation and visualization using immunofluorescence (Li, X.,
Traganos,
F., Melamed, M. R., and Darzynlciewicz, Z. (1995). Single-step procedure for
labeling DNA
strand brealcs with flourescein-or BODIPY-conjugated deoxynucleotides:
detection of
apoptosis and bromodeoxyuridine incorporation. Cytometry 20, 172-180).
A1t11ough this
method is highly specific for apoptosis, the procedure cannot be done on-chip
due to the
fixation - and incubation steps. Briefly, microscale chips were run under
specified
experimental conditions, the cell chips were removed from their housing units,
fixed in 1%
paraformaldeliyde and processed with the APOPTAGTM kit using the
manufacturer's
protocol.
[0284] Microscale Chip Fabrication and Experimental Methods. Microscale chips
were fabricated as follows: A pattei7i using a computer assisted design (CAD)
software
(Cadence) was designed and a clirome photomask using a GCA/Mann 3600F Optical
Pattern
Generator was created. This high-resolution pattern was then transferred to a
silicon wafer (3
inch diameter) containing a thin coat (-l m) of positive photoresist (Shipley
1813) by
exposing the wafer to UV light through the photomask using a Karl Suss MA6
Contact
Aligner. Following exposure, the photoresist was developed, tllus exposing the
silicon
through the photoresist layer in the defined pattern. The exposed silicon was
etched to- a
specified depth (20 to 100 m) using a PlasmaTherm SLR 770 ICP Deep Silicon
Etch
Systeni. The photoresist was stripped from the wafer with acetone. Individual
22 inm square
microscale chips were diced from the wafer, washed in Nanostrip (Cyantek),
rinsed in
distilled water, and dried in a drying oven at 170 C.
[0285] The surface of the silicon in the organ compartments was treated with
collagen to facilitate cell attachment. Approximately 10 l of a 1 mg/mi
solution of collagen
Type I was deposited onto the surface of the microscale chip and incubated at
room
temperature for 30 minutes. The collagen solution was removed and the organ
compartments
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were rinsed with cell culture medium. Cells were dissociated from the tissue
culture dishes,
cell number was determined, and the concentration was adjusted such that there
would be a
confluent monolayer of cells in each cell conzpai-tYllent. For exaniple, for
the microscale chip
described in FIG. 2 (hereinabove), 10 l of a 2,400 cells/ l suspension of
the L2 cells was
deposited onto the lung chamber of the cell chip and 15 l of a 3,400 cells/
1 suspension of
the H4IIE cells was deposited onto the liver chaniber. Cells were allowed to
attach in a C02
incubator overnight. Once the cells were attached, the chip was assembled in
aciylic chip
housings. The top of the housings contain fluid interconnects to provide cell
culture medium
to the chip. Stainless steel tubes are connected to micro-bore pump tubing and
inserted into a
small hole in the top of a micro-centrifuge tube containing culture medium
with or without
test compound. The pump tubing is connected to the peristaltic pump, primed
with this
solution, and connected to the inlet ports of the chip housing. A small
section of pump tubing
with a stainless steel tube connected to the end is connected to the outlet
port and the tube is.
inserted into a small hole in the top of the micro-tube, thus completing the
re-circulation fluid
circuit. The entire instrument is placed in a COZ incubator at 37 C. A
schematic diagram of
this setup is presented in FIG. 22.
EXAMPLE 1
Calculations for a System Replicating a Rat
[0286] In designing the chip 1000 all necessary chambers were fit onto a
silicon
chip no larger than 2 cm by 2 cm. This size of chip is easy to manufacture and
is compatible
with the sizes of oonnective tubing and pumping devices intended for use to
direct fluid flow.
There were also several other important factors constraining the design of the
device listed
below, along- with acceptable values for each variable. This one embodiment of
the device
consists of a two compartment system, one compartment representing the liver
of a rat and
one conipartment representing the lung of a rat. The total size of the chip is
2 cm by 2 cm and
consists of an interconnected array of 20 parallel channels 40 m wide, 10 m
deep and 5
min long to serve as the "lung" chamber and two parallel channels 100 nz
wide, 20 in deep
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and 10 cm long in a serpentine shape to serve as the "liver" chamber. The two
organ
conzpartnients are connected by a channel 100 .in wide and 20 m deep. There
are many
other possible geometries, dimensions, number of chanibers, etc. This design
was chosen as
one example.
TABLE 1
Constraining variables in device desiQn.
Constraining variable Acceptable values
Chip size 2 cm x 2 cm
"Lung" liquid residence time 1.5 seconds
"Liver" liquid residence time 25 seconds
"Other tissues" liquid residence time 204 seconds
Number of each cell type >10,000
Cell shear stress 8-14 dyne/cn12
Charmel liquid-to-cell volume ratio 1 to 2
[0287] Sample Calculations
[0288] Channel or Chamber Calculations: -
[0289] These calculations assume we have obtained a flow rate from a previous
iteration by the method described above with respect to chip 1000 for system
1100.
- [0290] By this, Q=8.05x105 m3/trencli-second.
[0291] The liquid residence time in a trench was then calculated in the
following
manner:
V VCharoiel
R ~
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[0292] Next, the number of cells in a"cell-length" was calculated
(40,uni) - (10,um) = (5000,ctm)
VR - 5 ,tan3
(8.05x10 )
sec
vR = 2.48 sec
Channel Width 2= Wall _ Height
NL~"g'J' Cell Dianzetey~ + Cell Diameter
N 40,cusa + 20,um
L~ g''' = 7.41,can 7.41,um
NLe,Zgtj, = 7 Cells (Each term is separately rounded down)
[0293] Then, a channel/chamber cell-length volume was calculated,
VTcL =(Cell Diameter)=(Trench Cross Sectional Area)
VTcL = (7.41 ,urn)=(7.41 ,cnn')
VTCL = 2960 ,tnn3
[0294] The cell-length volume was also determined.
(NLe grh (VCe11)
VccL = 2
VCCL (7Cells) = [32o3]
2cell
VccL = 1120 M23
[0295] The liquid cell-length volume is sinlply the cell cell-length volume
subtracted from the channel/chamber cell-length volume. The ratio of the cell
cell-length
volume and the liquid cell-length volume gives the liquid-to-cell volume ratio
for the system:
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Liquid-to-cell ratio VLCL
= ( ) Vcci
Ratio = 2960,uin3 -1120,uin3 )
(
1120,tun3
Ratio =1.65
[0296] The shear forces on individual cells associated witli a given flow rate
were
detemiined. Based on the liquid cell-length volume and cell diameter, an
average surface area
available for liquid to flow through was calculated.
Average Liquid-Surface Area = VL~L
Dce(!
_ (1844,ctna3 )
ALS 7.4l,can
ALS = 249,uy7a Z
[0297] An average linear velocity of fluid in the channel was then calculated.
Vavg = Q
ALs
(8.05x105 'C n3 )
sec
VRvg = 249pm2 2
Vvti = 3.23x103 Pn
sec
[0298] Assuming laminar flow, Stokes' law was used for calculating the drag on
a
sphere to estimate the total shear force experienced by an individual cell,
(37e77Dcelt VAvg )
rs Acerr
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(3 = ;c (9.6x10-4 N-2ec (7.41,can) .(3.23x103 icm))
rs= in sec
~ (7.4~,cnn)Z
rs =12.6 dyn3
cnz
[0299] Next, the actual residence time of the liquid in a channel/chanlber was
verified and calculated to total number of cells in the chaimel/chamber,
LTrench * NTrencHes * NLength
Ncelrs - Dcerr
(5000,um) = (20trenches) = (7Cells)
Ne~ns - (7.41,tan)
Ncerrs = 9=45x104 Cells
[0300] I. B. Membrane Oxygenation Calculations:
[0301] The area of silicone membrane for oxygenation was detemlined in the
following manner:
[0302] First, approximate the Oxygen Uptake Rate (OUR) for the cells:
OUR=qo, - X
OUR = (7.00 11g02 ) = (2x10s Cells)
10~ cells - ht~
OUR = 4.4x10-5 mmolO2
hr
[0303] Then calculate the partial pressure of oxygen on the inside of the
membrane to determine if it is sufficient to re-oxygenate the liquid medium.
This was done
using an equation for the flux of a gas tlirougli a porous membrane, wliere Q
is the membrane
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permeability. J represents the flux of gas into the cells, and z is the
thickness of the
membrane:
Qo, ' (Poõour - -PoõI,r )
Jo, AMeõt,,,Y,ne = OUR =
z
mmolO2 8[cm3 (STP) = cm] z (1'oõoi,r -16cmHg)
(4.4x10- ) = (5.OOx10 = (55mm ) =
hr (cmZ = s = cmHg) 0.05cm
Poõotrt =15.5cmHg
[0304] This pressure is sufficient to saturate the liquid medium with oxygen
in the
200 seconds it is in contact with the menlbrane. The area of inembrane was
determined in an
iterative manner so as to maximize the inside oxygen partial pressure.
Principle Design Calculations
Rat-Mode1:
Primary cell characteristics Lung (L2) Liver (H4IIE)
Surface area (cm2/organ) 4890 21100
Cell volume ( m3/cell) 320 4940
Plating area (m2/cell) 320 988
Cell Diameter ( m) 7.41 18.5
Stokes' law: 3 Tc9DU = FD
(Plating area is the inverse of experimentally detennined saturation densities
for L2 and
H411E cells.)
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[0305]
LUNG CELL CALCULATIONS:
Calculation of cell and liquid volumes in one cell-length of channel/chamber:
Cell diarneter 7.41 M (a cell-length
Cell volume 320 m3/cell included the diameter
Channel widtll 40 m of the cell as well as
Channel depth 10 m spacing on either side
Spacing between channels 30 m equal to the "distance
Channel X-sectional area 400 m2 between cells")
Cells across channel 5
Cells on side of channel 1
Total cells in one cell-length 7
Channel cell-length volume 2964 m3
Cell cell-length volume 1120 m3
Liquid cell-length volume 1844 m3
Liquid-to-cell volume ratio 1.65
Detem7ination of liquid velocity and shear on individuals cells:
Viscosity of cell plasma 9.60E-04 N-s/m2
medium
Number of channels 20 (this number
picked to give
adequate # of
cells and
feasible flows)
Liquid flow rate per cliannel 8.05E+05 m3/sec (tliis number
picked to give
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a stress of 12
dyne)
Average liquid surface area 249 ni2
Average liquid linear Velocity, 3.23E+03 M/SEC
U 3.23E-03 M/SEC
Drag force on individual cell 1.08E-10 Newtons (for a half-
1.08E-04 N sphere)
1.08E-05 dyne
Surface area of individual cell 8.63E+01 m2 (for a half-
8.63E-07 cm2 sphere)
Shear stress on individual cell 12.6 dyne/cm2 (This result
assumes
smooth half-
spherical
geometry for
the cells; it is
likely the
actual number
is small due to
larger surface
area or surface
irregularities)
Total flow rate 1.61E+07 m3/sec
Desired residence time 1.5 seconds
Chaimel length 5 mm (t11is number is
chosen to give
the desired
residence time)
Total Channel liquid volume 2.49E+07 m3
Actual Residence time 1.55 seconds
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Total nuniber of cells 9.45+04 cells
[0306]
LIVER CELL CALCULATIONS:
Calculation of cell and liquid volumes in one cell-length of chamzel/chamber
Cell diameter 18.5 m
Cell volume 4940 m3/cell
Channel width 100 m
Channel depth 20 m
Spacing between channels 50 m
Channel X-sectional area 2000 m2
Cells across channel 5
Cells oii side of channel 1
Total cells in one cell-length 7
Channel cell-length volume 36918 m3
Cell cell-lengtli volume 17290 m3
Liquid cell-length volume 19628 m3
Liquid-to-cell volunie ratio 1.14
Determination of liquid velocity and shear on individual cells:
Viscosity of cell plasma medium 9.60E-04 N-s/m2
Total liquid flow rate from 1.61E+07 m3/sec (from above
Lung Calcs. calcs.)
Nuniber of channels 2
Liquid flow rate per channel 8.05E+06 m3/sec
Average liquid surface area 1063 m2
Average liquid linear U 7.57E+03 m/sec
velocity
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7.57E-03 m/sec
Drag force on individual cell 6.32E-10 Newtons Stokes' law:
6.32E-05 dyne 3 TtrlDU = FD
Surface area of individual 535.24 m2
cell 5.35E-06 cm3
Shear stress on individual 11.81 dyne/cm2
cell
Desired residence time 25 sec
channel length 100 mm
Total Channel liquid volume 4.OOE+08 m3
Actual Residence time 24.86 sec
Total number of cells 7.58E+04 cells
[0307]
Residence Time Calculations
Actual (target) residence times in rat tissues:
Lung 1.5 sec
Liver 25 sec
Otlier Tissues 204 sec
Actual organ characteristics:
Volume
Blood Flow Rate (mL/min) (mL)
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Lung 73.3 1.2
Liver 18.3 7.4
Otlier Tissues 55 190
Preliminary flow rate 0.85 L/min
0.0142 L/sec
Unit Conversions:
l m l L
0.000001 m 1.00E-06 L
1.00E-09 m3
1.OOE+09 m3
[0308]
Calculations using seipentine pattenling:
Preliminary Residence Time Calculations for Liver/Lung:
Channel Depth 310 m
Channel Width 500 m
Channel X-sectional Area 0.155 mm2
155000 m2
Cells per area 3200 cells/mm2
Channel Surface
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Residence Volume Channel Area Max #
Time (sec) ( L) Length (mm) (mm2) cells
Lung 1.5 0.02125 0.1 6.85E+01 2.58E+04
Liver 25 0.4 2 1.14E+03 3.66E+06
Preliminary Residence Time Calculations for Other Tissues:
Channel Depth 50 m
Channel Width 2000 m
Channel X-sectional Area 0.1 mm2
100000 m2
Residence CHANNEL VOLUME Channel Length Surface Area
Time (sec) ( L) (mm) (mm2)
204 2.89 29 57.8
EXAMPLE 2
A Four Organ Compartment Chip
[0309] A chip was designed to consist of four organ compartments--a "liver"
compartnlent to represent an organ responsible for xenobiotic metabolism,
a"lung"
compartment representing a target tissue, a "fat" compartment to provide a
site for bio-
accumulation of hydrophobic compounds, and an "otller tissues" compartment to
assist in
mimicking the circulatory pattern in non-metabolizing, non-accumulating
tissues (FIG. 15).
These and other organ compartments (e.g., kidney, cardiac, colon or muscle)
can be fully
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modularized as CAD files and can be fabricated in any configuration or
combination. The
device itself can be produced in any number of substrates (e.g., silicon,
glass, or plastic).
[0310] Once the cells were seeded in the appropriate coinpartments, the cllip
was
assembled in a Lucite manifold. This manifold holds four cliips and coiitained
a transparent
top so the cells could be observed in situ. The top contained fluid
interconnects to provide
cell culture medium to the chip. The culttue medium was pumped through the
chip using a
peristaltic pump at a flow rate of 0.5 l/min. Culture mediunl was re-
circulated in a closed
loop consisting of a fluidic reservoir (-15 to 50 l total volume), micro-bore
tubing, aild the
compartments and channels of the chip.
[0311] Using a three compartment system with human HepG2-C3A cells in the
liver compartment and HT29 colon cancer cells in the target tissues
compartment, it was
found that cells remain viable under continuous operation for greater than 144
hours. HepG2-
C3A cells are a well characterized human liver cell line lcnown to express
various liver
metabolizing enzymes at levels comparable to fresh primary human hepatocytes.
In these
experiments, cells were seeded in the appropriate compartments and a specially
formulated
cell culture medium was re-circulated through the system for up to 144 hours.
At various
time points, the culture medium was switched to PBS containing LIVE/DEAD
fluorescent
reagent (a dual fluorescent stain, [Molecular Probes, Inc., Eugene, Oreg.,
USA]) for 30
minutes. Cells were visualized under a fluorescent microscope and fluorescent
images of
identical fields were obtained using the appropriate filter sets. Living cells
fluoresced green
whereas dead cells were red (data not shown).
EXAMPLE 3
Drug Metabolism in the Chip
[0312] The metabolism of two widely used prodrugs, tegafur and sulindac
sulfoxide, was studied using a microscale chip comprising three compartments,
liver, target
tissue, and other tissues. Both prodrugs require conversion to an active
metabolite by
enzyines present in the liver, and have a cytotoxic effect on a target organ.
For the prodrug
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sulindac sulfoxide, its anti-inflammatory and cancer chemopreventive
properties are derived
from its sulfide and sulfone metabolites, catalyzed by the liver enzyme
sulfoxide reductase.
The sulfide metabolite (and a second sulfone metabolite) have been
demonstrated to induce
apoptosis in certain cancer cells (e.g., colon cancer).
[0313] A proper treatment regimen requires administration of its prodrug,
tegafur
[5-fluoro-l-(2-tetrahydrofuryl)-2,4(IH,3H)-pyrimidi-nedione] as 5-FU itself is
quite toxic to
nonnal cells. Unlike sulindac however, tegafur is converted to 5-FU in the
liver primarily by
cytochrome P450 2A6.
[0314] To test the efficacy of sulindac, the microscale chip was seeded with
HepG2-C3A cells in the liver compartment and HT29 liuman colon cancer cells in
the target
tissue compartment. One hundred micromoles of Sulindac (need manufacturer) was
added to
the re-circulating medium for 24 hours and the chip was treated as described
above--living
cells fluoresced green and dead cells fluoresced red (data not shown). In the
absence of the
HepG2-C3A liver cells, minimal levels of cell deatli (similar to vehicle
control) was
observed. These results demonstrate that a di1ig can be metabolized in the
liver compartment
and consequently circulate to a target where its metabolite(s) induce a
biologicaleffect much
as it would in a living animal or human.
[0315] The cancer therapeutic pro-drug tegafur was tested in the microscale
chip
system. For efficacy, tegafur requires metabolic activation by cytochrome P450
enzyines
present in the liver to its active form, 5-fluorouracil (5-FU) (Ilceda, K.,
Yoshisue, K.,
Matsushima, E., Nagayama, S., Kobayashi, K., Tyson, C. A., Chiba, K., and
Kawaguchi, Y.
(2000). Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytoclirome
P-450 2A6 in
human liver microsomes iri vitro. Clin. Cancer Res., 6, 4409-4415; Komatsu,
T., Yamazaki,
H., Shimada, N., Nakajima, M., and Yokoi, T. (2000). Roles of cytochromes P450
1A2, 2A6,
and 2C8 in 5-fluorouracil formation fiom tegafur, an anticancer prodrug, in
human liver
microsomes. Drug Met. Disp., 28, 1457-1463; Yamazaki, H., Komatsu, T.,
Takemoto, K.,
Shimada, N., Nakajima, M., and Yokoi, T. (2001). Rat cytochrome P450 1A and 3A
enzymes
involved in bioactivation of tegafur to 5-fluorouracil and autoinduced by
tegafur liver
microsomes. Drug Met. Disp., 29, 794-797. A proper therapeutic regimen
requires
administration of its pro-di-ug, tegafur, as 5-FU itself is veiy toxic to
normal cells. 5-FU is
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currently the most effective adjuvant tlierapy for patients with colon cancer
(Hwang, P. M.,
Bunz, F., Yu, J., Rago, C., Chan, T. A., Murphy, M. P., Kelso, G. F., Smith,
R. A. J., Kinzler,
K. W., and Vogelstein, B. (2001). Ferredoxin reductase affects p53-dependent,
5-
fluorouracil-induced apoptosis in colorectal cancer cells. Nat. Med., 7, 1111-
1117.) Like
most chemotherapeutic agents, 5-FU induces marlced apoptosis in sensitive
cells through
generation of reactive oxygen species (Hwang, P. M., Bunz, F., Yu, J., Rago,
C., Chan, T. A.,
Murphy, M. P., Kelso, G. F., Smith, R. A. J., Kinzler, K. W., and Vogelstein,
B. (2001).
Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced in
colorectal cancer cells.
Nat. Med., 7, 1111-1117).
[0316] To measure the cytotoxic effects of tegafur against colon cancer cells,
the
microscale chip was prepared with HepG2-C3A cells in the liver compartment and
HCT- 116
human colon cancer cells in the target tissue compartment. Tegafur was added
to the re-
circulating medium at various concentrations for 24 hours and the cells
labeled with Hoechst
33342, a membrane peimeable DNA dye, and ethidium homodimer, a membrane
impermeable DNA dye (see Methods Section). All cells fluoresce blue, but dead
cells were
marlced by the fluorescent red ethidium homodimer (data not shown). Tegafur
was cytotoxic
to HCT- 116 cells in a dose-dependent fashion in this microscale chip system,
while it was
ineffective with the traditional cell culture assay (FIGS. 16A and 16B). In
addition, while 5-
FU triggered cell death in the traditional cell culture assay, cytotoxicity
was not observed
until after 48 hours of exposure compared to 24 hours of exposure to tegafur
with the
microscale chip.
[0317] To- demonstrate that the liver compartment was responsible for the bio-
activation of tegafur, the microscale chips were seeded with HCT- 116 cells
only. No cells
were in the liver compartment. Tegafur or 5-FU was added to the re-circulating
culture
medium for 24 hours and the chip was treated as described above (data not
shown). Tegafur
did not cause significant cell death of the HCT-1 16 cells in the absence of a
liver
compartYnent wllile the active metabolite 5-FU caused substantial cell death.
Furtlzer, when
HT-29 colon cancer cells are substituted for HCT- 116, tegafur was ineffective
(data not
shown). This was likely due to the mutant p53 present in HT-29 cells, wllich
is necessary for
5-FU cytotoxicity. Together, these experiments demonstrate that tegafur, lilce
sulindac, was
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metabolized to an active drug in the liver conipartment where it circulated to
another organ
coinpartnient to eliminate the cancer cells. These effects were
mechanistically distinguishable
witli the chip--sulindac was effective even in the absence of an active p53,
whereas tegafiir,
,
was not.
EXAMPLE 4
Multiple Cell Cultures in a Single Organ Conlparhnent
[0318] It is also possible to use a mixture of multiple cell types in a single
organ
compartment. In one study, the hepatocyte cell line HepG2/C3A (from ATCC) is
used in the
liver compartment. The cells are propagated in McCoy's 5A medium with 1.5 mM L-
glutamine 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. To more
closely mimic
an in vivo organ, a mixture of primary hepatocytes and fibroblasts can be used
at a 1- to 2
ratio along with macrophages (Kupffer cells).
[0319] In anotller example, a mixture of cells or cell lines derived from lung
epitlielial cells is used to more closely mimic the lung tissue. This includes
a mixture of type
I epithelial cells, type II epitllelial cells (granular pneumocytes),
fibroblasts, macrophages and
mast cells.
EXAMPLE 5
Optimization of Tissue Culture Conditions in the Chip-based System
[0320] A tissue culture medium compatible witli two different rat cell culture
lines, H4IIE (a rat liver cell line) and L2 (a rat lung cell line) was
developed. Preliminary
experiments indicated that a 1:1 mixture of DMEM and Hams F12K medium
supplemented
witl-i 2 mM L-glutamine, 1 mM sodium pyiuvate and 10% fetal bovine serum (FBS)
maintained the viability of botll H4I1E cells and L2 cells for up to 20 hours
of continuous
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operation in a microscale chip. This media forniulation was used for all rat-
based microscale
chip studies.
[0321] The proper human liver cell line that realistically mimics lnunan liver
fitnction was selected Additionally the optimum cell culture medium
forinulation for
maintaining hunian cell lines on a microscale chip was detennined. The basal
expression
levels of tliree key cytochrome P450 (CYP) isofoi7ns (1A2, 3A4, and 2D6) in
HepG2 and
HepG2/C3A (a HepG2 subclone) cell lines were examined. CYP-1A2, 2D6, and 3A4
were
examiiied because they account for the metabolism of 80-90% of al11n1own drugs
(Hodgson,
J., (2001). ADMET--turning chemicals into drugs. Nat. Biotech., 19, 722-726.
The C3A
subclone of the HepG2 liver cell line was examined as this cell line has been
reported to be a
highly selected cell line exhibiting more "liver-like" characteristics,
particularly much higller
CYP expression compared to the parental cell line (Kelly, J. H. (1994).
Peimanent human
hepatocyte cell line and its use in a liver assist device (LAD). U.S. Pat. No.
5,290,684). The
RT-PCR analysis confirmed that basal CYP levels in HepG2/C3A cells were
significantly
greater than HepG2 parentals and comparable to adult liuman liver (FIG. 23).
[0322] HepG2/C3A cells were used as a liver surrogate in all subsequent
experiments. To select a common media for use during microscale cllip
experiments, the
components of a number of media were compared (DMEM, McCoy's 5a, RPMI 1640,
MEM,
F12, F12K, Waymouth's, CMRL, MEM, and Iscove's modified Dulbecco's medium).
Analysis of the inorganic salt, glucose, amino acid composition, and vitamin
content
suggested that EMEM, DMEM, McCoy's 5a and RPMI were the most suitable "common"
media of the media examined. After several passages, cells were then split
and_ sub-cultured
in the following media:
[0323] Eagle's Minimum Essential medium (EMEM) with Earle's balanced
salts solution, 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0. 1 mM nonessential
amino aids, 1.5 g/L sodium bicarbonate, and 10% fetal bovine serum.
[0324] Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-
glutamine, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% fetal bovine
serum.
[0325] McCoy's 5a medium (McCoy's) with 1.5 mM L-glutamine 1.5 g/L
sodium bicarbonate and 10% fetal bovine serum.
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[0326] RPMI 1640 medium (RPMI) wit112 mM L-glutamine, 4.5 g/L glucose,
1.0 mM sodium pyruvate, 1.5 g/L sodium bicarbonate.
[0327] Growth curves for both cell lines in each media were then determined as
described in the Methods section (FIG. 24) DMEM was found to be inappropriate
for the
HepG2/C3A cells, as significant changes in cellular morphology and adliesion
after N5
passages were observed (not shown). Similarly, a significantdecrease in
HepG2/C3A and
HCT116 viability and growth after 3 days in RPMI was noticed. Both cell lines
grew well in
McCoy's and EMEM coinpared to their preferred medium.
[0328] Next, the expression levels of these CYP isoforms in HepG2/C3A cells
growing in either EMEM or McCoy's using RT-PCR were investigated (see Metllods
section)
(FIG. 25). The results indicated that EMEM was superior to McCoy's for
maintaining CYP
expression and the preferred media for HepG2/C3A. The effect of different
growth substrates
on CY-P expression was studied (FIG. 26). A comparison of silicon treated
witli eitlier poly-
D-lysine or collagen as the attachment substrate against cells grown on
standard tissue culture
treated polystyrene was performed. Togetlier, the results indicated that EMEM
supported the
growth of both HepG2/C3A and HCT116 cells and that collagen was the preferred
substrate
based on RT-PCR CYP expression analysis.
[0329] Using these conditions, the long term cell viability of these cells,
HepG2/C3A and HCT116, was studied under continuous operation in the microscale
chip
system. Using a three compartment system with human HepG2/C3A cells in the
liver
compartnient and HCT1 16 colon cancer cells in the target tissues compartment,
it was
demonstrated that cells remain viable under continuous operation for greater
than 144 hours.
In these experinients, cells were seeded in the appropriate compartments and
EMEM was re-
circulated through the systenl for up to 144 hours. At various time points (6,
24, 48, 72, 96,
120 and 144 lir), total live or dead cells were visualized using LIVE/DEAD
stain (data not
shown). Cells were visualized under a fluorescent microscope and fluorescent
images of
identical fields were obtained using the appropriate filter sets. Living cells
fluoresced green
whereas dead cells were red (data not shown).
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EXAMPLE 6
Assay for Detection of Cytotoxicity oii a Microscale Chip
[0330] Trypan blue is the most comn7on stain used to distinguish viable cells
from nonviable cells; only nonviable cells absorb the dye and appear blue.
Conversely, live,
healthy cells appear round and refiactile without absorbing the blue dye.
Experiments were
performed using trypan blue to determine cell viability in a microscale chip.
Altliougll tiypan
blue (see Methods section) is easy to use and requires only a light microscope
to visualize,
viable cells will absorb trypan blue over time, which can affect results. hi
addition, trypan
blue has a higher affinity for serum proteins than for cellular proteins, thus
the background is_
dark when using serum-containing media. Therefore, alternative methods to
distinguish
viable cells fiom dead cells were studied.
[0331] The LIVE/DEAD assay was optimized (see Methods section) using cells
grown on glass coverslips. Briefly, HepG2/C3A cells were seeded onto poly-D-
lysine treated
glass coverslips and treated with and without 1 M staurosporine for 24 hours.
Staurosporine
is a broad-spectrum protein kinase inhibitor and is known to induce apoptosis
in a variety of
cell types (Smyth, P. G., Bennan, S. A., and Bursztajn, S. (2002). Markers of
apoptosis:
methods for elucidating the mechanism of apoptotic cell death from the nervous
system.
Biotechniques, 32, 648-665). Coverslips were washed with phosphate buffered
saline (PBS)
and LIVE/DEAD reagents were added and incubated at room temperature for 30
minutes.
The coverslips were removed and visualized (data not shown). Staurosporine was
found to
clearly cause cell death of HepG2/C3A cells (data not shown).
[0332] The assay for detection of cytotoxicity on the nlicroscale chip system
was
then optimized. Microscale chip cell chips were seeded with HepG2/C3A cells in
the liver
compart7nent and HCT116 cells in the target tissues compartment as described
in the
Methods section. Cell chips were loaded onto the microscale cliip system and
treated with
and without 1 M staurosporine as described above. After a 24-hour incubation,
the
recirculating medium was switched to PBS, allowed to flow through the system
to waste for
30 minutes, then switched to PBS containing the LIVE/DEAD reagents and flowed
through
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the systein for an additional 30 minutes. The aciylic housing containing the
cell chips was
removed from the system and placed under a stereofluorescence microscope and
the cell chip
was visualized tlirough the transparent top of the housing (data not shown).
Cells were
visualized under a fluorescent microscope and fluorescent images of identical
fields were
obtained using the appropriate filter sets. Living cells fluoresced green
wliereas dead cells
were red (data not shown). Significant cell death of the HCT116 cells was
caused by 1 M
staurosporine after a 24 hour treatment conlpared to untreated control cell
chips (data not
shown).
EXAMPLE 7
Chip-Based Assays to Detect the Occurrence of Cell Death and Distinguish
Between
Apoptosis or Necrosis
[0333] Two different assays to detect apoptosis were investigated. The first
assay
was the immunofluorescence-based terminal deoxynucleotidyl transferase BrdU
nick end
labeling (TUNEL) technique available in kit form as APOPTAG (Intergen Co., MA)
(see
Methods section). The assay was first optimized using cells grown on glass
coverslips.
Briefly, HepG2/C3A cells were seeded onto poly-D-lysine treated glass
coverslips and treated
with and without staurosporine. Coverslips were processed as described (see
Methods
section). Various staurosporine concentrations and treatment times were
tested, and the
results indicated that 1 M staurosporine-caused significant apoptosis
compared to untreated
controls after a 24-hour incubation (data not shown). Next, the assay for
detection of
apoptosis on the microscale chip system was optimized and a comparison of the
APOPTAG
nzetllod to the LIVE/DEAD staining technique was performed. The microscale
cell chips
were seeded with HepG2/C3A cells in the liver compartment and HCT1 16 cells in
the target
tissues conlpartment as described in the Methods section. Cell chips were
loaded onto the
microscale chip system and treated with and without 1 M staurosporine as
described above.
After a 24-hour incubation, the recirculating medium was switched to PBS for
30 minutes.
Half the cell chips were removed from the housing and the APOPTAGTM assay was
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perfoinied as described above. The other cell chips were left in the
microscale chip system
and subjected to the LIVE/DEAD staining technique as previously described.
Cells were
visualized under a fluorescent microscope and fluorescent images of identical
fields were
obtained using the appropriate filter sets. Living cells fluoresced green
wliereas dead cells
were red (data not shown). Botli techniques produced veiy similar results,
i.e., a 24 hour
exposure to 1 M staurosporine induced significant apoptosis (or cytotoxicity)
to the
HCT116 cells compared to untreated controls (data not shown).
[0334] The annexin V-FITC was used to detect apoptosis in the microscale chip
system as described in the Methods section. Briefly, the microscale chip cell
chips were
seeded with HepG2/C3A cells in the liver coinpartment and HCT116 cells in the
target
tissues compartment. Cell chips were loaded onto the microscale chip system
and treated
with and witlzout 1 M staurosporine as described above. After a 6-hour
incubation, the re-
circulating medium was switched to PBS containing Annexin V-FITC and Hoechst
33342
and allowed to flow through the system for 30 minutes. Cell chips were removed
from the
acrylic housing and visualized under a fluorescent microscope. Cells were
visualized under a
fluorescent microscope and fluorescent images of identical fields were
obtained using the
appropriate filter sets. Living cells fluoresced green whereas dead cells were
red (data not
shown). 1 M staurosporine caused significant apoptosis after a 6-hour
treatment compared
to untreated control cell chips (data not shown).
EXAMPLE 8
Use of Naphthalene as a Model Toxicant
[0335] Naphthalene was used to study toxicology because enzymatic conversion
in the liver is required for lung toxicity. Therefore, the effects of
naphthalene on a rat lung
cell line were studied. These experiments used a three-conipartment (liver,
lung, and other
tissues) rat-based microscale cliip with H4IIE cells in the liver compartment
and rat L2 cells
in the lung compartment. Microscale chips were fabricated and prepared for
experiments as
described in the Metliod section.
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[0336] The microscale chip system was operated for 20 hours in the presence or
absence of 250 ghnl naphthalene before switching to PBS containing tiypan
blue. This
solution was re-circulated tllrough the cell chip for 30 minutes and the chip
visualized under
a liglit microscope (see Metliods section). Naphthalene caused significant
cell death of the rat
L2 cells in the lung compartnient of the cell chip while no cell deatli was
observed in the
absence- of naphtlialene (data not shown). No cell death was obseived in the
H4I1E- cell
coinpartment with or without naphthalene or in the L2 cell coinparttnent in
the absence of
H4IIE cells (data not shown).
[0337] These results demonstrate that naphthalene is activated in the "liver"
compartnlent and the toxic metabolites circulate to the "lung" and cause cell
death. These
results are consistent with data obtained with the benchtop CCA device and
expected from
the PBPK model (Sweeney, L. M., Shuler, M. L., Babish, J. G., and Ghanem, A.
(1995). A
cell culture analogue of rodent physiology: application of napthalene
toxicology. Toxicol. in
Vitro, 9, 307-316).
EXAMPLE 9
A Human Microscale Chip Prototype
[0338] [0238] A human biochip prototype was prepared that contained
compartments for lung, target tissues, and other tissues. The dimensions of
the compartments
and channels were as follows:
[0339] Inlet: 1 n1m by 1 mm
[0340] Liver: 3.2 mm wide by 4 mm long
[0341]. Target Tissues: 2 nini by 2 mm
[0342] Other Tissues: 340 gm wide by 110 mm long
[0343] Outlet: 1 mm by 1 min
[0344] Channel Connecting Liver to Y connection: 440 m wide
[0345] Channel from Y connection to Target Tissue: 100 m wide
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[0346] The human biochip prototype is fabricated as described previously. The
placement of the organ conipartments is intended to simulate exposure to a
compound (diug)
that has been ingested orally. When a coinpound is orally ingested it is
absorbed into the
blood from the small or large intestine. From here it circulates directly to
the liver via the
hepatic portal vein then gets distributed througliout the body (FIG: 27).
Therefore, witli this
design, the liver is the first organ coinpartliient, followed by a split to
other tissues a
compartment and a chamber for the target tissue. The other tissues
conipartment representsd
distribution and hold-up of blood in the body, the target tissue compartment
represents the
therapeutic target of interest (e.g., colon cancer cells representing a colon
tumor.
Conclusion
[0347], The invention provides a pharnlacokinetic-based culture device and
systems, usually including a first cell culture chamber having a receiving end
and an exit end,
and a second cell culture chamber having a receiving end and an exit end, and
a conduit
connecting the exit end of the first cell culture chanrber to the receiving
end of the second cell
culture chaniber. Preferably the device is chip-based, i.e., it is microscale
in size. A. culture
medium can be circulated through the first cell culture chamber, through the -
conduit and
through the second culture chamber. The culture medium may also be oxygenated
at one or
more points in the recirculation loop.
[0348] The device may include a mechanism for communicating signals from
portions- of the device to a position off the chip, e.g., with a waveguide to
communicate
signals from portions of the device to a position off the chip. Multiple
waveguides can be
present, e.g., a first waveguide communicating signals from the first chamber,
and a second
waveguide communicating signals from a second chamber, and so forth.
[0349] In one embodiment, at least one of the first cell culture chamber and
the
second cell culture chamber is tllree dimensional. In another embodiment, both
the first cell
culture chamber and the second cell culture chamber are three dimensional.
[0350] The device for inaintainirig cells in a viable state also includes a
fluid
circulation mechanism, may be a flow tlirough fluid circulation mechanism or a
fluid
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circulation mechanism that recirculates the fluid. The device for maintaining
cells in a viable
state also includes a fluid path that connects at least the first compartment
and the second
compartment. In an embodiment, a debubbler renioves bubbles in the flow path.
The device
can fui-ther include a pumping mechanism. The pumping mechanism may be located
on the
substrate.
[0351] A method is provided for sizing a substrate to maintain at least two
types
of cells in a viable state in at least two cell chambers. The method includes
the steps of
determining the type of cells to be held on the substrate, and applying the
constraints from a
physiologically based pharmacokinetic model to determine the physical
characteristics of the
substrate. The step of applying the constraints from a physiologically based
pharmacokinetic
model includes deteimining the type of chamber to be formed on the substrate,
which may
also include determining the geometry of at least one of the cell chambers and
determining
the geometry of at a flow path interconnecting two cell chambers. The step of
applying the
constraints from a physiologically based pharmacokinetic model may also
include
deterinining the flow media composition of the flow path.
[0352] All publications and patent applications cited in this specification
are
herein incorporated by reference as if each individual publication or patent
application were
specifically and individually indicated to be incoiporated by reference.
[0353] It is to be understood that the above description is intended to be
illustrative, and not restrictive. Many other embodiments will be apparent to
those of skill in
the art upon reviewing the above description. The scope of the invention
should, therefore, be
determined with reference to the appended claims, along with the full scope of-
equivalents to
which such claims are entitled.
[0354] One embodiment of the invention relates to a microscale permeable
material. While certain embodiments of the invention describe the permeable
material as a
biological barrier associated with a microscale device, it is to be understood
that the
microscale permeable material could exist in a wide variety of context and
devices.
[0355] One example of a suitable microscale device includes one or more
microscale features dimensioned to maintain biological material under
conditions that
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provide a value of at least one phaz7nacokinetic paranleter in vitro that is
comparable to the
value of at least one pharmacokinetic parameter -found in vivo. Details
regarding formation
and operation of various embodiments of the microscale features are disclosed
above. For
the purpose of description hereinbelow, "microscale" can niean a dimension in
a range of
approximately 0.1 m to approximately 500 m. Thus, a microscale feature can
be
dimensioned so that at least one of its dimensions falls within the microscale
range. It will
also be understood that various embodiments of the present disclosure can be
implemented in
a larger scale than the above-defined microscale level. For the purpose of
description
hereinbelow, "millimeter-scale" can mean a dimension in a range of
approximately 0.1 mm
to approximately 100 mm. Thus, one or more features of the present disclosure
can be a
millimeter-scale feature where at least one of its dimensions falls within the
millimeter-scale
range. It will be understood that some features may have a conlbination of
dimensions where
one is a microscale and another is a millimeter-scale. Such features can be
characterized as
either of the two scales. Moreover, various features of the present disclosure
can be
implemented in dimensions outside of the above-defined ranges. For example, in
one
embodiment, a microscale feature can-have a dimension less than-0.1 gm, or
greater than 500
m. Likewise, in one embodiment, a millimeter-scale feature can have a
dimension less than
0.1 mm, or greater than 100 mm.
[0356] In other embodiments, the microscale permeable material facilitates
interactions between different fluidic systems. For example, a drug taken
orally enters the
gastrointestinal (GI) system. One or more compounds associated with the dnig
can pass from
the GI system to blood of the circulatory system via the lining of the small
intestine. The
drug compound in the blood can reach and affect various organs and/or systems.
For
example, the drug compound can pass from the blood to the brain fluidic system
to thereby
affect the brain.
[0357] In another example, the drug compound can pass from the blood to the
biliary system in the liver and enter the enterohepatic recirculation cycle.
The dnig
compound can remain in the enterohepatic circulation for a prolonged time and
result in high
concentration in the liver, and thus can become unexpectedly hepatotoxic.
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[0358] Thus, one can see that accounting for passage of drug compounds or
their
metabolites between different systems can allow better understanding of
pharmacokinetics of
the drug involved.
[0359] Figure 31 shows that in one eilibodiment, an interaction 3100 between
first
and second fluidic systems 3102, 3140 can be provided and maintained in vitro
under
conditions with physiological paranzeter values similar to those_ found in
vivo. For the
puipose of description, the first fluidic system 3102 includes one or more
microscale features,
and the second fluidic system 3104 also includes one or more microscale
features.
[0360] As further shown in Figure 31, the interaction 3100 between the first
and
second systems 3102, 3104 can involve passage of one or more compounds from
the first
system 3102 to the second system 3104 (depicted by an arrow 3106), and/or
passage of one
or more compounds from the second system 3104 to the first system 3102
(depicted by an
arrow 3108).
[0361] Figure 32 shows a block diagram of an example biological system 3110
having some example fluidic systems that can be formed using microscale
features. Blood
circulatory system 3112, GI system 3114, biliary system 3116, and brain fluid_
system 3118
are some non-limiting exaniples that can be simulated using microscale
features.
[0362] In one embodiment, at least one inter-system interaction is provided
between the microscale feature based systems. Various inter-system
interactions are
described below in greater detail.
[0363] Figures 33A-33D show non-limiting examples of various interaction
configurations that can be arranged for two or more fluidic systems. In one
embodiment, as
shown in Figure 33A, a two-system configuration 3120 can include an
interaction 3172
between two systems "A" and "B" (3162 and 3164). Figure 33B shows that in one
embodiment, a three-system configuration 3130 can include an interaction 3174
between A
and B (3162 and 3164), as well as an interaction 3176 between B and "C" (3164
and 3166).
Figure 33C shows that in one embodiment, a four-system configuration 3140 can
include an
interaction 3182 between B and "D" (3164 and 3168), in addition to
interactions 3178 and
3180 that are similar to the interactions 3174 and 3176 of Figure 33B.
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[0364] In one einbodiment, the pharmacolcinetic dynamics associated with the
interactions 3178 and 3180 (Figure 33C) may be substantially same as that of
the interactions
3174 and 3176 (Figure 33B). In another embodiment, the presence of the
additional
interaction 3182 (Figure 33C) can significantly alter the pharinacolcinetic
dynamics
associated with the interactions 3178 and 3180 from that of the interactions
3174 and 3176
(Figure 33B).
[0365] Figure 33D sliows that in one embodiment 3150, multiple systems (for
example, three) can be configured to provide and simulate recirculation
functionality. In the
example shown, systems A and B (3162 and 3164) are shown to be interacting via
interaction
3184; systems B and C (3164 and 3166) via interaction 3186; and systems C and
A (3166 and
3162) via interaction 3188.
[0366] Specific examples of the configurations shown in Figures 33A-33D are
described below in greater:detail. Also, other configurations are possible.
[0367] Figures 34A-34C show various views of one embodiment of a two-fluidic
system configuration 3200. Figure 34A shows a partially exploded view of the
assenibled
view of Figure 34B, and Figure 34C shows a top view. A first system is shown
to include a
layer 3220 that defines one or more compartments (depicted as compartment
3222). As
shown, the compartment 3222 can be supplied with fluid for pharmacokinetic
study via an
input flow (indicated as an arrow 3250) through an input pathway 3212 (defined
through a
cover layer 3210) and an input channel 3260. The fluid from the compartment
3222 can exit
through an output channel 3262- and througli an output pathway 3214 (defined
through the
cover layer 3210) as an output flow (indicated as an arrow 3252).
[0368] A second system is shown to include a layer 3230 that defines one or
more
compartments (depicted as compartments 3232, 3234, 3236). As shown, the
compartments
3232, 3234, and 3236 can be supplied with fluid for pharmacolcinetic study via
an input flow
(indicated as an arrow 3254) through an input pathway 3242 (defined through a
cover layer
3240) and an input channel 3270 that is connected witli the compartment 3232.
The fluid
from the conlpartment 3232 can be supplied to the otlier compartments 3234 and
3236 via
channels 3272, 3274, and 3278. The fluids from the compartments 3234 and 3236
can exit
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tlirough output channels 3276 and 3280 and through an output pathway 3244
(defined
through a cover layer 3240) as an output flow (indicated as an arrow 3256).
[0369] In one einbodiment; formation of the compartments, input and output
pathways, and various channels of the first and second systems can be formed
by various
techniques disclosed above. Also, circulation of the fluids for the two
fluidic systems can be
effectuated by various techniques disclosed above.
[0370] As shown in Figures 34A-34C, the two-fluidic system configuration 3200
includes a permeable material 3224 positioned between at least one of tlie
compartments of
the first system 3220 and at least one of the conzpartments of the second
system 3230. In the
example shown, the permeable material 3224 is depicted as being positioned
between the
compartments 3222 and 3232, thereby allowing for fluidic interaction between
the first and
second systems 3220 and 3230. The permeable material 3224 is described below
in greater
detail.
[0371] In Figures 34A-34C, the compartments 3222 and 3232, and the pemieable
material 3224 are depicted as having different dimensions. This is simply for
the purpose of
clarity in illustration. The permeable material 3224 can be dimensioned to be
smaller than,
larger than, or generally same as either or both of the compartments 3222 and
3232. In one
embodiment, the permeable material 3224 can be situated partially or
substantially inside of
either of the compartments, or between the compartments 3222 and 3232.
[0372] Figure 34D shows a partially exploded view of one embodiment 3200 of a
variation of the example configuration shown in Figure 34A. As shown, the two-
fluidic
system configuration 3200 can include a first module 3902 having a first
culture system that
includes one or more cell culture compartments (depicted as compartnlent 3914)
and/or one
or more biological barriers (depicted as barrier 3916).
[0373] As shown, the two-fluidic system configuration 3200 can include a
second
module 3904 having a second culture systenl that includes one or more cell
culture
compartments (depicted as compartments 3918 and 3920). In one embodiment, the
second
module 3904 can also include one or more biological barriers (not shown).
[0374] In one embodiment, as shown, the two-fluidic system configuration 3200
can include fluid interconnects 3910 that facilitates flow of fluid for the
first culture system
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3902. Ixi one elnbodiment, a housing top 3900 can be positioned above the
first module 3902
and define fluid patliways of the fluid interconnects 3910.
[0375] Similarly, fluid interconnects 3922 facilitates flow of fluid for the
second
culture system 3904. In one embodiment, a housing bottom 3906 can be
positioned below
the second module 3904 and define fluid pathways of the fluid interconnects
3922.
[0376] For the purpose of description herein, a"penneable" material includes
any
biological or non-biological material that allows passage of one or more
materials in a
selective manner as found in or sinlulating biological systems. Thus, a
permeable material as
used herein can include a semi-permeable material.
[0377] The foregoing two-system configuration 3200 can provide an in vitro
environment for pharmacokinetic studies for combinations such as, but not
limited to, GI-
blood, blood-biliary, blood-brain, blood-tissue, and blood-urinary.
[0378] Figures 35A and 35B show partially exploded and assembled views of one
embodiment of a three-fluidic system configuration 3290. A first system is
shown to include
a layer 3300 that defines one or more con7partments (depicted as compartnient
3304). A
second system is shown to include a layer 3320 that defines one or more
compartments
(depicted as compartinents 3322, 3324, and 3328). A tliird system is shown to
include a layer
3340 that defines one or more compartments (depicted as compartment 3342).
[0379] In one embodiment, the first system 3300 can supplied with fluid flow
(aiTows 3350 and 3352) througli pathways 3302a and 3302b. The third system
3340 can be
supplied with fluid flow (arrows 3354 and 3356) through pathways 3344a and
3344b. The
second system 3320 can have circulation that provides coupling between the
first and second
systems 3300 and 3340. The compartment 3322 that interacts with the first
system 3300 can
be interconnected via channels (not shown) and pathways 3326a and 3326b with
the
compartment 3328 that interacts with the third system 3340.
[0380] As shown in Figures 35A and 35B, the three-fluidic system configuration
3290 includes two peimeable material assemblies 3310 and 3330. The first
permeable
material assenibly 3310 is shown to be configured so that permeable material
3312 is
positioned between compartments 3304 and 3322 of the first and second systems
3300 and
3320. The second permeable material assembly 3330 is shown to be configured so
that
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permeable material 3332 is positioned between compai-tnieiits 3328 and 3342 of
the second
and third systems 3320 and 3340.
[0381] In the exaniple configuration 3290 shown in Figures 35A and 35B, the
penneable materials 3312 and 3332 are depicted as being parts of separate
layers 3310 and
3330. In one embodiment, the peniieable materials 3312 and 3332 can be forined
so as to be
part of one of their neighboring layers. For exaniple, the permeable material
3312 can be
foimed as part of either of the layers 3300 and 3320 sucll that the permeable
material 3312
separates the comparthnents 3304 and 3322. Similarly, the permeable material
3322 can be
forined as part of eitlier of the layers 3320 and 3340 such that the permeable
material 3322
separates the coinpartments 3328 and 3342.
[0382] In one embodiment, the pemieable materials 3312 and 3322 can be
configured so as to facilitate their respective inter-system interactions. The
permeable
materials 3312 and 3322 are described below in greater detail.
[0383] In one embodiment, a three-system configuration can be implemented in a
manner described above in reference to Figures 35A and 35B. Figure 36 shows a
block
diagram of an example 3360 of such a three-fluidic system. A drug deliveiy
system 3362 can
be represented by the first system 3300 (Figures 35A and 35B); an organ
systenz 3364 can be
represented by the second system 3320; and brain 3366 can be represented by
the third
system 3340. An interaction 3370 between the drug delivery system 3362 and the
organ
system 3364 can be represented by the permeable material assembly 3310; and an
interaction
3372 between the organ system 3364 and the brain 3366 can be represented by
the peimeable
material assenzbly 3330.
[0384] In the example application 3360 of the three-system configuration, the
drug delivery system 3362 can include a GI system, and the organ system can
include various
organs (other than the brain) and the blood circulatory system. Thus, the
interaction 3370 can
include passage of one or more compounds associated with the drug from the GI
system into
the blood; and the interaction 3372 can include passage of one or more
compounds associated
with the drug from the blood to the brain's fluidic system.
[0385] It will be understood that other three-system conflgurations are
possible.
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[0386] Figure 37 shows a block diagram of an exaniple configuration 3380
involving a liver 3384. The liver 3384 is shown to interact with a GI tract
3382 via an
enterohepatic circulation (depicted as arrows 3390 and 3392). The liver 3384is
also shown
to interact with a urinaiy system 3388 (depicted by an aiTow 3396) and tissues
3386 (depicted
by an arrow 3394). The interaction 3396 between the liver 3384 and the urinary
system 3388
can be facilitated by blood circulation system acting as an intennediary.
Similarly, blood
circulation system can facilitate the interaction 3394 between the liver 3384
and the tissues
3386.
[0387] Figure 38 shows that blood circulatoiy system 3406 can also facilitate
the
enterohepatic circulation process involving the liver 3384 and the GI tract
3382. As shown,
biliary system 3402 (of the liver 3384) interacts (a~.-row 3410) witli GI
system 3404, that in
turn interacts (ai'row 3412) with the circulatory system 3406. The circulatory
system 3406
interacts (arrow 3414) with the biliary system 3402, tliereby forn7ing a
recirculation process.
[0388] As is generally lcnown, liver produces bile acids that are delivered to
the
small intestine to aid in digestion. In the digestive tract, bile acids are
converted to
conjugated bile- salts (primary or secondary), and these salts are absorbed -
either actively or
passively - in to the hepatic portal circulation to be recycled by the liver.
Typically, each bile
salt molecule is reused about twenty times in the enterohepatic cycle.
[0389] One of the consequences of the foregoing recycling process is that
drugs or
components thereof can remain in the enterohepatic circulation for a prolonged
period of
time. Thus, some molecules that would otherwise not be toxic can accumulate in
the liver
and become toxic. Thus, pharmacokinetics associated with the enterohepatic
recirculation
process can provide important understanding on toxicity (or non-toxicity) of
drugs being
tested.
[0390] As described above, various features of the foregoing interactions
between
different fluidic systems can be facilitated by one or more types of permeable
materials. In
some embodiments, such pernieable materials can be part of a microscale
permeable device.
[0391] As described below in greater detail, one or more features of the
present
disclosure can, on its own, or in combined form, provide various systems and
methods. For
example, an apparatus can have at least one feature dimensioned to maintain
biological
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material under conditions that provide a value of at least one pharmacokinetic
parameter in
vitro that is comparable to the value of at least one pharmacolcinetic
parameter found in vivo,
and a penneable material. The permeable material is described below in greater
detail. In
one einbodiment, the at least one feature includes a inicroscale feature.
[0392] In one embodiment, the at least one feature can be configured to
represent
at least portions of one or more of the following non-limiting example
systems: central
nervous, circulatory, digestive, biliary, pulmonaiy, urinary, ocular,
olfactory, epidemlal, and
lymphatic systems.
[0393] In one embodiment, as described herein, the apparatus can further
include
at least one microfluidic channel connected to the permeable material. Such a
channel, can
facilitate flow of fluid in, through, or in proximity to the permeable
material so as to provide
the at least one pharmacokinetic parameter. In one embodiment, the
characteristics of such
fluid flow can be based on a mathematical model such as a physiologically-
based
pharmacokinetic ("PBPK") model.
[0394] In one embodiment, the at least one feature and/or the permeable
material
can be integrated into a chip format.
[0395] In one embodiment, the permeable material can be located in or external
to
the device. In one embodiment, the permeable material can include a
microporous material
coated at least in part with an organic material.
[0396] In one embodiment, cells can be located in, on or near both sides of
the
permeable material. In one embodiment, the device having such cells can
facilitate
detemiination or estimation of parameters such as absorption characteristics,
metabolic
enzynie activity and/or expression levels. In one einbodiment, the cells on
eitlier side of the
permeable material can be of the same type or of different types.
[0397] Figure 39 shows one embodiment of microscale pemzeable device 3420
having permeable material 3430 that can facilitate one or more interactions
between two
fluidic systems. Some non-limiting examples of the permeable material 3430 can
include the
following: a membrane, a porous membrane, porous silicon, microporous silicon,
a semi-
permeable menibrane, a microporous polymer, a porous polycarbonate membrane,
alginate,
collagen, MATRIGEL, cells, cellular material, tissue, and pieces of tissue.
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[0398] In one enibodiment, the peimieable material 3430 can include organic or
inorganic material in, on or near a microporous surface of the permeable
materia13430.
[0399] In one embodiment, the peimeable material 3430 includes a microporous
material. Some non-limiting examples of the microporous material can include
the
following: organic or inorganic material cultured, deposited, or inserted in,
on or near the
microporous surface of the microporous material.
[0400] In one embodiment, the permeable material 3430 can be configured to
simulate at least one of a biological barrier, passage of substances in or
through a biological
barrier, or absorption of substances in, through or by a biological barrier.
In one
embodiment, the biological barrier can include at least one of the following:
a
gastrointestiiial baiTier, a blood-brain barrier, a pulmonary barrier, a
placental barrier, an
epidermal barrier, ocular baiTier, olfactory barrier, a gastroesophageal
barrier, a mucous
menibrane, a blood-urinary baiTier, air-tissue barrier, a blood-biliary
barrier, oral barrier, anal
rectal barrier, vaginal barrier, and urethral barrier.
[0401] In one embodiment, the permeable material 3430 can facilitate
determination of various pharmacokinetic parameters while accounting for one
or more inter-
system interactions. These pharmacokinetic parameters can include at least one
the
following: tissue size, tissue size ratio, tissue to blood volume ratio, drug
residence time,
interactions between cells, liquid residence time, liquid to cell ratios,
metabolism by cells,
shear stress, flow rate, geometry, circulatory transit time, liquid
distribution, interactions
between tissues and/or organs, and molecular transport by cells.
[0402] hi one embodiment, the permeable material 3430 can facilitate
determination of absorption, metabolism, or distribution of a substance in,
through or by the
permeable material.
[0403] In one embodiment, the permeable material 3430 can be formed in,
contained in, inserted, assembled, made, or constituted in a device that
include a plurality of
microscale features representative of two or more fluidic systems.
[0404] In one embodiment, either or both sides of the permeable material 3430
can be configured to allow culturing, attaching or positioning of cells or
cellular materials.
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Such a configuration can allow for deterinination of parameters such as
absorption
characteristics, metabolic enzyme activity and/or expression levels.
[0405] In one enibodiment, the permeable material 3430 can include a cell line
capable of formiilg a confluent monolayer and polarizing. -
[0406] In one embodiment, the permeable assembly 3430 can include a
microscale pemieable material 3432. In one embodiment, the microscale
permeable material
3432 can include a microporous substrate having a plurality of pores. hi some
embodiments,
the pores generally have dimensions less than approximately 10 m. In some
embodiments,
the microporous substrate inhibits passage of particles having dimensions
larger than
approximately 10 m.
[0407] In one embodiment, the microporous substrate can be formed from porous
silicon having pores with dimensions in a range of approximately 0.1 to 10 gm.
The
thickness "T" for such a substrate can be in a range of approximately 5 to 100
gm. In one
embodiment, the microporous substrate can be formed from a porous
polycarbonate
membrane having pores with dimensions in a range of approximately 0.4 m. The
thickness
"T" for such a substrate can be in a range of approximately 100 m: In one
embodiment, the
microporous substrate can be formed from porous low stress silicon nitride
material having
pores with dimensions in a range of approximately 0.2 to 1 m. The thiclcness
"T" for such a
substrate can be in a range of approximately 2 to 5 m.
[0408] In one embodiment, the lateral dimension "L" (perpendicular to the
direction defining the thiclmess) can have any value relative to the lateral
dimension of a
conipartment 438. For a given thickness, a larger surface area (and thus
larger lateral
dimension(s)) will likely provide greater amount of interaction between two
fluidic systems.
Thus, the aniount of passage of materials between the two systems can be
controlled by
providing different laterally sized surface area. Thus, the lateral dimension
L can be less
than, substantially equal to (as shown in the example of Figure 39), or
greater than the
corresponding lateral dimension of the compartnzent 343 8.
[0409] In one embodiment, the microporous substrate has lateral dimensions in
a
range of approximately 0.1 to 10 mm. In embodiment, the lateral dimensions are
approximately 3.4 mm x 4 mm.
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[0410] As further shown in Figure 39, the penneable asseinbly 3430 can
furtlier
include one or more function-specific cells 3434 positioned on, within, and/or
about the
microscale permeable material 3432. The function-specific cells 3434 are
described below in
greater detail by way of example for interaction between blood and biliaiy
systems. It will be
understood, however, that different ftmction specific cells can be positioned
with respect to
the microscale permeable material 3432 to provide desired fiinctionalities for
different inter-
system interactions.
[0411] In one embodiment, the permeable assembly 3430 can further include one
or more binders 3445 that facilitate binding of the cells 3434 to the surface
of the microscale
permeable material 3432. Examples of the binders 3445 are described below in
greater
detail.
[0412] In one embodiment, the permeable assembly 3430 can further include one
or more features 3436 that provide functionality similar to fibroblast cells.
In one
embodiment, the function-specific cells 3434 can be distributed on the surface
of the
microscale permeable material 3432, and the fibroblasts 3436 can fill the
areas on the surface
of the microscale permeable material 3432 not occupied_ by the cells 3434. In
such a
configuration, the fibroblasts can provide a sealing functionality such that
passage of
materials through the permeable assembly 3430 occurs mostly via the cells
3434.
[0413] In one embodiment, the fibroblasts 3436 can provide a favorable
environment for growth and maintenance of the cells 3434. In one embodiment,
the
fibroblasts 3436 can provide both functionalities - cell growth and
maintenance, as well as
sealing of the permeable materia13430.
[0414] In one embodiment, the permeable assembly 3430 can be formed as part of
a layer 3422 so as to define the compartinent 3438 on at least one side of the
penneable
assembly 3430. In other embodiments, both sides of the peimeable assembly 3430
can define
their respective conipartments. For such a configuration, the permeable
assembly 3430 can
have the cells 3434 and the binders 3434 on either or both sides of the
permeable assembly
3430.
[0415] Figures 40A and 40B show various example situations where the
permeable assembly can be provided to allow interactions between different
fluidic systems.
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The example inter-system interactions" are based on the enterohepatic
recirculation process.
However, other inter-system interactions can be facilitated in a similar
manner.
[0416] Figure 40A shows one embodiment of an interaction configuration 3440
between the blood flow system and the bile flow system. In one embodiment, a
pernleable
assenibly 3442 can be interposed between the blood flow and the bile flow, and
can include a
microscale peiiiieable material 3444. In some embodiments, the microscale
permeable
material 3444 can be formed and dimensioned in a similar manner as described
above in
reference to Figure 39.
[0417] In" one embodiment, the permeable assembly 3442 can further include one
or more function-specific cells 3446. For the blood-bile interaction, the
cells 3446 can
include hepatocyte cells.
[0418] Hepatocytes of the liver can be polarized cells; and different surfaces
of
differentiated hepatocytes can have unique functions. In one einbodiment,
sinusoidal
membrane of the basolateral surface and the bile canalicular membrane of the
apical surface
in the liver can be simulated in the following manner. Isolated hepatocytes
generally are not
polarized. Hepatocytes generallybecome polarized when they physically contact -
adjacent
hepatocytes. Bile canaliculi can be formed between two or more of such
juxtaposed cells.
[0419] External cues can be important for epithelial cell polarization, and
the
physical contact between two adjacent hepatocytes appears to be the signal for
such
hepatocyte polarization. Hepatocytes can form connections with adjacent
hepatocytes
through the binding of junction or adhesion proteins, and the interaction of
these proteins
appears to be an important signal for bile canalicular morphogenesis.
[0420] As shown in Figure 40A, these proteins can act as binders 3445 that
facilitate binding of the hepatocytes 3446 to the microscale substrate 3444
and polarization of
the hepatocytes. In some embodiments, these proteins can include gap junction
proteins (e.g.,
connexiii 32), tight junction proteins (e.g., occludin, claudin-1, ZO-1, ZO-
2), adherens
junction proteins (e.g., E-cadherin and beta-catenin), and cell adhesion
molecules (e.g.,
uvomorulin).
[0421] In one embodiment, one or more of these proteins attached to the
microscale permeable substrate 3444 can in effect mimic a plasma membrane
surface for an
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adjacent hepatocyte. When isolated hepatocytes bind to this surface, the
hepatocytes can be
induced to polarize, such that the apical surface or bile canaliculi (3449b)
can be formed at
the surface of the microscale peiineable substrate 3444, and the basolateral
or sinusoidal
surface (3449a) can be formed on the opposite surface.
[0422] In one enibodiment, the hepatocytes 3446 can be seeded at an
appropriate
density to inhibit cell-cell interactions. Once the hepatocytes 3446 are
attached to the
microscale pernleable substrate 3444, fibroblasts 3448 or other appropriate
cells can be
cultured on the surface to substantially seal the microscale permeable surface
at areas not
occupied by the hepatocytes 3446, thus forming a "blood-biliary" barrier,
and/or to provide a
favorable environment for hepatocyte growth.
[0423] In one embodiment, one or more selected compounds of interest can be
introduced to flow over the hepatocytes 3446. Such a compound caii be
transported via the
hepatocytes 3446 across the microscale permeable surface into the bile
suirogate flow of the
device 3440. The presence of the compound or its rnetabolites can be measured
in the bile
surrogate flow to deternline biliary excretion.
[0424] Once the bile is transferred into the GI system and reabsorbed into the
blood system, "bile" in the GI system can include the following compounds:
bile salts
(chenodeoxycholic, hyodeoxycholic, cholic, a-nluricholic, and Bbeta;-
muricholic acids);
phospholipids (phosphatidylcholine (-82%), trace amounts of
phosphatidylinositol,
phosphatidylserine, and sphingomyelin); bile alcohols (5 beta-cholestane-3
alpha,7 alpha,12
alpha,26-tetrol); and amino acids.
[0425] In one embodiment, the bilialy flow can be coupled to the GI flow to
further mimic the enterohepatic recirculation. hi one embodiment, the bile can
be mixed with
the GI fluid. Such mixing can be achieved, for example, in a manner described
below in
greater detail.
[0426] hi one embodiment 3460 as shown in Figure 40B, the GI flow can be
coupled to the blood flow to further mimic the enterohepatic recirculation.
The interaction
3460 can include a permeable assenlbly 3462 that has a microscale permeable
substrate 3464
and a surface defined by one or more function-specific cells 3466. In one
embodiment, the
function-specific cells 3466 can include intestinal epithelial cells. In one
embodiment, Caco-
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2 cells 3468 can be provided adjacent the cells 3466 so as to facilitate in
vitro absoiption of
coYnpounds from the GI flow to the blood flow.
[0427] In one embodiment, the pernieable material 3462 can include a layer of
gastrointestinal enterocytes cultured on the microscale permeable substrate
3464. h1 one
embodiment, at least a portion of the layer of gastrointestinal enterocytes
can be positioned in
the device 3460 such that fluid may flow along either side of but not tlirough
the layer. In
one embodiment, at least a first microscale feature located on a first side of
the layer of
gastrointestinal enterocytes can represent the gastrointestinal tract, and at
least a second
microscale feature located on a second side of the monolayer can represent a
circulatory
system. hi one embodiment, a third microscale feature can be provided and
configured to
contain the same or a different type of biological material.
[0428] Figures 41A and 41B show partially exploded and assembled views of an
example enzbodiment-of a device 3700 that can provide phaimacokinetic
simulation of the
enterohepatic recirculation process described above. The device 3700 can
include a GL
surrogate module 3720 that can provide GI-blood interaction functionality
similar to that
described above in reference to -Figure 40B. The device 3700 can also include
an organ
system module 3730 that can provide blood-biliary interaction functionality
similar to that
described above in reference to Figure 40A. Housing caps 3710 and 3760 can
provide
housing for the device 3700, and can also provide pathways for various fluid
flows.
[0429] As shown, GI flow to (arrow 3770) and from (arrow 3772) the GI
surrogate module 3720 can be provided by respective pathways 3712 and 3714.
Similarly,
blood flow to (arrow 3774) and frorii (arrow 3776) the blood side of the organ
system module
3730 can be provided by respective pathways 3762, 3750 and 3752, 3768.
Similarly, bile
flow to (arrow 3778) and from (3780)-the biliaiy side of the organ system
module 3730 can
be provided by respective pathways 3764 and 3766.
[0430] As shown, the GI surrogate module 3720 can include a compartment 3722
that includes a permeable assenlbly having a microscale permeable substrate
3724. The
microscale permeable substrate 3724 can be formed from any one or coinbination
of
materials described above in reference to Figure 39. The peimeable assenibly
can also
include intestinal epithelial cells 3726 formed on the microscale permeable
substrate 3724.
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In one eznbodiment, the GI side of the conlpartment 3722 can include Caco-2
cells 3728
adjacent the cells 3726. As is generally lcnown, Caco-2 cells can facilitate
in vitro absorption
of compounds from the intestine to the blood.
[0431] Conipounds absorbed tlirough the peizneable assembly of the GI
suiTogate
module 3720 can enter the blood system at a compartment 3732 of the organ
system module
3730. Blood can circulate between the compariment 3732 and one or more other
compartnients. For the purpose of description, a compartment 3734 having a
permeable
assembly for blood-biliary interaction and a coinpartment 3744 simulating a
target organ (via
target cells 3746) are shown. In one enlbodinlent, target organ 3744 can
include organs or
tissues that may be affected by drug activity. For example the target organ
3744 can be a
heart when testing cardiac medications. In another exaniple, the target organ
can be pancreas
when testing for drug toxicity.
[0432] The permeable assembly of the compartment 3734 is shown to include a
microscale permeable substrate 3736. The microscale permeable substrate 3736
can be
formed from any one or combination of materials described above in reference
to Figure 39.
The peimeable assembly can also include hepatocytes 3738 formed on the
microscale
permeable substrate 3736. In one enibodiment, the hepatocytes 3738 can be
bound to the
microscale penneable substrate 3736 via binders in a manner described above in
reference to
Figure 40A. In one embodiment, the permeable assembly can further include
fibroblasts
3740 to provide functionality as described above in reference to Figure 40A.
[0433] The permeable assembly of the organ system module 3730 can facilitate
the blood-biliary interaction between the blood flow (in the space 3742 of the
compartment
3734) and the bile flow (on the other side of the permeable assembly). The
bile flow can then
be circulated via the pathways 3764 and 3766, and bile can be re-introduced
(not shown) into
the GI flow.
[0434] Figure 41C, shows another partially exploded view of the organ system
module 3700 similar to that shown in Figure 41A. In Figtue 41C, the
comparhnent 3734
having the permeable assembly for blood-biliaiy interaction is shown in
greater detail by the
callout. In one enibodiment, the permeable assembly of the compartment 3734
can be similar
to that described above in reference to Figure 39. Thus, the permeable
assenibly 3430 can
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include a permeable material 3432 and cells or cellular materials 3434 foimed
on either or
botlz sides of the permeable material 3432. Iii one einbodiment, the cells
3434 can be
hepatocytes that can be bound as described herein. In one enibodiment where
hepatocyte
cells are used, the pexxneable assembly 3430 can fiu-ther include fibroblasts
3436.
[0435] Figure 42 depicts an exaniple schematic 3800 of various fluid flows
that
can be implemented in the example enterohepatic recirculation device of
Figures 41A and
41B. hi one enibodiment, a GI fluid flow 3802 (depicted as a dashed line) can
be provided to
flow through a GI tract compartment 3810 having a GI-blood barrier 3812 as
described
herein. The GI fluid flow 3802 can be made to flow from a GI fluid reservoir
3850 to anotlier
reservoir (not shown). In one embodiment, the GI fluid flow 3802 does not
recirculate.
[0436] As shown in Figure 42, a GI-biliary interaction can be facilitated by
the
GI-blood barrier 3812. Blood flow 3804 is depicted as solid lines. The blood
flow indicated
as 3804a interacts with the GI flow 3802 in the GI tract compartment 3810 via
the barrier
3812, and is directed to a liver compartment 3820. A blood-biliary barrier
3822 (as described
herein) can facilitate interaction of the blood flow 3804a with a bile flow
3806. In one
einbodiment, the bile flow 3806 to the livercompartmeiit 3820 can be provided
from a bile
fluid reservoir 3860. In one enlbodiment, the bile flow 3806 from the liver
compartment
3820 can be mixed with the GI flow 3802 at a location that is upstream of the
GI tract
comparlinent 3810, thereby providing the recirculating functionality of the
bile from the liver
compartment 3820.
[0437] In one embodiment, the blood flow 3804a from the liver compartment
3820 can be directed to one or more other compartments. For example, a blood
flow 3804c
(via 3804b) is shown to provide blood to a target tissue compartment 3830, and
a blood flow
3804e (via 3804b) is shown to provide blood to other-tissue compartment 3840.
Blood flows
3804d and 3804f fiom the compartments 3830 and 3840 are can be recombined into
a blood
flow 3804g that can become part of the blood flow 3804a at a location that is
upstream of the
GI tract coinpartment 3810.
[0438] Figures 43A to 43E show various stages of fabrication of one embodiment
of the microscale permeable device described above. Figure 44 shows one
embodiment of a
process 3520 that can perform the fabrication of the device of Figures 43A to
43E.
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[0439] As shown in Figure 43A, an opening 3502- can be formed on a substrate
3500. Such formation of the opening can be achieved in a process block 3522.
[0440] As shown in Figure 43B, a microscale permeable substrate 3504 can -be
forined in the opening 3502. Sucli formation of the microscale permeable
substrate 3504 can
be achieved in a process block 3524.
[0441] As shown in Figure 43C, one or more binders 3506 can be positioned on
the microscale permeable substrate 3504. Providing of such binders 3506 can be
achieved in
a process block 3526.
[0442] As shown in Figure 43D, one or more function-speciflc cells 3508 can be
bound to the inicroscale permeable substrate 3504 via the binders 3506. Such
binding of the
function-specific cells 3508 can be achieved in a process block 3528.
[0443] As shown in Figure 43E, one or more fibroblasts 3510 can be introduced
between the function-specific cells 3508 so as to provide sealing and/or to
facilitate growth
and maintenance of the cells 3508. Such introduction of the fibroblasts 3510
can be achieved
in a process block 3530.
[0444] In one enlbodinient, the microscale peiineable substrate 3504 can be
foimed via the following non-limiting example. A microporous surface can be
formed from
silicon by etching with HF (hydrofluoric acid) under an applied bias. A
microporous surface
can also be formed from low-stress silicon nitride thin films by using
standard
photolithography and etching techniques for pore sizes greater than about 0.4
microns in
diameter or electron beam lithography and etching for pore sizes less than
about 0.4 microns
in diameter.
[0445] In one non-limiting example embodiment, binder proteins can be
micropatterned on the microporous surface by utilizing microcontact printing
techniques. A
silicone elastomer "rubber stamp" can be produced using replica molding
tecliniques. The
rubber stanip can be dipped in a solution of binder proteins and these binder
proteins can then
be deposited onto the surface of the microporous material thus producing a
micropattern of
binder proteins. This process is commonly known as micro-contact printing.
[0446] In one non-limiting example embodiment, the hepatocytes can be allowed
to attach to the binder proteins and once attached, fibroblasts can be
introduced to the surface
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and allowed to attached to substantially all areas of the microporous surface
not occupied by
the hepatocytes.
[0447] Other fabrications tecluiiques can be utilized.
[0448] In one enibodiment, a microscale permeable material (such as 3432 in
Figure 39) and at least one binder (such as 3506 in Figure 43C) can define a
device. The at
least one binder can be configured to polarize a substance, the substance
manifests at least
one characteristic of liver function.
[0449] In one embodiment, the substance can be one or more hepatocytes. In one
embodiment, the substance can be a genetically engineered biological material.
[0450] In one enzbodiment, the binder can bind and polarize hepatocytes to the
microscale permeable material.
[0451] In one embodiment, a device can include a microscale permeable material
(such as 3432 in Figure 39), and at least one substance configured to manifest
at least one
characteristic of liver function, where molecules processed by the substance
can be directed
to pass through at least a portion of the microscale permeable material.
[0452] Figure 45 shows non-limiting examples of various combinations of
systems that can be coupled using one or more techniques of the present
disclosure. A
microscale permeable device 3540 can allow interaction between blood and
biliary systems.
A microscale penneable device 3542 can allow interaction between-blood and GI
systems. A
selected coupling (depicted as an arrow 3544) can allow interaction (for
example, by mixing
at a selected location) between biliary and GI systems. A microscale permeable
device 3546
can allow interaction between blood and brain systems. A microscale permeable
device 3548
can allow interaction between blood and urinary systems.
[0453] It will be understood that otlier inter-system interactions are
possible via a
microscale permeable device. Thus in general, as shown in Figure 46, a
microscale
pemleable device 3550 can allow interaction between a first fluidic system and
a second
fluidic system.
[0454] In the description above, various enibodiments of the microscale
permeable device are depicted as being part of a layer that is either part of
a system layer or a
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separate layer. For such configurations, coinpartments associated with
different systems are
depicted as being formed on different layers.
[0455] In some embodiments, this is not necessarily a requirement. For
exaniple,
in one embodiment, an organ system module (3730 in Figures 41A and 41B) can be
formed
on one side of a layer, and a GI sui-rogate module (3720 in Figures 41A and
41B) can be
fornzed on the other side of the same layer.
[0456] In another example embodiment, a microscale penneable device can be
formed on a given layer so as to define two compartments, witli each
coinpartmenf
representing a separate system. Thus, as shown in an exaniple embodiment 3560
of Figure
47, a microscale permeable device 3562 can be forined on a layer so as to
define and separate
two compartments 3564 and 3566. . Thus, the first compartment 3564 can
represent a first
fluidic system, and the second compartment 3566 can represent a second fluidic
system. The
microscale permeable device 3562 can provide the interactionbetween the first
and second
fluidic systems. A more complex system such as that shown in Figures 41A and
41B can be
formed accordingly.
[0457] Although the above-disclosed embodiments have shown, described, and
pointed out the fundamental novel features of the invention as applied to the
above-disclosed
embodiments, it should be understood that various omissions, substitutions,
and chaiiges in
the form of the detail of the devices, systems, and/or methods shown may be
made by those
skilled in the art without departing from the scope of the invention.
Consequently, the scope
of the invention should not be limited to the foregoing description, but
should be defined by
the appended claims.
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