Note: Descriptions are shown in the official language in which they were submitted.
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
SYSTEMS, DEVICES AND METHODS FOR MICROFLUIDIC CULTURING,
MANIPULATION AND ANALYSIS OF TISSUES AND CELLS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional App. No.
61/894,298, filed Oct. 22,
2013. This application is related to U.S. Application No. 13/682,710, filed
Nov. 20, 2012,
which claims priority to U.S. Provisional App. No. 61/561,907, filed November
20, 2011, and
U.S. Provisional App. No. 61/677, 157, filed July 30, 2012. This application
is also related to
International App. No. PCT/US2011/055444, filed October 7, 2011 and
designating the U.S.,
which claims priority to U.S. Provisional App. No. 61/391,340, filed October
8, 2010. The
teachings of each of these applications are incorporated by reference.
FIELD
[0002] Systems, methods, and devices related to the field of medical
testing/diagnostics, cell-
based assays, and compound discovery are provided herein. In various aspects,
systems, devices,
and methods are provided for the determination of the growth, and/or oncogenic
potential,
migration rate, and/or metastatic potential of mammalian cells or patient's
cells (e.g., cells
obtained from biopsy). In some aspects, microfluidic tissue disassociation,
cell, protein, and
particle separation, cell manipulation, and assay devices and methods for
using the same are
provided. Exemplary applications include but are not limited to diagnostic and
cell based assays.
BACKGROUND
[0003] Primary cell culture that allows the study of native tissue samples
derived from an
organism. Culturing cells derived from organisms, can be useful and necessary
for applications
such as medical diagnostics, cell-based assays, for compound discovery and
characterization.
[0004] For example, cancer diagnosis and identification of compounds for
treatment of cancer
are of great interest due to the widespread occurrence of the diseases, high
death rate, and
recurrence after treatment. According to National Vital Statistics Reports,
from 2002 to 2006 the
rate of incidence (per 100,000 persons) of cancer in Caucasians was 470.6, in
people of African
descent 493.6, in Asians 311.1, and Hispanics 350.6, indicating that cancer is
wide- spread
among all races. Lung cancer, breast cancer and prostate cancer were the three
leading causes of
death in the US, claiming over 227,900 lives in 2007 according to the NCI.
[0005] Survival of a cancer patient depends heavily on detection. As such,
developing
technologies applicable for sensitive and specific methods to detect cancer is
an inevitable task
for cancer researchers. Existing cancer screening methods include: 1. the
Papanicolau test for
women to detect cervical cancer and mammography to detect breast cancer, 2.
prostate-specific
antigen (PSA) level detection in blood sample for men to detect prostate
cancer, 3. occult blood
1
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
detection for colon cancer, and 4 endoscopy, CT scans, X-ray, ultrasound
imaging and MRI for
various cancer detection. These traditional diagnostic methods however are not
very powerful
when it comes to cancer detection at very early stages and give little
prognostic information.
Moreover, some of the screening methods are quite costly and not available for
many people.
[0006] Likewise, existing methods for cancer staging are often qualitative and
therefore limited
in applicability. For example, diagnoses made by different physicians or of
different patients
using existing methods can be difficult to compare in a meaningful manner due
to the subjective
nature of these methods. As a result, the subjectivity of the existing methods
of cancer staging
often results in overly aggressive treatment strategies. By way of example, in
the absence of
better data, the most drastic, potentially invasive, strategy is often
recommended, which can lead
to overtreatment, poor patient quality of life, and increased medical costs.
[0007] One method to detect and/or characterize cancer, for example, is to
directly assess living
tissue derived from small biopsy samples taken from suspicious tissue. To get
a relevant and
useful sense of the biological characteristics of tissue, one would be well
served by being able to
culture biopsy tissue in vitro.
[0008] Therefore, the development of technology that is specific and reliable
for culturing
primary human tissue and/or detecting and characterizing a cancer (e.g.,
determining the growth,
oncogenic, migration rate, and/or metastatic potential of cells obtained from
a patient) is an area
of significant importance. Likewise, there remains a need for improved
systems, methods, and
devices for diagnostic cell-based assays and compound discovery.
SUMMARY
[0009] The systems, methods, and devices described herein generally involve
medical
testing/diagnostics, cell-based assays, and/or compound discovery. In various
aspects,
microfluidic devices, systems, and methods disclosed herein can provide
clinical and/or research
purposed diagnostics and assay platforms that enable tissue disassociation,
cell, protein, and
particle separation, and cell manipulation. The systems and devices disclosed
herein can provide,
for example, the culturing of a small number of cells in environments that can
approximate in
vivo conditions, while allowing for a determination of the cells' growth,
and/or oncogenic
potential, migration rate, and/or metastatic potential. A determination of
these characteristics can,
among other things, facilitate treatment decision steps taken by a physician
for patients having
symptoms of cancer and/or aid in the discovery of therapeutics that alter
and/or perturb a cell's
characteristics that engender its cancer-like, oncogenic, and/or metastatic
phenotype.
2
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
[0010] For example, quantitative prognostic metrics according to aspects of
the present
disclosure can improve the accuracy of diagnosis by supplementing a
physician's decision-
making process with clinical data to support the available treatment options.
As a result,
embodiments of the present disclosure can provide numerous advantages, for
example, reduced
healthcare costs, reduced risk associated with treatment, improved patient
quality of life, and
increased patient survival.
[0011] As will be described in detail below, one exemplary aspect of the
present disclosure
provides cell processing systems and devices, including uses thereof, that
include microfluidic
channels and a substrate to process (e.g., culture, filter, image) cells
derived, for example, from a
biopsy. In other aspects, the systems and devices enable diagnostic imaging,
cell-based assays
such as metabolic testing, and/or compound discovery.
[0012] In one exemplary embodiment, a system for cell processing is provided.
The system can
include at least one microfluidic cell dissociation module and at least one
microfluidic cell-
processing module fluidly coupled to at least one cell dissociation module.
The cell dissociation
module can be configured, for example, to dissociate one or more tissue
fragments received
therein into one or more of single cells and/or smaller tissue fragments. The
microfluidic cell-
processing module can receive at least a portion of said one or more single
cells and/or smaller
tissue fragments.
[0013] In various embodiments, one or more cell-processing modules of the
system can be
configured to perform various cell processing functions. In various aspects,
microfluidic systems
can incorporate one or more of the following exemplary individual microfluidic
modules and/or
substrates:
= a cell dissociation module, which can receive mammalian tissue and
separate the
tissue into smaller clumps and/or single cells, e.g., via enzymatic,
mechanical,
and/or shear forces;
= a cell separation module, where cells and extra-cellular components such
as
proteins and other particles can be segregated and sorted;
= perfusion chambers, in which single cells and/or clumps of cells can be
adhered
to specialized micro- and nano-featured substrates. When functionalized with
protein coatings, these specialized substrates can create a permissive surface
for
cell adhesion and subsequent examination via microscopy techniques. Cells can
also be cultured in such an environment;
= metabolic assay, compound discovery, and titration modules integrated as
part of
the above perfusion chamber, whereby cells adhered to various substrates can
be
subjected to various compounds for assay or therapeutic applications. The
cells
can then be monitored via microscopy techniques for their response. Titrations
3
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
can also be conducted in the titration module and can be similarly inspected
via
microscopy; and
= various specialized substrates for cell adhesion and also for testing
cellular
properties such as invasion potential.
[0014] In frequent embodiments, an extracellular matrix (ECM) formulation is
used to
functionalize the microfluidic device or substrates contained therein. The
formulation, for
example, improves primary cell culture growth conditions by closely
replicating an in vivo
environment (rather than a plain polymer substrate to which cells often poorly
attach). This
allows cells from biopsy samples to be cultured in vitro. In their natural in
vivo environments,
cells interact with other cells and the surrounding ECM. A cell's external
environment can
greatly influence it. Additionally, the mechanisms by which cells respond to
external stimuli
shed light onto the properties of a cell's underlying state. For example,
cells can sense the
stiffness of their surroundings and induce distinct and irreversible
remodeling of the ECM and
ECM-cell contacts. Overall tissue stiffness or tissue stiffness gradients can
be integral in tumor
progression and other diseases.
[0015] In an exemplary embodiment, a cell dissociation module can include one
inlet port for
receiving one or more tissue fragments, several cell dissociation chambers, an
outlet port for
extracting fluids, dissociated cells, and other particles to be transmitted to
one or more
downstream modules for further processing, and a channel fluidly coupled to
said chambers,
inlet, and an outlet to allow fluid to be displaced through the chamber. A
pump can be coupled
to the inlet or outlet to cause movement of the fluid through the channel
and/or chambers.
[0016] The cell dissociation module can have various configurations and
dimensions. By way of
example, the fluidic pathway can take on a serpentine, spiral shape, or
configured for back and
forth movement of the fluid within a dissociation chamber. In some aspects, a
plurality of
microstructures disposed in the channel can facilitate dissociation of said
one or more tissue
fragments. The microstructures can have a variety of dimensions. By way of
example, the
microstructures can range from about 1 micron to 1 millimeter in length and
vary in post-to-post
gap distance from 100 micron to lmm. The microstructures can have a variety of
configurations
to facilitate dissociation, e.g., through mechanical perturbation. For
example, the microstructures
can be rectangular.
[0017] As noted above, systems and devices in accord with the present
teachings can include a
cell-processing module. In one exemplary embodiment, the cell-processing
module includes a
perfusion chamber to culture cells, image samples, and perform metabolic
assays. The chamber
can include a sample inlet for receiving one or more samples, a reagent inlet
for receiving one or
4
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
more metabolic reagents, an imaging/culturing chamber for culturing, imaging,
and analyzing
cells, and a waste outlet for extracting waste fluid, and a channel fluidly
coupled to said chamber,
inlets, and outlet to allow fluid to be displaced through the chamber.
[0018] In some embodiments, the cell-processing module can be adapted for
hands free, long
term cell culture by coupling a media reservoir to the imaging/culture
chamber. In addition, the
channels entering the imaging/culturing chamber can have baffles to encourage
even flow
distribution within said channel.
[0019] In various aspects, the perfusion chamber can take on various
configurations and
dimensions. By way of examples, the imaging/culturing chamber can be
hexagonal, and the
module can have a number of perfusion chambers for parallel processing.
[0020] In another exemplary embodiment, the cell-processing module can perform
adhesion-
based cell sorting of heterogeneous cell populations. By way of example, cell
sorting is
performed by fluidly linking a number of perfusion chambers from the above
description, each
perfusion chamber functionalized with a different type of substrate with micro-
and nano-features
coated with proteins solutions specialized for attachment of one particular
subset of cells in the
heterogeneous mixture of cells in the sample. In another example, the cell
sorting module
consists of several sorting chambers, a cell inlet, several reagent inlets,
and several waste outlets.
The chambers are also functionalized with different types of substrates. In
some aspects, the
cell-sorting module can be adapted for cell culturing, imaging, and metabolic
assays.
[0021] In some aspects, the various modules described herein (or at least a
portion of the
modules such as the microfluidic channels) can be formed in a monolithic
substrate. For
example, a cell-dissociation module and the cell-processing layer of a cell-
processing module
can be formed in a monolithic substrate. Such devices can be fabricated and
operated with
techniques familiar to those skilled in the art of multi-layer soft
lithography, photolithography,
and microfluidic device fabrication and in light of the teachings herein.
[0022] In addition or in the alternative, because discrete functions can be
performed by the one
or more modules, individual modules can be coupled to one another and/or
combined to create
an integrated chip or platform that can be used for numerous biological and
chemical
applications, for example, but not limited to a cell-based assay for compound
discovery,
validation, testing, and or an in vitro diagnostic or prognostic test for
disease states such as
epithelial-born cancers, blood-born cancers, bone cancer, skin cancer, lung
cancer, prostate
cancer, breast cancer, pancreatic cancer, brain cancer, cervical cancer, colon
cancer, stomach
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
cancer, cardiac hypertrophy, cardiovascular diseases, and fibrotic diseases
such as fibrosis of the
kidney, and liver.
[0023] By disassociating and or culturing tissue and cells derived from an
organism using any
combination of the devices and substrates described herein, it can be possible
to create powerful
experimental and diagnostic tools with immediate research, pharmaceutical,
biotechnology, and
clinical development applications.
[0024] In accordance with various aspects of the present teachings, methods
for producing the
exemplary devices and systems described herein are also provided. By way of
example, in some
aspects a method of manufacturing is provided by producing a rigid substrate
within at least one
of the imaging and sorting chambers having a fixed height. In a related
aspect, the rigid
substrate can comprise a plurality of microstructures. In various aspects, the
rigid substrate can
be produced using photo-polymerization. In accord with various aspects, rigid
substrate having
regions of different stiffness can be produced within an imaging chamber, for
example, by
modulating the intensity of light during the photo-polymerization process.
Additionally or
alternatively, a rigid substrate can be produced in a two layer imaging
chamber, for example,
through the use of a dissolvable membrane.
[0025] These and other embodiments, features, and advantages will become
apparent to those
skilled in the art when taken with reference to the following more detailed
description of various
exemplary embodiments of the present disclosure in conjunction with the
accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The skilled person in the art will understand that the drawings,
described below, are for
illustration purposes only.
[0027] Figure 1 is a schematic representation of a microfluidic tissue
dissociation chamber;
[0028] Figure lA is a schematic representation of a microfluidic tissue
dissociation chamber;
[0029] Figure 2 is a schematic representation of a microfluidic tissue
dissociation chamber;
[0030] Figure 3 schematic representation of a microfluidic device designed for
the introduction
and adhesion of cells for imaging of cells via techniques such as optical-
based microscopy, and
introduction of reagents to perform various biochemical assays;
[0031] Figure 3A schematic representation of a microfluidic device designed
for the
introduction and adhesion of cells for imaging of cells via techniques such as
optical-based
microscopy, and introduction of reagents to perform various biochemical
assays;
6
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
[0032] Figure 3B schematic representation of a microfluidic device designed
for the
introduction and adhesion of cells for imaging of cells via techniques such as
optical-based
microscopy, and introduction of reagents to perform various biochemical
assays;
[0033] Figure 4 is a schematic representation of a microfluidic device
designed for the
introduction and adhesion of cells for imaging of cells via techniques such as
optical-based
microscopy, introduction of reagents to perform various biochemical assays,
and long term
culturing of cells;
[0034] Figure 4A is a schematic representation of a microfluidic device
designed for the
introduction and adhesion of cells for imaging of cells via techniques such as
optical-based
microscopy, introduction of reagents to perform various biochemical assays,
and long term
culturing of cells;
[0035] Figure 5 is a schematic representation of a microfluidic device
enabling adhesion-based
cell sorting from a heterogeneous population of cells, imaging of cells via
techniques such as
optical-based microscopy, and introduction of reagents to perform various
biochemical assays;
[0036] Figure 6 is a schematic representation of a microfluidic device
enabling adhesion-based
cell sorting from a heterogeneous population of cells;
[0037] Figure 7 is a schematic representation of an integrated microfluidic
device featuring
inlets, tissue dissociation module, cell sorting module, and perfusion array;
[0038] Figure 8 is a schematic representation of an integrated microfluidic
device featuring
inlets, tissue dissociation module, cell sorting module, and perfusion array;
[0039] Figure 9 is a schematic representation of a multilayered, integrated
microfluidic device
featuring inlets, tissue dissociation module, cell sorting module, flow
dividing module, cell
imaging module, and outlets;
[0040] Figure 9A is a schematic representation of a microfluidic tissue
dissociation module
[0041] Figure 9B is a schematic representation of a microfluidic device
enabling adhesion-based
cell sorting from a heterogeneous population of cells;
[0042] Figure 9C is a schematic representation of a microfluidic device
enabling division of a
single sample source into multiple samples of smaller volumes;
[0043] Figure 9D is a schematic representation of a microfluidic device
enabling imaging of
cells;
7
CA 02927947 2016-04-18
WO 2015/061458
PCT/US2014/061782
[0044] Figure 9E is a schematic representation of a microfluidic device
enabling collection of
waste fluid;
[0045] Figure 10 is a schematic representation of reversible connections of
one microfluidic
device to another in the z-direction;
[0046] Figure 11 is a schematic representation of a multilayered, integrated
microfluidic device
featuring inlets, tissue dissociation module, cell sorting module, flow
dividing module, cell
imaging module, and outlets;
[0047] Figure 11A is a schematic representation of an integrated microfluidic
device enabling
tissue dissociation and cell sorting; and
[0048] Figure 11B is a schematic representation of an integrated microfluidic
device enabling
sample dividing and cell imaging;
[0049] Figure 12 is an exemplary process for producing rigid substrates in
accordance with
various aspects of the applicant's present teachings; and
[0050] Figures 13 and 13A depict a side view and top view, respectively, of an
exemplary
microfluidic chip produced utilizing an exemplary method for substrate
polymerization in
accordance with various aspects of the present teachings.
[0051] Figure 14 presents an exemplary curve of adherent cell numbers versus
time, indicating
that devices of the present disclosure can process diagnostic results within 3
days of receiving
sample.
[0052] Figure 15 depicts a variety of the aspects capable of interrogation
using the devices and
according to the methodologies described herein, which presents an innovative
suite of
biomarkers to better assess tumor aggressiveness and biological behavior
leading to improved
patient risk stratification.
[0053] Figure 16 presents exemplary data indicating that oncologic potential
and metastatic
potential are used to distinguish between normal and malignant tissue and low-
and high-risk
patient samples by analyzing individual cells within a sample. (A) represents
cell distribution
for a normal tissue, and (B) represents cell distribution for a malignant
tissue.
[0054] Figure 17 presents a chart indicating the stratification of patients
into 4 zones, predicting
indolent (PxP Zone 1), local growth potential (PxP Zone 2), metastatic
potential (PxP Zone 3),
and both local growth and metastatic growth potential (PxP Zone 4).
8
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
[0055] Figure 18A depicts (A) the distribution of a number of samples on the
0P3 vs MP2
algorithm graph, and (B) is a closeup of the boundaries of the
indolent/malignant zones.
[0056] Figure 18B depicts (A) the plot of a number of samples determined to be
negative for
seminal vesicle invasion, and (B) a number of samples determined to be
positive for seminal
vesicle invasion.
[0057] Figure 18C depicts (A) the plot of a number of samples determined to
not have positive
margins during surgery, and (B) a number of samples with positive margins
during surgery.
[0058] Figure 18D depicts (A) the plot of a number of samples that do not
exhibit vascular
invasion, and (B) a number of samples which exhibit vascular invasion.
DETAILED DESCRIPTION OF THE VARIOUS EMBODIMENTS
[0059] As used herein, the term "a" or "an" means "at least one" or "one or
more."
[0060] As used herein, "Oncogenic potential" (OP) refers to a quantitative
prediction of a
tumor's growth potential.
[0061] As used herein "Metastatic potential" (MP) refers to a quantitative
prediction of whether
a tumor will invade other tissues.
[0062] Any sample suspected of containing cells relevant to the therapeutic
indication being
evaluated can be utilized in the devices and according to the methods of the
present disclosure.
By way of non-limiting example, the sample may be tissue (e.g., a prostate
biopsy sample or a
tissue sample obtained by prostatectomy), blood, urine, semen, cells (such as
circulating tumour
cells), cell secretions or a fraction thereof (e.g., plasma, serum, exosomes,
urine supernatant, or
urine cell pellet). In the case of a urine sample, such is often collected
immediately following an
attentive digital rectal examination (DRE), which causes prostate cells from
the prostate gland to
shed into the urinary tract. The patient sample may require preliminary
processing designed to
isolate or enrich the sample for the markers or cells that contain the
markers. A variety of
techniques known to those of ordinary skill in the art may be used for this
purpose. As used
herein "sample" is used generically and is intended to refer to any of a raw
patient sample, a
preliminarily processed sample, and/or a processed patent sample, including
that derived from
prostate, bladder, colon, breast, lung, kidney, or another tissue.
[0063] The following detailed description should be read with reference to the
drawings. The
drawings, which are not necessarily to scale, depict selected embodiments and
are not intended
to limit the scope of the present disclosure. The detailed description
illustrates by way of
example, and is not intended to limit the scope of the present disclosure.
9
CA 02927947 2016-04-18
WO 2015/061458
PCT/US2014/061782
[0064] The teachings herein generally provide microfluidic systems, devices,
and methods for
dissociating, culturing, assaying, inspecting, and/or otherwise manipulating
cells, and can have
application in medical testing/diagnostics, cell-based assays, and/or compound
discovery. In
various aspects, the exemplary microfluidic devices, systems, and methods can
provide clinical
and research purposed diagnostics and assay platforms that enable tissue
disassociation, cell,
protein, and particle separation, and other cell manipulation. By way of
example, the present
teachings can enable the culturing of a small number of cells in environments
that can
approximate in vivo conditions, while allowing for a determination of the
cells' growth, and/or
oncogenic potential, migration rate, and/or metastatic potential.
[0065] As will be described in detail below, exemplary cell processing systems
and methods in
accordance with the present teachings enable a variety of cell processing
procedures, cell-based
assays, and/or experiments (e.g., compound discovery) to be performed within
the various
microfluidic modules described in detail below. Though particular cell-
processing functions are
generally described with reference to individual cell-processing modules, it
will be appreciated
that the various exemplary modules and/or their functions can be combined to
form a cell-
processing system for performing multiple cell-processing functions. By way of
example, it will
be appreciated that various exemplary modules described herein can be combined
in a single
device (e.g., in a lock-and-key manner or combined in a monolithic
microfluidic chip) to enable
a specific clinical, diagnostic, and/or experimental workflow. Accordingly,
the following
description provides exemplary modules that can be incorporated into various
systems in accord
with the present disclosure.
Tissue dissociation
[0066] In one aspect, a microfluidic tissue dissociation module can be
provided. Tissue
dissociation involves in the progressive isolation of smaller and smaller
clusters of tissue and
cell clumps into a single cell suspension. The process of dissociation can be
accomplished via
anumber of methods and combinations of methods including, but not limited to,
enzymatic
treatment, mechanical agitation, stress, and shear forces.
[0067] Schematic representations of microfluidic devices for tissue
dissociation are shown in
Figure 1, 1A, and 2. In an exemplary depiction, as seen in Figure 1, tissue
fragments (e.g.,
minced tissue, sliced biopsy tissue, etc) can be inserted in inlet port 1. The
tissue fragments may
be mixed beforehand with dissociation enzymes such as trypsin, DNase, papain,
collagenase
type I, II, III, IV, hyoluronidase, elastase, protease type XIV, pronase,
dispase I, dispase II, and
neutral protease. As will be appreciated by a person skilled in the art, the
tissue fragments can
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
range in size, for example, in a range of about 0.1mm to lmm. In some aspects,
tissue samples
can be injected into the device via needles, pipettes, or integrated and
exterior fluidic handling
mechanisms, such as plastic tubes. Six dissociation chambers 2 through 7 are
utilized to reduce
the input tissue samples to progressively smaller clumps of tissue until
ultimately an
approximately single cell suspension is achieved. As will be appreciated by a
person skilled in
the art, 6 dissociation chambers are shown here, although more or less
chambers (e.g., n) are
possible The dissociated cells can then be extracted from outlet 8 via the
same, similar, or
different methods from those used to introduce the samples.
[0068] Once loaded within the device, a positive pressure from inlet 1 or a
vacuum source from
outlet 8 can be used to displace the fluid mixture within the device at
varying flow rate. As will
be appreciated by a person skilled in the art, any pressure generators known
or developed
hereafter and modified in accord with the teaching herein can be utilized to
displace the fluid
mixture within the device.
[0069] As will be appreciated by a person skilled in the art, the fluidic
pathway of the tissue
dissociation module may take on different configurations to transport the
tissue samples and
associated fluid to the various dissociation chambers depending on the usable
space available on
the microfluidic chip. By way of example, the fluidic pathway of lA takes on a
spiral shape as
opposed to the serpentine shape as depicted in Figure 1. In another exemplary
depiction, as seen
in Figure 2, the tissue fragments can enter the device via any port, including
sample inlet 9, and
pass back and forth through dissociation chamber 10 via alternating positive
pressure source
between pressure inlets 11 and 12. The dissociated cells can then be extracted
from any port,
including cell outlet 13.
[0070] With reference now to Figures 1, 1A, and 2, various microstructures can
be incorporated
into the dissociation module to facilitate mechanical perturbation. In the
depicted embodiments,
for example, a plurality of microstructures is present at the top or bottom of
the chamber to aid
in the mechanical perturbation of the tissue samples. As will be appreciated
by a person skilled
in the art, microstructures 15, 16, and 17 can have various dimensions,
configurations, and
geometries. By way of example, the microstructure 17 can range from about 1
micron to 1
millimeter in length and vary in post-to-post gap distance from 100 micron to
lmm. Their
presence, along with the varying chamber geometries, flow rates, and finally
the presence of
dissociation enzymes in the formulation, can enable the reduction of tissue
fragments to single
cells.
11
CA 02927947 2016-04-18
WO 2015/061458
PCT/US2014/061782
[0071] Upon completion of tissue dissociation into single cells or small
clumps of cells, the
dissociation module can, for example, transfer the cells and the associated
fluid and other
particles to one or more downstream modules for further processing such as
cell sorting,
culturing, adhesion, and imaging via one or more of the various embodiments
described below.
Perfusion chamber for metabolic assays, cell culturing, and imaging.
[0072] As noted above, various cell-processing modules can be utilized to
perform various
functions. In various exemplary embodiments, a perfusion chamber module can be
provided.
Specialized substrates with micro-and nano-features coated with proteins
solutions such as
fibronectin, laminin, vitronectin, and collagen can be incorporated within the
perfusion chamber
module so as to create permissive surfaces upon which mammalian cells can
preferentially
adhere and be cultured when they otherwise would be unable. Examples of these
substrates are
described, for example in PCT/U52011/055444 filed October 7, 2011, the
contents of which are
incorporated herein by reference.
[0073] Several of biomarkers noted above are based on cell-ECM interactions.
In those
interactions, the primary mechanotransducers between the cell and the ECM are
integrins. The
integrins recruit proteins to the sites of cell¨ECM contacts, forming
aggregates known as focal
adhesions. The adhesions connect the external matrix to the cytoskeletal
structure of the cell.
Both focal adhesion proteins and integrins have been shown to be involved in
the ontogeny of
many epithelial cancers, including prostate cancer or cancer of or derived
from colon, breast,
lung, kidney, or bladder tissues. Focal adhesion proteins and integrins also
participate in cell-
ECM mediated events, and ECM additionally plays a role in the development of
diseases. In
addition, preclinical data and other emerging evidence suggest that focal
adhesion¨actin
coupling regulates more than simple motility events. The coupling may, for
example, regulate
cellular growth and proliferation. In examining cell-ECM interactions overall,
frequent
biomarkers are related to or provide measures of focal adhesion, actin
dynamics, and/or cellular
force generation. Research indicates that metrics derived from these
biomarkers are able to
differentiate between healthy and cancer cells. One such metric comprises a
Traction Force
Index (TFI). This metric was measured in multiple cell lines: including
healthy, wild-type
human cell lines and human cancer cell lines. When plotted against doubling
time and
migration rate, TFI has approximately linear and parabolic correlations,
respectively. The
inventors have found that healthy cell types exhibit low values (<10), while
cancer cell lines
exhibited higher ones (>10).
12
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
[0074] With reference now to the exemplary module depicted in Figure 3, the
single perfusion
chamber can enable the introduction of mammalian cells through sample inlets
18, 19, 20, and
21, their adhesion to the specialized substrate region in the
imaging/culturing chamber 22, 23, 24,
and 25, introduction of various reagents for metabolic assays through reagent
inlets 26, 27, 28,
and 29, and subsequent inspection via techniques such as optical-based
microscopy. Waste fluid
can be collected and removed via waste outlets 30, 31, 32, and 33. As noted
above, this substrate
region can be comprised of micro- and nano-structures to enable investigation
of the cell's
characteristics (e.g. motility). Examples of fabrication methods, materials,
and dimensions are
described in PCT/US2012/066162 filed November 20, 2012, the contents of which
are
incorporated herein by reference.
[0075] On an exemplary device, cells are seeded, grown, and imaged with
limited operator
interaction. Prior to seeding cells, the device is often sterilized and coated
with an ECM
formulation. Selected biomarkers are periodically measured, for example, via
microscopy. In
certain embodiments, a single device can simultaneously analyze 1000 cells.
The device's
structure also often plays a role in biomarker measurement. The structure is
preferably
comprised of substrates, and the stiffness of a substrate is often controlled,
for example, through
the use of micropillars. One biomarker, cellular force generation, can be
measured by analyzing
the deflection of a micropillar by a cell placed on the device.
[0076] The perfusion chamber can also be adapted for hands free, long-term
cell culturing
purposes. With reference now to Figure 4, the media reservoir 36 provides
storage of cell culture
media in an environment separate from the sample and reagent inlet, and
delivers nutrients to
cells placed previously in perfusion chamber 37 via passive diffusion. Baffles
38 provide a
method to create even flow distribtuion into the perfusion chamber.
[0077] As will be appreciated by a person skilled in the art, the inlet
placements, chamber
dimensions, channel connections between the ports and perfusion chambers, and
the number of
perfusion chambers may take on various forms depending on the usable space
available on the
chip, device, or substrate. By way of examples, Figure 3A has a hexagonal-
shaped chamber as
opposed to a rectangular shaped as depicted in Figure 3; Figure 3B has a
rising channels 35 to
deposit samples and reagents from the middle of the chamber; Figure 3A and 3B
has 2 perfusion
chambers as opposed to 4 perfusion chambers; Figure 4A has a media inlet
placement at the
center of the media reservoir as opposed to the end of the media reservoir in
Figure 4; Figure 4B
has an elongated and smaller chamber to negate the baffles 38 and encorage
even flow
distribution.
13
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
Adhesion-based Cell Sorting
[0078] Arrays of the perfusion chamber described above can be interconnected
to form a cell
sorting module. With reference to Figure 5, multiple perfusion chambers
modules are linked in
series. As will be appreciated by a person skilled in the art, 3 perfusion
chamber modules are
shown here, although more or less perfusion chambers (e.g., n) are possible. A
tissue sample
(which has been homogenized beforehand or can be introduced from a tissue
dissociating
module as described with reference to Figures 1, lA and 2) consisting of a
heterogeneous group
of cells is introduced through cell inlet 39 and 40, and travels into the
first perfusion/sorting
chamber, which was initialized beforehand with a substrate with micro-and nano-
features coated
with proteins solutions specialized for attachment of one particular subset of
cells in the
heterogeneous mixture of cells in the sample. Examples of these substrates are
described, for
example in PCT/US2011/055444 filed October 7, 2011, the contents of which are
incorporated
herein by reference, and examples of fabrication methods, materials, and
dimensions of the
micro- and nano-features are described in PCT/US2012/066162 filed November 20,
2012, the
contents of which are incorporated herein by reference. The cells that are not
captured by the
substrate in the first perfusion chamber can exit the chamber and enter the
bridge connector 41,
which can transfer the remaining cell population into further chambers, each
containing
substrates specific to a unique subset of cells in the sample. The remaining
waste fluid exits
through the outlet port 42 and 43. Once the desired cells are captured in each
perfusion chamber,
the cell sorting module can be separated by removing the bridge connectors,
and each perfusion
chamber can function independently for cell culturing, imaging, and metabolic
assays.
[0079] With reference now to Figure 6, another embodiment of the cell sorting
module is
depicted on one microfluidic chip. Fluid containing substrates specific to
different cells are
introduced to the chip via inlets 44, 45, and 46, which enter the sorting
chambers 47, 48 and 49
independently and coat the bottom of the chamber. Excess fluid from the
substrate-coating
process exit via outlet 50, 51, and 52. As will be appreciated by a person
skilled in the art, 3
inlets, sorting chambers, and outlets are shown here, although more or less
inlets, sorting
chambers, and outlets (e.g., n) are possible. After the substrate coating step
is performed, the
tissue sample consisting of a heterogeneous population of cells is entered
from cell inlet 53.
Similar to the method described above, the first sorting chamber 46 captures a
subset of cells to
which the substrate is specific, and the remaining cell population flow into
further chambers.
The waste fluid exits via outlet 54. The attached cells can be release by
introducing release
agents via inlets 44, 45, and 46 and extracting from outlets 50, 51, and 52,
which can be
14
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
connected to an imaging stage off-chip or transferred to the perfusion chamber
module as
described above.
[0080] The microfluidic chip depicted in Figure 6 can further be purposed as
the perfusion
module by flowing media through inlet 53. To run assays, inlets 44, 45, and 46
can be
repurposed as reagent inlets, and imaging can be performed over the large
sorting chambers.
Rigid Substrate Integration
[0081] Bottom surfaces of imaging and cell sorting chambers can express
different rigidities or
stiffness in order to promote cell attachment and survival. These rigidities
are achieved through
specifically engineered biocompatible polymer gels including but not limited
to polyacrylamide,
polyethylene glycol, and polydimethylsiloxane. The rigidity of these polymer
gels can be
controlled by a number of factors such as: ratio of pre-polymer reagents,
quantity of the initiator,
time of polymerization, and intensity of light (e.g., intensity of light can
be relevant for photo-
polymerization methods). The manufacturing of these gels can be done in many
mechanisms
including but not limited to photo-polymerization and chemical polymerization.
[0082] With respect to Figure 12, an exemplary embodiment of photo-
polymerization method of
creating rigid substrates involves the creation of the gel substrates prior to
bonding of the device
is shown. In this exemplary method, the bottom layer of the microfluidic
device (glass or plastic)
can be silanized. Next, a solution containing the pre-polymer reagents and
photoinitiator can be
spin-coated onto the bottom layer. The spin-coating can achieve a uniform
thickness of the
solution. A photomask and light source can then be used to selectively
introduce light (at the
wavelength necessary for the photoinitiator) to regions of the bottom layer
that correspond to the
locations of the imaging or sorting chamber, which can result in
polymerization of the gels only
in the locations of the imaging or sorting chambers. The unpolymerized
solution will be washed
away during the development step leaving the bottom layer with rigid
substrates in the location
of the imaging chamber. The different layers of the device can then be bonded
together.
[0083] In addition to the photo-polymerization method described above, a
method may be used
that will result in varying intensities of light being introduced to the pre-
polymer solution, which
can result in a gel that can have regions of different stiffness. An exemplary
embodiment of this
method can utilize a photomask having varying levels of transparency in the
region of the
imaging chamber in place of the photomask described above. The utilization of
this method can
allow for the gel substrate within the imaging chamber to have regions of
varying stiffness for
the cells to interact with.
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
[0084] In some aspects, a polymerization method for creating rigid substrates
can be achieved
after a chip has already been manufactured. For example, with reference now to
Figures 13 and
13A, which depict a top and side view of an exemplary microfluidic chip, the
imaging chamber
can be separated into two separate layers by a dissolvable membrane. Due to
the nature of the
device inlets, any injected liquids in the reagent inlet can travel to the
bottom layer of the
imaging chamber. To create the rigid substrate at a fixed height, a solution
of pre-polymer
reagents and initiators can be injected into the reagent inlet 88 until the
lower imaging chamber
89 is filled. The solution can then be allowed to polymerize for a determined
period of time
dependent on the reagents used. Once the polymerization is completed, a
solution can be
injected into the upper imaging chamber 91 that will dissolve the dissolvable
membrane 92
resulting in a device that is ready for use. Excess fluid can exit the chamber
via waste outlet 93.
An example of the solution of pre-polymer reagents and initiators is a
solution containing
acrylamide, bis-acrylamide, TEMED, and ammonium persulfate in order to create
a poly-
acrylamide gel.
Device Integration
[0085] As noted above, though particular cell-processing functions are
generally described with
reference to individual cell-processing modules, it will be appreciated that
the various exemplary
modules and/or their functions can be integrated and/or combined to form a
cell-processing
system for performing multiple cell-processing functions. As will be
appreciated by a person
skilled in the art, all of the microfluidic device embodiments described above
can be integrated
in a modular fashion depending upon the desired applications of the device. By
way of example,
it will be appreciated that various exemplary independent modules described
herein can be
coupled to one another (e.g., in a lock-and-key manner) such that the
microfluidic channels of
each module can be coupled to one another. Alternatively, as will be discussed
in detail below,
various microfluidic cell-processing modules can be formed in a single
monolithic structure (e.g.,
a microfluidic chip) to enable a specific clinical, diagnostic, and/or
experimental workflow.
Accordingly, the following description provides exemplary modules that can be
incorporated
into various systems in accord with the present disclosure. As will be
appreciated by a person
skilled in the art in light of the teachings herein, the various exemplary
modules can be utilized
and integrated in various combinations depending upon the desired
applications.
[0086] For example, in one exemplary embodiment, the described modules can be
fully
integrated using into a microfluidic system "chip" that can be used with
existing microscopy
platforms. In this particular embodiment, various modules are integrated onto
a 96 well plate
format. As schematically depicted in Figure 7, device operation can proceed by
first introducing
16
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
two samples into inlet 56 and 57. The samples then travel through zone 58 for
tissue dissociation,
then travel to zone 59 for adhesion-based cell sorting, cell
culturing/imaging, and metabolic
assays. Due to the orientation and fluidic pathway of the integrated module,
the port 60
originally described as an inlet in Figure 5 is now repurposed as a waste
outlet.
[0087] With reference now to Figure 8, another embodiment of the integrated
device is depicted
on one microfluidic chip in an optical disk format. Device operation can
proceed by first
introducing two samples into inlet 61 and 62. The samples travels through zone
63 and 64 for
tissue dissociation, then enter zone 65 and 66 for adhesion-based cell
sorting, cell
culturing/imaging, and metabolic assays. Two cell sorting modules are used to
provide duplicate
tests for one sample, and two integrated modules allow for comparison between
experimental
groups.
[0088] The integrated device can also exploit space in the z-direction to
create a multilayer
microfluidic device. In one exemplary embodiment, Figure 9 illustrates a
perspective view of the
multilayer device with five different layers on a 96 well plate format. Figure
9A-E depicts an
exemplary tissue dissociation layer, cell sorting layer, flow dividing layer,
cell imaging layer,
and outlet layer, respectively. Device operation can proceed by first
introducing samples into
inlets 67. The samples enter zone 68 for tissue dissociation, then exit the
tissue dissociation layer
via outlets 69 and enter the cell sorting layer via cell inlets 70. As will be
appreciated by a
person skilled in the art, three dissociation chambers and three independent
fluidic pathways are
shown here, although more or less chambers and pathways can be used in
accordance with the
present teachings. The cells travel through sorting chambers 71 for cell
sorting and brief
attachment to the substrate. Excess fluid can exit the cell sorting layer via
waste outlet 72. After
sorting and some culturing, the cells can be detached from the substrate
through introduction of
cell detachment reagents such as trypsin through reagent inlets 73. After cell
detachment, the
detachment reagent can be removed from the sorting chamber via reagent outlets
74. Valves 75
can prevent cells from exiting the reagent outlets during the removal of
detachment reagents.
The cell then leaves the cell sorting layer via cell outlets 76. Three sorting
chambers and
associated inlets/outlets are shown in each fluidic pathway, although more or
less chambers are
possible. The cell outlet is connected to the flow dividing layer via cell
inlet 76. The cell
suspension can be distributed into flow dividing channels 78 to reduce the
sample volume upon
imaging. Each flow path can exit the flow channel via cell outlet 79, and into
the imaging layer
via cell inlet 80. Four dividing channels are shown in a diverging tree
configuration, although
more or less divisions in fluidic pathways and other channel configurations
are possible. The
cells can be fed into the cell imaging chamber 81, and can then be cultured
and imaged. Each of
17
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
the four replicate imaging chambers can be functionalized with the same or
different substrates
and micro features in order to measure different biomarkers on the same sample
of cells. In one
embodiment, two of the replicate imaging chambers may be functionalized with
substrates of
different rigidity, a third replicate imaging chamber may be functionalized
with a micro-pillar
array, and a fourth may be functionalized with a different ECM formulation.
Excess fluid can
exit the cell imaging layer and into the outlet layer via fluidic connection
between waste outlets
82 in the cell imaging layer and waste inlets 83 in the outlet layer. The
waste fluid can be
collected in a common channel and removed from the chip via outlet 84.
[0089] The various microfluidic layers can be fluidically coupled via
reversible or irreversible
connections between inlets and outlets. By way of example, press fit ports 85
and 86 are
depicted in Figure 10, allowing for addition and removal of a fluidic layer
during operation of
the device.
[0090] As will be appreciated by a person skilled in the art, the functional
features found on the
multilayer device can take on various configurations and number of features
depending on
operational need and available space on the chip. By way of example, Figure 11
depicts an
integrated multilayer microfluidic device with two functional layers as
opposed to five layers as
depicted in Figure 9, the top layer being a combination of tissue dissociation
and cell sorting
functionalities, and the bottom layer being a combination of the flow dividing
layer, cell
imaging layer, and outlet layer. With reference to Figure 11A, two independent
fluidic pathways
are shown as opposed to three in figure 9A. With reference to Figure 11B, the
flow divider 87
takes on a radial configuration as opposed to the diverging tree configuration
as depicted in the
flow dividing channels 77 in Figure 9C.
[0091] In various embodiments discussed above, given the inputs of mammalian
tissue, the
device, in an automated, systematic fashion, can dissociate, segregate, sort,
enrich, manipulate,
and assay cells for biomarker quantification. These quantified biomarkers,
which can be based
on physical properties of the cells or biochemical / metabolic properties of
the cells or associated
extracellular components, can then be used as inputs into algorithms to output
quantifiable
metrics regarding the aggressiveness, or oncogenic potential, of a cancer, or
the invasion,
motility, or metastatic potential of a cancer. Examples of these algorithms
can be found, for
example in PCT/US2011/055444 filed October 7, 2011, the contents of which are
incorporated
herein by reference.
[0092] The present inventors have developed innovative microfluidic devices.
Based on the
quantification of biomarkers in such devices, metrics of Oncogenic Potential
and Metastatic
18
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
Potential were developed to aid physicians in treatment decisions and
supplement the qualitative
Gleason score with a sensitive, specific, and quantitative metrics. The
devices and methods
described and contemplated herein represent, for example, a personalized
diagnostic solution
capable of predicting aggressiveness to better guide therapy selection.
Moreover, the inventors
have cultured prostate cells from clinically relevant patient samples in
vitro.
[0093] The presently described devices, methods and clinical measures can, in
certain
embodiments, be utilized along with the traditional Gleason Scores in
evaluating patients, which
adds critical information to the evaluation of patients having Gleason scores
of, for example, 6-8.
OP and MP allow the presently described technology to mitigate the current
state of over-
treatment in prostate cancer, inform the choice between local and systemic
therapy, and identify
aggressive tumors earlier during watchful waiting or active surveillance
periods.
[0094] On one exemplary protocol, biopsied cells are introduced (e.g.,
injected) into
microfluidic devices of the present disclosure. The cells are then analyzed on
the chip using, for
example, automated light/fluorescent microscopy, and images are uploaded to,
or accessed in a
database by, a program that utilizes machine vision image analysis to
calculate and return OP
and MP values. In such an exemplary protocol, the following steps are
characterized the use of
one or more technologies selected from the group consisting of ECM
formulation, a microfluidic
device, a biomarker suite, machine vision software, and prognostic algorithms.
Frequently, raw
images are generated that require processing. After processing and then
analysis, the resulting
data is often synthesized into distinct, meaningful outputs that can be
delivered to physicians.
Though prostate samples are often utilized, the presently described
technologies and methods
are readily applied to bladder, kidney, breast, colon, and lung tissues and
cells.
[0095] In one exemplary protocol, a patient sample is processed as noted above
and OP and MP
values or information are provided to a physician within about five days in
addition to
confidence intervals to gauge the sensitivity for each patient's results
(e.g., from a biopsy).
Thereafter, the physician can provide more informed treatment options to
patients with increased
confidence (e.g., radical prostatectomy or active surveillance).
[0096] In certain embodiments, the present devices and methods provide the
ability to
differentiate between low-risk (low-grade) and high-risk (high-grade) prostate
cancer as
correlated with the reference standard of the Gleason Score. The present
devices and methods
also often provide a stratification of low-risk, intermediate-risk, and high-
risk patients as
correlated with the reference to Gleason Score standards. In addition, the
present devices and
methods provide the ability to differentiate between different types of
intermediate risk patients
19
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
(Gleason 6 or 7) ¨ risk stratifying within the intermediate patient prostate
cancer population,
segregating patients as having indolent, locally aggressive, or metastatically
aggressive types of
cancer. Also, the present devices and methods provide the ability to act as a
therapy guide,
differentiating patients who should be treated via active surveillance,
surgery or radiation, and /
or adjuvant therapy. In certain embodiments, the present devices and methods
also provide the
ability to facilitate compound validation and therapeutic pipeline
acceleration to bridge
conversations with strategics towards exploratory and co-development deals. In
frequent
embodiments, the present devices and methods also provide the ability to
distinguish between
normal and cancer samples, predict aggressive potential of disease, stratify
patients by risk
category, wthin patients that are intermediate risk (clinically ambiguous),
identify patients with
local growth potential and/or metastatic potential, control for biopsy sample
heterogeneity,
provide high signal to noise biomarker analysis, and return clinically
actionable metrics
[0097] The microfluidic chips and related methods of the present disclosure
have been
successfully applied to diagnostic processes in the clinic. By way of example,
figures 14-18D
provide results at various stages of the diagnostic process obtained from
samples run through an
exemplary microfluidic device. Figure 14 demonstrates the microfluidic
device's ability to
prepare the sample for analysis before any in vitro transformation occurs.
Figure 15 provide
select images of various biomarkers obtained from the diagnostic process
operated within an
exemplary device. With reference to Figure 16, the oncogenic potential (OP)
and metastatic
potential (MP) metrics derived from biomarkers obtained from within an
exemplary device can
distinguish the difference in cancerous/non-cancerous cells distribution
between normal and
malignant tissue. Figure 17 to Figure 18B depict the ability of representative
sample diagnostic
results according to the present disclosure to be translated to relevant
patient clinical information
with various iterations of OP and MP. Figure 17, for example, demonstrates the
ability of
devices and methods according to the present disclosure to stratify patients
into 4 zones that
predict indolent (PxP Zone 1), local growth potential (PxP Zone 2), metastatic
potential (PxP
Zone 3), and both local growth and metastatic growth potential (PxP Zone 4).
Figure 18A is an
example of the 0P3-prime and MP2-prime algorithms distinguishing Gleason 6
samples from
Gleason 7s, 8s, and 9s with 79.5% sensitivity and 85% specificity (n=56).
Figure 18B
demonstrate the 0P4 and MP11 algorithms's ability to predict samples that will
invade seminal
vesicles. Figure 18C is an example of the 0P3 and MP10 algorithms predicting
samples that
will exhibit positive margins during surgery. Finally, Figure 18D demonstrates
the ability of the
0P8 and MP4 algorithms to predict samples that will exhibit vascular invasion,
resulting in
metastasis into the bloodstream. Further examples of OP and MP metrics can be
found, for
CA 02927947 2016-04-18
WO 2015/061458
PCT/US2014/061782
example in PCT/US2011/055444, filed October 7, 2011, the contents of which are
incorporated
herein by reference.In frequent embodiments, a microfluidic device is provided
for processing
tissue, comprising: a cell inlet port for receiving a tissue fragment, a cell
dissociation chamber
comprising a plurality of microstructures, an outlet port for extracting a
cell suspension, a
channel fluidly coupled to the inlet port, the chamber, and the outlet port to
allow sequential
flow through the device, and a pump coupled to the inlet port and/or outlet
port to cause
displacement of a fluid through the channel and chamber.
[0098] In other frequent embodiments, a microfluidic device is provided for
processing tissue,
comprising: a cell inlet port for receiving a tissue fragment, a cell
dissociation chamber
comprising a plurality of microstructures, a plurality of pressure inlet ports
for circulating a fluid
back and forth within the cell dissociation chamber; an outlet port for
extracting a cell
suspension, a channel fluidly coupled to the inlet port, the chamber, and the
outlet port to allow
controlled flow of the fluid through the device, and a pump coupled to the
cell inlet port,
pressure inlet ports, chamber, and/or outlet ports to circulate the fluid back
and forth through the
dissociation chamber.
[0099] In certain embodiments the microstructures comprise posts and/or are
diamond or
rectangular in shape. Often the device comprises two or more cell dissociation
chambers,
wherein each of the cell dissociation chambers comprises a plurality of
microstructures having a
differing gap distances. Often, each of the plurality of the microstructures
is separated from the
other microstructures by a distance defined as a gap distance, and wherein the
chamber
comprises multiple gap widths. Frequently, the gap distance is between 1
micron and 1
millimeter in distance.
[00100] In
certain embodiments a microfluidic device is provided for processing tissue,
comprising: a cell inlet port for receiving a tissue fragment and/or a cell
suspension, a perfusion
chamber for culturing, imaging, and/or assaying a cell, a reagent inlet for
receiving assay
reagents, the reagent inlet being in fluid communication with the perfusion
chamber, an outlet
for extracting excess fluid, a channel fluidly coupled to the cell inlet,
perfusion chamber, reagent
inlet, and outlet port to allow controlled flow through the device, and a pump
coupled to the cell
inlet and/or the reagent inlet to cause displacement of fluid through the
channel, chamber, and
the outlet. Often the perfusion chamber comprises a cell adhesion surface.
Also often, the
device further comprises a perfusion layer comprising a channel disposed
therein and positioned
relative to the cell adhesion surface to allow diffusion of a gas and/ora
nutrient to a cell adhered
to the cell adhesion surface. The cell adhesion surface is optionally
functionalized with a
reagent suitable to facilitate a preferential adhesion of a cell to the
surface. Often the reagent
21
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
comprises one or more of fibronectin, collagen, laminin, or vitronectin. The
device itself may be
comprised of a thermoplastic, a thermoset, or an elastomer. Often a device
composition material
comprises an epoxy, a phenolic, polydimethylsiloxane (PDMS), glass, silicone,
nylon,
polyethylene, and/or polysterene.
[00101] In frequent embodiments, the perfusion chamber comprises an
optically
transparent portion, wherein the optically transmissive portion is positioned
relative to the cell
adhesion surface to permit optical interrogation of a cell adhered to the cell
adhesion surface.
Frequently, a plurality of perfusion chambers are provided that are fluidly
coupled. Often a
surface of the optically transparent portion is functionalized with a reagent
suitable to prevent
adhesion of a cell to the surface. The cell adhesion surface is optionally
planar or substantially
planar. Frequently, the cell adhesion surface comprises a microstructure.
[00102] In certain embodiments the device further comprises a media
reservoir in fluid
communication with the perfusion chamber to passively diffuse a nutrient
and/or reagent (or
multiple nutrients or reagents) into the perfusion chamber.
[00103] In certain embodiments a plurality of perfusion chambers are
fluidly coupled by
bridge connectors. Often, the bridge connectors are removable from the device.
[00104] In certain embodiments, each of the plurality of perfusion chambers
comprises a
cell adhesion substrate, wherein each substrate is configured to selectively
capture a designated
subset of cells within a heterogeneous cell population in the sample. Often,
the cell adhesion
substrate comprises a microstructure and/or a protein formulation configured
to preferentially
capture a designated subset of cells.
[00105] In certain embodiments, a microfluidic device is provided for
processing tissue
and/or cells, comprising: a reagent inlet for introducing a reagent input, a
sorting chamber for
selectively capturing a designated subset of a heterogeneous cell population
culturing, imaging,
and/or assaying one or more cells, a reagent outlet for extracting excess
fluid introduced into the
reagent inlet, a cell inlet for introducing a cell suspension, a cell outlet
for extracting excess fluid
from the cell inlet, a channel in fluid communication with thethe reagent
inlet, cell inlet, sorting
chamber, reagent outlet, and cell outlet for controlling and/or confining
fluid flow therethrough,
and a pump coupled to the reagent inlet and cell inlet to cause displacement
of a fluid through
the channel, chamber, reagent outlet, and cell outlet. Often, a substrate, a
release reagent, and/or
an assay reagent are introduced to or comprised in the reagent input.
[00106] In certain embodiments, the sorting chamber is configured to
culture, image,
and/or assay one or more cells.
22
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
[00107] In one embodiment, a microfluidic device is provided for processing
tissue and/or
cells, comprising: a tissue dissociation module, a cell sorting module, a
channel fluidly coupled
to the tissue dissociation module and the cell sorting module for allowing
sequential flow
therethrough, and a pump for effecting displacement of a fluid through the
tissue dissociation
module and cell sorting module. Often, the tissue dissociation module
comprises: a cell inlet
port for receiving a tissue fragment, a cell dissociation chamber comprising a
plurality of
microstructures, and an outlet port for extracting a cell suspension, wherein
the channel is
fluidly coupled to the outlet port. Also often, the pump is configured to
provide an
unidirectional flow of the fluid through the cell dissociation chamber. In
certain embodiments,
the pump is configured to circulate the fluid back and forth through the cell
dissociation
chamber.
[00108] In certain embodiments, the cell sorting module comprises: a cell
inlet port for
receiving a cell suspension from the tissue dissociation module, a perfusion
chamber for
culturing, imaging, and/or assaying a cell, a reagent inlet for receiving an
assay reagent, the
reagent inlet being in fluid communication with the perfusion chamber, an
outlet for extracting
excess fluid from the perfusion chamber.
[00109] In certain embodiments, the perfusion chamber comprises a cell
adhesion surface.
Often the cell adhesion surface is functionalized with a reagent suitable to
facilitate preferential
adhesion of the cell to the surface.
[00110] In certain embodiments, the tissue dissociation module comprises a
plurality of
perfusion chambers fluidly coupled to one another. Often, each of the
plurality of perfusion
chambers comprises a cell adhesion substrate, wherein each substrate is
configured to selectively
capture a designated subset of cells within a heterogeneous cell population in
the sample.
[00111] In certain embodiments, a microfluidic device is provided for
processing tissue
and/or cells, comprising: an inlet for receiving an input, one or more, or two
or more, layers
selected from the group consisting of: a tissue dissociation layer, a cell
sorting layer, a flow
dividing layer, an imaging layer, and an outlet layer, a plurality of
microfluidic channels
connecting the two or more layers for allowing fluid flow between the layers,
and an outlet for
extracting an output. Frequently, the layers are vertically arranged or
positioned relative to one-
another. Often in such arrangements, one layer partially or completely
overlaps another layer of
the same device. The microfluidic channels often act as fluid conduits
connecting multiple
layers on the same or different vertical levels. Frequently, the plurality of
microfluidic channels
connecting the two or more layers allows for reversible fluid communication
therebetween.
Often, the plurality of microfluidic channels connecting the two or more
layers allows for one-
23
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
way fluid flow therebetween. Also often, the device further comprises a pump
for causing
displacement of a fluid through the two or more layers. The pump can
optionally be coupled to
a sample inlet of the tissue dissociation layer to cause displacement of a
fluid through the two or
more layers.
[00112] Often, the device comprises multiple vertically arranged layers,
including the
tissue dissociation layer, the cell sorting layer, the flow dividing layer,
the imaging layer, and the
outlet layer. Also often, the device comprises two or more of the tissue
dissociation layer, the
cell sorting layer, the flow dividing layer, the imaging layer, or the outlet
layer. In frequent
embodiments, the device comprises a plurality of vertically arranged layers,
each vertically
arranged layer comprising two or more of the tissue dissociation layer, the
cell sorting layer, the
flow dividing layer, the imaging layer, and/or the outlet layer.
[00113] Often, the tissue dissociation layer comprises: a cell inlet port
for receiving a
tissue fragment, a cell dissociation chamber comprising a plurality of
microstructures, and an
outlet port for extracting a cell suspension, wherein at least one of the
microfluidic channels is
fluidly coupled to the outlet port. Often ,the cell sorting layer comprises: a
cell inlet port for
receiving a cell suspension from, if present, the tissue dissociation layer,
another layer, or the
inlet, a perfusion chamber for sorting a cell, a reagent inlet for receiving
an assay reagent, the
inlet being in fluid communication with the perfusion chamber, an outlet for
extracting excess
fluid from the perfusion chamber. Also often, the cell inlet port of the cell
sorting layer is
fluidly coupled to an outlet port of the tissue dissociation layer.
[00114] In certain embodiments, the device further comprises a valve
configured to
control the flow of a fluid through the cell sorting layer and optionally a
cell outlet for extracting
sorted cells.
[00115] Often, the flow dividing layer comprises: a cell inlet port for
receiving a
suspension of sorted cells from, if present, the cell sorting layer, another
layer, or the inlet, a
flow divider for reducing a sample volume, a cell outlet for extracting cells,
and a channel for
fluidic coupling the cell inlet, flow divider, and cell outlet for controlling
fluid flow therethrough.
[00116] Also often, the imaging layer comprises: a cell inlet port for
receiving a
suspension of sorted cells, an imaging chamber for imaging cells disposed
therein, a waste outlet
for extracting a waste fluid, and a channel for fluidic coupling the cell
inlet, imaging chamber,
and waste outlet for controlling fluid flow therethrough. In certain
embodiments, the device
further comprises a reagent inlet for introducing a reagent to a cell within
the imaging chamber.
[00117] Often, the outlet layer comprises: a waste inlet for receiving a
waste fluid
generated in the tissue dissociation layer, the cell sorting layer, the flow
dividing layer, and/or
24
CA 02927947 2016-04-18
WO 2015/061458 PCT/US2014/061782
the imaging layer, a waste outlet for removing the waste fluid from the
microfluidic device, and
a channel in fluid communication with the waste inlet and the waste outlet for
controlling or
containing fluid flow therethrough. Often the device further comprises a waste
reservoir for
storing the waste fluid disposed between the waste inlet and the waste outlet.
[00118] In certain embodiments, a method of manufacturing a device
described herein is
provided, comprising: producing a rigid substrate within the device or portion
thereof having a
fixed height. Often, the rigid substrate comprises a plurality of
microstructures. Frequently, the
rigid substrate is produced within the device or portion thereof, including
surfaces of modules,
channels, or layers thereof, using photo-polymerization. Often, the rigid
substrate is produced
having regions of different stiffness within the device or portion thereof by
modulating an
intensity of light during a photo-polymerization process. In certain
embodiments, the device
comprises an imaging chamber or layer, and the method comprised producing a
rigid substrate
in the imaging chamber or layer through the use of a dissolvable membrane. The
imaging
chamber or layer often comprises a two-layer imaging chamber or layer. In
certain
embodiments, such devices are configured in an optical disc format.
[00119] In frequent embodiments, methods are provided for evaluating a
cell, tissue, or
patient, comprising introducing a cell to a microfluidic device functionalized
with an
extracellular matrix formulation, imaging the cell, and stratifying the cell
based on oncologic
potential and metastatic potential. Machine vision is often utilized to image
the cell. Also, often
the cell is exposed to a biomarker or suite of biomarkers in the device. In
certain embodiments
the evaluation comprises determining the potential of the cell to invade a
seminal vesicle,
determining the potential of the cell to invade the vasculature of a patient,
or determining the
likelihood that a tumor from which the cell was derived will exhibit positive
margins during
surgery. In certain embodiments, a method is provided for evaluating a cell,
tissue, or patient,
comprising introducing a cell to a microfluidic device described hereinabove
functionalized with
an extracellular matrix formulation, imaging the cell, stratifying the cell
based on oncologic
potential and metastatic potential, and/or stratifying the cell based on or in
reference to a
Gleason score. In the methods of the present invention, assaying cell types
such as prostate,
colon, lung, bladder, kidney, and/or breast cells or cellular extracts or
components thereof is
contemplated.
[00120] One skilled in the art will appreciate further features and
advantages of the
presently disclosed methods, systems and devices based on the above-described
embodiments.
Accordingly, the presently disclosed methods, systems and devices are not to
be limited by what
has been particularly shown and described, except as indicated by the appended
claims. All
CA 02927947 2016-04-18
WO 2015/061458
PCT/US2014/061782
publications and references cited herein are expressly incorporated herein by
reference in their
entirety.
26
