Note: Descriptions are shown in the official language in which they were submitted.
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PERFUSION BIOREACTOR FOR CULTURING CELLS
FIELD OF THE INVENTION
The present invention relates generally to the field of bioreactors, and, more
particularly, to a
system and method for culturing cells under perfusion flow, in a single
chamber or in a high
throughput format.
BACKGROUND OF THE INVENTION
Recent developments in cell/tissue engineering have recognized benefits to
growing and
studying cells in dynamic environments. Spinner flasks, rotary devices,
perfusion
bioreactors, or fluid sheer chambers have all been used to enhance nutrient
and metabolite
diffusion to and from cells. The mechanical aspects of fluid sheer forces have
also been
shown to trigger second messenger signals and alter cellular gene expression.
While these
new culture conditions have been recognized to affect cell functions (growth,
signaling,
morphology, differentiation, etc.), devices for studying these environments
have not been
translated to high throughput platforms. Furthermore, systems that incorporate
three-
dimensional scaffolds with highly aligned pores for long-range control over
fluid flow paths
have also not been established.
Fluid flow was first established as a regulator of cellular gene expression in
two-dimensional
culture systems with flowing culture medium over cells adherent to glass
slides. Cells
respond to the fluid sheer by aligning in the direction of the force, and
altering their gene
expression. These two-dimensional devices are now commercially available from,
for
example, Flex-Cell International, as well as other vendors. Fluid flow studies
have recently
been translated to three-dimensional scaffolds, and it has been established
that fluid sheer is
another important factor in maintaining hepatocyte and bone differentiation.
The true
importance of fluid flow as an environmental signaling factor, however, has
not been fully
appreciated because it is difficult to screen against or study in conjunction
with a plethora of
other environmental cues that are known to alter cell function including but
not limited to
signaling factors such as growth factors, ECMs, cytokines, media factors, and
small
molecules to name a few. For example, to date, all of the devices designed to
study how
these forces affect cell cultures are one-pot or single chamber devices. These
devices may
be utilized to study how rotation or fluid sheer forces affect cells under one
condition at a
time, but not under different or varying conditions, which greatly limits the
utility of these
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devices. Accordingly, current devices are not suitable for performing medium
or high
throughput experimentation for optimization of conditions for controllable
cell phenotype, or
for testing substances such as molecules of unknown function for altering
specific functions
in highly relevant cell or engineered tissue cultures.
Furthermore, in the field of drug discovery, the use of primary human cells to
study
ADMETox (ADMETox is an acronym for set of analyses that measure the
absorption,
distribution, metabolism, elimination and toxicity of a drug candidate)
properties of drugs is
highly desirable. This is due to the fact that whole animal studies are
expensive, and results
are not always predictive of responses in man. In vitro study of primary human
cells is
attractive due to the economics of the approach, and the fact that data from
liuman cells
should be more relevant than animal data. Unfortunately, the culture of
primary human cells
is extremely difficult for most cell types, and there are few model systems
that are capable of
creating relevant models of in vivo tissues and organs. As an intermediate
between whole
animals and primary cells, tissue or organ slices offer an alternative that
keeps cells in their
native setting (not dissociating-them from their microenvironment), while
allowing for in
vitro testing of xenobiotic effects on cell viability, metabolism, and other
ADMET-type
aspects that one desires. For exainple, liver slices are often utilized for
measuring liver-
specific drug toxicity, as well as CYP induction.
In vitro culture of tissue slices also has several challenges. For example,
one significant
challenge is the high metabolic rates and nutrient requirements that tissue
slices need in
vitro. Since the tissue slices require a large nutrient load, it is necessary
to culture these
slices in large quantities of medium. However, the more medium that one adds
to a culture
increases the diffusion distance of oxygen to the extent that the rate of
consumption by the
tissue is greater than the diffusion of oxygen, leading to hypoxic conditions
and cell death.
There is, therefore, a great need for bioreactor-type devices that enhance
nutrient and
metabolite transport while maintaining a medium-to-high throughput parallel
testing format.
A need exists for a system and method for culturing cells under fluid
perfusion in medium to
high throughput format, to test and/or discover how new environments alter the
ability of
cells to respond to other chemical or physical cues in the presence of fluid
sheer and to
facilitate the systematic and high througlhput discovery of dynamic cell
culture conditions for
cell growth and differentiation, and then utilizing these optimized
environments for creating
in vitro engineered tissues for therapeutic, diagnostic, or research purposes.
A need also
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exists for a perfusion system that allows for the dynamic and multiplexed
culture of a variety
of tissue or organ slices for ADMET and tissue culture applications.
SUMMARY OF THE INVENTION
The present invention is directed to a bioreactor system including a perfusion
unit, a
pumping unit in fluid communication with the perfusion unit, and a fluid
source unit in fluid
communication with the pumping unit. The perfusion unit includes an array of
cell wells
configured to contain cell cultures and the fluid source unit includes an
array of media wells
configured to contain cell culture media. The pumping unit includes an array
of pumping
elements in fluid communication with the cell wells and media wells and is
configured to
pump cell culture media from the media cells to the cell wells.
In a preferred embodiment, each of said cell wells is adapted and configured
to contain a
scaffold having a porous structure. In one embodiment, the scaffold is a two-
dimensional
scaffold. In another embodiment, the scaffold is a three-dimensional scaffold.
In one
embodiment, the three-dimensional scaffold may include directionally aligned
pores.
In one embodiment, the fluid is deliverable directly into the internal
structure of said
scaffold. In another embodiment, a return pathway is provided for the fluid to
flow from the
array of cell wells to the array of media wells. In a preferred embodiment,
each pathway is
in fluid communication with a single cell well and a single media well.
In another embodiment, the perfusion unit is removably couplable to the
pumping unit and
each pumping element may comprise a fluid stem having a fluid port therein.
Each stem
may be configured to extend into the cell wells. In another aspect of the
invention, each cell
well may include a scaffold coupled thereto configured to receive a portion of
the stem
internal thereto. The fluid source unit is also removably couplable to the
pumping unit.
The present invention is also directed to a method of growing cells,
comprising pumping cell
culture media from a first array of wells of a fluid source unit into a second
array of wells of
a perfusion unit, wherein each well of the perfusion unit is adapted and
configured to house a
cell adherent structure. In one embodiment the method further comprises the
step of
perfusing the media into and through a scaffold. In one embodiment, the cell
adherent
structure comprises a two-dimensional scaffold, and in another embodiment the
cell adherent
structure comprises a three-dimensional scaffold. The first array of wells is
in fluid
communication with the second array of wells for the return of media to the
second array of
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wells. In another method, each well of the first array of wells is in singular
fluid
communication with a corresponding well of the second array of wells.
The present invention is also directed to a perfusion bioreactor system,
including an array of
bioreactor units. Each bioreactor unit includes a cell adherent structure in
fluid
communication with a fluid stored in a fluid reservoir and, in operation,
fluid flows from the
fluid source directly into and through the cell adherent structure. In one
variation, the cell
adherent structure is a three-dimensional scaffold having a porous structure,
and in another
variation the cell adherent structure is a two-dimensional scaffold. In
another preferred
embodiment, the cell adherent structure is fluidly interconnected to the fluid
reservoir by a
pumping unit.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. 1 is an exploded view of a first embodiment of a bioreactor system
according to the
present invention;
FIG. 2 is a perspective view of one embodiment of a perfusion unit of the
bioreactor system
shown in FIG. 1;
FIG. 3 is a perspective view of one embodiment of a purriping unit of the
bioreactor system
shown in FIG. 1;
FIG. 4 is a cross-sectional exploded view of a bioreactor unit of the system
of FIG. 1;
FIG. 5 is a cross-sectional view of another embodiment of a bioreactor system
according to
the present invention;
FIG. 6 is a cross-sectional view of another embodiment of a bioreactor system
according to
the present invention;
FIG. 7 is a cross-sectional view of another embodiment of a bioreactor system
according to
the present invention;
FIGS. 8-10 are cross-sectional views of another embodiment of a bioreactor
system
according to the present invention;
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FIGS. 11-12 are cross-sectional views of another embodiment of a bioreactor
system
according to the present invention;
FIG. 13 is a graphical representation of one example of a cell biology
experiment performed
according to the invention showing hepatocytes growth on scaffolds with
perfusion;
FIGS. 14-16 are graphical representations of additional examples of cell
biology
experiments performed according to the invention;
FIG. 17 is a side view of one einbodiment of a system according to the present
invention;
FIG. 18 is a perspective view of one embodiment of a carrier of the system of
FIG. 17;
FIG. 19 is a partial perspective view of the carrier of FIG. 18 depicting a
single well as seen
from the bottom;
FIG. 20 is a partial perspective view of the carrier of FIG. 18 depicting a
single well as seen
from the top; and
FIGS. 21 and 22 are partial side views of a single well of the carrier of FIG.
18 shown
without and with a screen.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to bioreactors generally, and, more
particularly, to a system and
method for culturing cell specimens under perfusion flow, in a single chamber
or in a high
throughput format for the high throughput discovery of complex enviromnents
for
controlling cell function and engineered tissue development. The present
invention may also
be utilized for creating highly relevant cell cultures and systems for direct
drug testing on
cells in dynamic cell cultures, for drug discovery, drug testing, or ADMETox
applications.
Referring to FIG. 1, a preferred embodiment of a bioreactor system 5 generally
includes a
multi-well platform comprising an array of bioreactor units 10 wherein in each
bioreactor
unit, an independent cell study or experiment may be performed. As shown in
FIG. 1, the
bioreactor system 5 comprises a perfusion unit 12 and a fluid source unit 14
fluidly
interconnected by a pumping unit or station 16.
In a preferred embodiment, perfusion unit 12 is a multi-well plate including a
plurality of
main chambers or wells 18 configured to house or contain a cell culture.
Similarly, the fluid
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source unit 14 may comprise one or more separate multi-well plates including a
plurality of
fluid reservoir chainbers or wells 20 to store fluid, such as cell culture
media. In operation,
each main chamber or well 18 is in fluid communication with a corresponding
individual
fluid reservoir chamber 20. In a preferred embodiment, top perfusion unit 12
and fluid
source unit 14 include 24 chambers or wells, however, in alternative
embodiments any
number of chambers or wells may be provided. For example, the wells of top
perfusion unit
12 and the fluid source unit 18 may be miniaturized to comprise 48 wells per
plate, 96 wells
per plate, or smaller. Similarly, pumping components may be miniaturized to
comprise a
smaller bioreactor system with a similar footprint, or increasing the
footprint to have more
individual perfusion units on one system. Referring to FIG. 2, one example of
a multi-well
perfusion plate 12 is shown wherein each well includes a passage or hole 15
extending
through the base of the well to permit the passage of fluid therethrough. A
triangulated post
structure 17 is fixed onto a base portion of each well 18 and extends above
hole 15. Post
structure 17 facilitates the attachment of a cell adherent structure or
scaffold 22 (shown in
FIG. 4) to grow cell cultures. Each well may also contain a fluid return
pathway 19. In one
preferred embodiment, perfusion unit 12 and fluid source unit 14 may be made
from
polystyrene, polycarbonate, polypropylene, other plastic, or any other
suitable material, and
may be injection molded in parts or in their entirety.
In another preferred embodiment, perfusion unit 12 and fluid source unit 14
are preferably
configured and dimensioned to be removably coupled to pumping unit 16.
Accordingly,
perfusion unit 12 and fluid source unit 14 may be interchangeable components
of the system,
such that a plurality of like units or plates may be exchanged or removably
coupled to
pumping unit 16 as desired. For example, the fluid source unit 14 is
configured to be
removably coupled to the pumping unit 16 such that the fluid source unit 14
may be re-
usable or disposable for media addition. Similarly, perfusion unit 12 may be
removed from
one pumping unit 16 to another to associate cell cultures with different
fluid/dynamic
environments.
Pumping unit 16 comprises an array of fluid connectors and/or hardware
components to
fluidly connect each main chamber 18 with each fluid reservoir chamber 20. In
a preferred
embodiment, pumping unit or station 16 may comprise any hardware components
suitable
for transferring or pumping fluid from the fluid source unit 14 to the
perfusion unit 12 such
as, for example, motorized pump(s), valves, tubes, pipes, or other devices or
means for
pumping or transferring the fluid. Generally, any type of pumping mechanism
may be used,
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including but not limited to peristaltic, centrifugal, vibrating, piezo, or an
air or fluid driven
pumping mechanism, or individual electronic pumps wherein each perfusion unit
could be
programmed with a different pumping rate. In a particular preferred
embodiment, shown in
FIG. 3, pumping unit or station 16 utilizes a peristaltic puinping mechanism
including an
array of pumping plates 30 mounted upon driving rods 32. Rods 32 are slidably
mounted to
housing 34 in bearings 36 and in operation are driven back and forth along the
axis of rods
32 by a motor attached to coupling plate 38. When rods 32 are driven, pumping
plates 30
squeeze flexible tubing 40 against static plates 42 to pump the fluid
contained in flexible
tubing 40. As best seen in FIG. 4, single direction valves 41 are provided on
either side of
flexible tubing 40 and interposed between an inlet tube 47 and an outlet tube
49 to pump or
direct fluid flow in one direction from the fluid reservoir chainbers 20 of
fluid source unit 14
toward the main chambers 18 of perfusion unit 12. A return pathway 19 is
preferably built
into each main chamber 18 of perfusion unit 12 which fluidly connects to
return pathway 25
pumping unit 16 to provide for fluid return to the fluid source unit 14 from
the perfusion unit
12, thereby creating a plurality or array of individual and separate
bioreactor units 10. In this
regard, when perfusion unit 12 and fluid source unit 14 are coupled to pumping
unit 16, each
bioreactor unit 10 is an independent fluidly self-contained entity.
Referring to FIG. 4, a cross-sectional view of an exemplary individual
bioreactor unit 10 is
shown, wherein each bioreactor unit generally includes a single main chamber
or we1118 in
fluid communication with the fluid source, housed for example in a single
fluid reservoir
chamber or we1120. A cell adherent structure or scaffold 22 is preferably
housed within
each main chamber 18 to facilitate high density cell culture growth. In this
embodiment the
cell adherent structure is a three-dimensional scaffold, such as a porous body
having a
plurality of three-dimensional cell adherent surfaces, however, in alternate
embodiments, the
cell adherent structure may be two-dimensional, such as a slide or plate
having a two-
dimensional cell adherent surface. In other alternate embodiments, the cell
adherent
structure may have varied shapes such as, for example, a tubular or
cylindrical shape, such
that a transplantable medical device/implant with a biological component may
be engineered
in a high throughput device. In this regard, cells and/or tissue may adhere or
grow upon the
tubular structure to grow cell or tissue containing tubes such as, for
example, vascular grafts,
stents, neural tubes, shunts, etc., for transplantation into the body of a
patient. In other
embodiments, cartilage and/or bone may be grown or engineered in a
predetermined shape.
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In a preferred embodiment, the cell adherent structure is coupled to the main
chamber about
a fluid port 44 such that the fluid flows directly into or about the cell
adherent structure. For
example, a three-dimensional scaffold 22 may be coupled, molded, bonded,
synthesized, or
otherwise attached to the main chamber 18 such that a stem or fluid port 44
extends into the
central portion or interior of the scaffold when, for example, perfusion plate
12 is coupled to
pumping unit 16. In another preferred embodiment, each main chamber 18 of
perfusion
plate 12 is configured to receive scaffolds that may be coupled, fastened, or
otherwise
connected to a portion of each main chamber 18 by any suitable means known to
those
skilled in the art. In one preferred embodiment, scaffold 22 may be releasably
plugged into
or attached to main chamber 18.
The scaffolds can be made from any type of polymer, ceramic, metal or mixture
of any type
suitable for adhering cells thereto. In a preferred embodiment, the scaffold
is made from a
hydrogel-based material, which may be synthesized from covalently crosslinked
alginate,
hyalrunic acid or a blend of the two polysaccharides at any mixing percentage
as desired.
For example, the mixing percentage may be tailored to achieve a desired
degradation profile
for the final application. In alternate embodiments, the scaffolds may be made
of other
suitable materials, such as those disclosed in U.S. Patent Publication No.
2004/0147016
entitled "Programmable scaffold and methods for making and using same", the
entire
contents of which are incorporated by reference. In one preferred embodiment,
the scaffold
may be a porous structure having randomly aligned pores. In alternative
embodiments,
scaffolds may be used that have directionally aligned pores such that a less
random pore
pattern may be attained and fluid flow may be further assured of navigating or
flowing
through all of the pores of the scaffold. In alternate embodiments, the
scaffolds may be
modified with any number or type of cell signaling or cell interacting
molecule, such as
those disclosed in U.S. Patent Publication No. 2004/0147016, entitled
"Programmable
scaffold and methods for making and using same," the entire contents of which
are
incorporated by reference.
In operation, fluid is pumped directly into the internal scaffold structure
and may perfuse or
flow from the interior 46 of scaffold 22 to the exterior 48 of scaffold 22. In
one preferred
embodiment, fluid is pumped at a rate ranging from about 10 to 0.1 milliliters
per minute. In
this regard, fluid may readily flow through the internal pores of the scaffold
as opposed to
circumventing the scaffold or flowing mainly along the exterior of the
scaffold. The
enhanced diffusion mass transport provided by the perfused fluid flow
advantageously
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allows metabolites and nutrients to diffuse into and out of scaffold 22. In
this regard,
perfusion culture permits long teim tissue engineering experiments allowing
growth of high
density cell cultures to mimic tissues.
In prior art devices where fluid is permitted to circumvent the scaffold,
severe oxygen
limitations may be caused because oxygen is consumed by the cells adhered upon
the
outside of the scaffold and cells adhered upon the inside of the scaffold may
be oxygen
starved.
Referring to FIG. 5, an alternative embodiment of a main chamber 18 is shown
wherein the
fluid flow 50 is directed from an inlet 52 through an alternative scaffold 54
and exits the
scaffold and chamber at an outlet 56 as opposed to flowing randomly throughout
the
scaffold.
Referring to FIG. 6, another alternative embodiment of a bioreactor unit 10 is
shown
wherein main chamber 18 includes a two-dimensional cell adlierent structure 62
with a cell
adherent upper surface. In this embodiment, the cell adherent structure 62 is
coupled to the
chamber 18 such that fluid may flow along path 63 through fluid port 44 and
across the two-
dimensional surface of structure 62 and returns through return pathway 64. A
plate 66
covers structure 62 and is spaced therefrom to contain the fluid such that the
fluid flows
directly over thecell adherent surface.
Referring to FIG. 7, another alternative embodiment of a main chamber 18 is
shown,
wherein a cell specimen 70 may be coupled, fastened, or otherwise connected to
a portion of
each main chamber 18 by any suitable means known to those skilled in the art.
In some
embodiments, cell specimen 70 may be a cell adherent structure or scaffold and
in other
embodiments cell specimen 70 may comprise portions or slices of tissue. For
example, cell
specimen 70 may comprise liver slices, pancreatic islets, liver spheroids, 3-D
tissue models
(such as those commercially available from Mattek, Inc. or Regenemed, Inc.), 3-
D cancer
models (such as those commercially available from Mina Bissell), cells on
microcarriers or
fiber disks (such as those commercially available from fibracell), or any
other cellular bodies
that may be grown in vitro.
In one embodiment, cell specimen 70 has a cylindrical or disc shape and may be
held in
place in main chamber 18, for example, between a pair of washers 72. Washers
72 include a
central opening to permit fluid flow therethrough. In operation, fluid may
flow through port
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44 and perfuse through cell specimen 70 and exit through the central opening
of the top
washer 72 and return via return pathway 75. In this regard, the present
embodiment is
configured to keep slices or cell specimens emerged at all times in media,
while exposing the
tissue or cell specimen to fluid flow similar to in vivo conditions and
enhancing gas and
nutrient transfer. In a preferred embodiment, the present system facilitates
the maintaining
of cell viability, and the maintaining of the specimens or tissue slices in a
format for drug
testing. The configuration of this embodiment may be advantageously utilized
with, for
example, tissue slices or scaffolds made of polymer or ceramic material or
other materials
that cannot be synthesized in place.
Referring to FIGS. 8-10, an additional embodiment of a bioreactor system 80 is
shown. In
general, bioreactor system 80 is a pneumatic system comprising three
disposable or reusable
pieces or components: a bottom reservoir plate 82 configured to contain media,
a pumping
device 84 that induces the motion of the media, and a top perfusion plate 86
that mates with
the bottom reservoir plate and pumping device, and is configured to maintain
the position of
the tissue slices or cell specimen 70 in the media flow and create a closed
fluid path for the
media to return to bottom reservoir plate 82. Both the bottom reservoir plate
and the top
perfusion plate generally include multiple wells or chambers 83 and each plate
may be
injection molded and may be disposable or reusable items. Also, the plates may
be sterilized
using any suitable sterilization method known to those skilled in the art.
Each well of the
perfusion plate 86 generally comprises an inlet portion 88 and an outlet
portion 90. Each
well of the bottom reservoir plate 82 generally comprises a fluid reservoir 92
and a pair of
one-way valves or check valves 94. In one embodiment, .the one-way valves may
be molded
into a one piece plate. Each of the pair of one-way valves is aligned with the
corresponding
inlet and outlet portions 88, 90 of the perfusion plate to direct and or allow
the fluid or media
to flow from fluid reservoir 92 into the inlet 88 and out of the outlet 90 and
return to the
fluid reservoir 92. In one embodiment, cell specimen 70 may be positioned
within the inlet
portion 88 of each well of the perfusion plate 86. In one variation, two mesh
discs and a
retaining ring may be used to retain the tissue slice or cell specimen 70 in
position on the
perfusion plate. The optimal geometry and orientation of the cell specimen may
vary .
depending on the tissue type. For example, the tissue may be oriented
vertically or
horizontally to the fluid flow.
The pumping device 84 of the present embodiment generally compiises a pressure
chamber
96 having an air inlet 98 and a flexible diaphragm 100 that interfaces with
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reservoir plate 82. As best seen in FIG. 9, in operation, air pressure is
introduced through
inlet 98 into the pressure chamber 96 and the flexible diaphragm 100 expands
and 'exerts
pressure on the fluid reservoirs 92 of the bottom plate 82 causing the upward
flow of media
or fluid. As shown in FIG. 10, when the air pressure is released through inlet
98, the
diaphragm 100 contracts, releasing pressure on the fluid reservoirs 92 and
drawing or,
inducing the downward or return flow of media or fluid. One advantageous
feature of the
present embodiment is that the media or fluid is self-purging or actively
drained as opposed
to gravity-driven. In this regard, once the well of the perfusion plate
contains media or fluid,
the pumping device 84 purges the perfusion plate well during operation. In one
embodiment, the pumping device may be disposable or reusable. Also, the
pumping device
may be sterilized using any suitable sterilization method known to those
skilled in the art.
Several variations of the multi-well plate pumping device may also be used,
including
electric, peristaltic, and other diaphragm pumping techniques known to those
skilled in the
art.
Referring to FIGS. 11-12, an additional embodiment of a bioreactor system 110
is shown. In
general, bioreactor system 110, similar to bioreactor system 80, is a
pneumatic system
comprising tliree disposable or reusable pieces or components: a bottom
reservoir plate 112
configured to contain media, a pumping device 114 that induces the motion of
the media,
and a top perfusion plate 116 that mates with the pumping device, and is
configured to
maintain the position of the tissue slices or cell specimen 70 in the media
flow and create a
closed fluid path for the media to return to bottom reservoir plate 112. Botli
the bottom
reservoir plate 112 and the top perfusion plate 116 are substantially similar
to plates 82, 86
described above and generally include multiple wells or chambers 113. Each
plate may be
injection molded and may be disposable or reusable items. Also, the plates may
be sterilized
using any suitable sterilization method known to those skilled in the art.
Each well of the
perfusion plate 116 generally comprises an inlet portion 118 and an outlet
portion 120. Each
well of the bottom reservoir plate 112 generally comprises a fluid reservoir
122 and a pair of
one-way valves or check valves 124. In one embodiment, the one-way valves may
be
molded into a one piece plate. Each of the pair of one-way valves 124 is
connected via
flexible passages or tubing 132 and another pair of one-way valves 125 with
the
corresponding inlet and outlet portions 118, 120 of the perfusion plate to
direct and or allow
the fluid or media to flow from fluid reservoir 122 into the inlet 118 and out
of the outlet 120
and return to the fluid reservoir 122. In one embodiment, cell specimen 70 may
be
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positioned within the inlet portion 118 of each well of the perfusion plate
116. In one
variation, two mesh discs and a retaining ring may be used to retain the
tissue slice or cell
specimen 70 in position on the perfusion plate. The optimal geometry and
orientation of the
cell specimen may vary depending on the tissue type. For example, the tissue
may be
oriented vertically or horizontally to the fluid flow.
The pumping device 114 of the present embodiment generally comprises a
pressure chamber
126 having an air inlet 128 and a plurality of flexible diaphragms 130 that
surround flexible
passages 132. Flexible passages 132 extend between the fluid reservoirs 122 of
the bottom
plate and a pair of one-way valves or check valves 125 aligned with the inlet
and outlet
portions 118, 120 of the perfusion plate 116. As best seen in FIG. 12, in
operation, air
pressure is introduced through inlet 128 into the pressure chamber 126 and the
flexible
diaphragms 130 expand and exert pressure on the flexible passages or tubing
132 causing the
upward flow of media or fluid through the inlet portion 118 of the perfusion
plate 116 and
the simultaneous downward flow of media or fluid out from the perfusion plate
116 through
the outlet portion 120 to the fluid reservoir 122. One advantageous feature of
the present
embodiment is that the media or fluid is self-purging or actively drained as
opposed to
gravity-driven. In this regard, once the well of the perfusion plate contains
media or fluid,
the pumping device 114 purges the perfusion plate well during operation. In
one
embodiment, the pumping device may be disposable or reusable. Also, the
pumping device
may be sterilized using any suitable sterilization method known to those
skilled in the art.
Several variations of the multi-well plate pumping device may also be used,
including
electric, peristaltic, and other diaphragm pumping techniques known to those
skilled in the
art. I
With respect to all of the aforementioned multi-well bioreactor systems,
different cell types
may be cultured in the same set of wells. For example, in the embodiment of
FIG.' 4, a
hepatocyte cell may be cultured in fluid reservoir chamber 20, while an islet
cell may be
cultured in main chamber 18. The cells are in fluid communication via the
media contained
within the bioreactor unit 10 of FIG. 4. In another variation, multiple
parallel wells may be
in fluid communication with each other. For example, referring to the
embodiment of FIG.
1, a hepatocyte cell may be cultured in well Al of FIG. 1, while an islet cell
may be cultured
in well D6 of FIG. 1. All of the wells may be fluidly connected together by
channels or
other fluid pathways, such that after a period of time, the media from wells
Al, D6, and as
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many of the wells in fluid communication, will mix with each other and may
come to a
steady state.
Utilizing the aforementioned multi-well bioreactor systems of the present
invention, unique
experiments may be studied that incorporate fluid flow. For example, multiple
parallel
experiments may be perfonned having substantially similar fluid flow
characteristics. In this
regard, highly complex environments may be created to perform experiments in a
medium
throughput or high throughput format. One skilled in the art could also create
cultures
consisting of several types of cell and tissue systems in fluid communication
for studying
complex metabolic diseases such as diabetes, obesity, and cardiovascular
diseases to name a
few. In one particular application for optimizing cell signaling environments,
a variety of
soluble and non-soluble signaling molecules consisting of growth factors,
cytokines,
extracellular matrix molecules, etc., may be tested at different
concentrations, different
mixing ratios, and at various times to facilitate the discovery of an optimal
combination of
factors to obtain a fully differentiated cell culture in vitro. These,
environments may be
created utilizing a variety of parenchymal cells and non-parenchymal cells
from tissues
including bone marrow, vasculature, skin, pancreas, liver, bone, cartilage,
smootli muscle,
cardiac muscle, skeletal muscle, kidney, etc. In another embodiment, cells
such as
endotlielial cells may be used to create vascularization with the host. In
alternate
embodiments, one skilled in the art could also create cultures consisting of
several types of
tissue systems for studying complex metabolic diseases such as the metabolic
syndrome. In
alternative applications, several cell types may be incorporated to study
fluid sheer and
perfusion, for example, to determine fluid flow that most likely promotes cell-
type '
segregation for vasculorgenesis and tissue development. In another
application, the cells or
tissue grown in the multi-well design may be used as a platform for testing
drugs in a
medium to high throughput format for direct drug testing on cells in dynamic
cell cultures,
either for drug discovery, drug testing, or ADMETox applications. Furthermore,
sensing
technology may be incorporated into the bioreactor system. For example,
biosensing
technology for sensing important cell culture variables such as glucose,
ammonia, urea, pH,
or general fluorescent detectors for monitoring metabolism of fluorescent
compounds may
be utilized with the system.
In one exemplary variation or application, a perfusion unit 12 may be used to
grow cell
cultures with preset conditions or particularly desirable characteristics
which can then be
later used for further experimentation and or discovery. The modularity and
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interchangeability of perfusion unit 12 advantageously permits the shipment
and or transfer
of a plurality of cell cultures which can be easily remounted on another
pumping station 16
or similar device to perform further experimentation and/or drug testing or
discovery.
Referring now to FIGS. 17-22, a system and method for manipulating or handling
scaffolds
in a platform for performing biological experiments in a high throughput
and/or parallel
screening environment is sh.own. As shown in FIGS. 17 and 18, a preferred
embodiment of
a scaffold handling system 201 generally includes a multi-well cartridge or
carrier 205
comprising an array of well units 210 wherein, in each well unit, an
independent scaffold
220 may be held and a biological experiment may be performed. As shown in FIG.
17, in
one embodiment, carrier 205 of scaffold handling system 201 comprises four
well units 211,
212, 213, and 214 and includes sidewalls or flanges 216 and 218 extending
distally from the
lateral ends of cross-member 217 to mate with a multi-well plate. In alternate
embodiments,
however, any number or multiple of well units 210 may be included in carrier
205. For
example, in one variation carrier 205 may have one well unit. In another
exemplary
embodiment, carrier 205 may have 8 well units. In yet another embodiment,
carrier 205 may
have 3 well units. .
Each well unit 210 generally comprises a frustoconical or tapered body 230
exetending
distally from the top of carrier 205 and includes a scaffold holding chamber
232 at the distal
end 234. A cell adherent structure or scaffold 220 is preferably housed or
held within each
well unit 210 to facilitate high density cell culture growth. In a preferred
embodiment, the
cell adherent structure is coupled or loaded into to the well unit 210 about a
distal end 234.
For example, a three-dimensional scaffold 220 may be coupled, molded, bonded,
'
synthesized, or otherwise attached to the distal chamber 232. In one preferred
embodiment,
scaffold 220 may be releasably plugged into or attached to chamber 232 for
example by
friction fit.
Referring to FIG. 19, a bottom perspective view of carrier 205 is shown
depicting one of the
well units 210. In one embodiment, scaffold holding chamber 232 is tapered,
i.e. wider at
the distal end of the well unit and narrower at the top or proximal end of the
chamber. This
tapered feature of chamber 232 may accommodate a range of scaffold sizes. For
example, in
one embodiment, chamber 232 may accommodate scaffolds with diameters ranging
from
about 4.8 mm to about 5.1 mm. In addition, one or more nubs or protrusions 236
may
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extend radially inward from the perimeter of chamber 232 to fiuther grip or
hold a scaffold
therein by friction.
Referring to FIG. 20, a top perspective view of carrier 205 is shown depicting
one of the
well units 210. The top or proximal end of each well unit 210 defines an
opening 237 to
permit physical and visual access to a scaffold 220 held therein. In addition,
a window 238
extends through the carrier 205 adjacent the well units 210 to provide access
to the bottom of
the well therethrough. In this regard, the open top of each well unit 210,
i.e. opening 237
and window 238, facilitate aspiration aspiration or pipetting within the well
unit. As best
seen in FIGS. 20 and 21, in one embodiment, a longitudinal slot, channel, or
opening 239
extends along a lateral portion of body 230. Opening 239 facilitates fluid
overflow and
permits perfusion circulation when carrier 205 is used in combination with a
perfusion
bioreactor as described in more detail below. As also can be seen in FIG. 21,
a ledge 241
may be provided adjacent the distal end of body 230 to accommodate a screen to
hold
scaffold 220 in a longitudinal direction, entrap cells or minimize particulate
flow. For
example, as shown in FIG. 22, screen 250 may be positioned and/or molded
adjacent ledge
241 to prevent movement of scaffold 220 in the proximal direction while
permitting fluid
flow therethrough.
Scaffold handling system 201 and carrier 205 of FIGS. 17 and 18 are configured
and
dimensioned to be used with a multi-well plate having a plurality of main
chambers or wells
to house or contain a cell culture or cell culture experiment. Multi-well
plates are well
known to those skilled in the art. Exemplary multi-well plates include the BD
FalconTM
multi-well plates, available in 24-well plates and 96-well plates. In this
regard, carrier 205
of the present embodiment is configured and dimensioned to be inserted into
and/or mate
with such a 24-well plate. In operation, carrier 205 may be placed across a
single row of the
24-well plate with each of the well units 211, 212, 213, and 214, extending
into a
corresponding well of the 24-well plate so that biological experimentation may
be
conducted. Multiple carriers 205 may be placed aver additional rows of the
multi-well plate
such that a scaffold may be held in each well of the multi-well plate. In
other words, for a
24-well plate, six carriers 205 may be utilized with the 24-well plate. Of
course, one skilled
in the art will appreciate that any number of arrays and configurations may be
utilized such
that the entire multi-well plate may include a cell adherent scaffold.
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Sidewalls or flanges 216, 218 of carrier 205 extend distally from the lateral
sides of carrier
205 and are configured and dimensioned to extend about the lateral outside of
the multi-well
plate to accurately mate carrier 205 with the 24-well plate. As best seen in
FIG. 18, flanges
216 and 218 may have a chamfered edge 219 for easy repositioning with respect
to the
multi-well plate. In addition, as best seen in FIG. 19, one or more nubs,
locating pins, or
protrusions 240 may be provided on the underside of carrier 205 to facilitate
the alignment
of carrier 5 with the individual wells of a multi-well plate. In this regard,
the combination of
protrusions 40, flanges 16, 18, and the geometry of carrier 2051ead to a
reliable and
repeatable system to hold scaffolds in place with respect to a multi-well
plate.
In yet another embodiment, scaffold handling system 201 and carrier 205 of
FIGS. 17 and 18
may also be used with a multi-well plate of the aforementioned perfusion
bioreactor. In this
regard, carrier 205 of the present embodiment is configured and dimensioned to
be inserted
into and/or mate with such a multi-well plate of a perfusion bioreactor. In
operation, carrier
205 may be placed across a single row of the multi-well plate of the perfusion
bioreactor in
the same manner as described above with respect to a 24-well plate with each
of the well
units 211, 212, 213, and 214, extending into a corresponding well of the multi-
well plate of
the bioreactor so that biological experimentation may be conducted. In this
regard, the
configuration and design of handling system 201 is advantageously configured
to permit
perfusion of cell culture media through the scaffolds. For example, the
reliable and
repeatable positioning of the carrier 205 is configured to hold the
scaffold(s) 220 in the flow
line of the perfusion bioreactor such that cell culture media flows through
the scaffold from
the distal end to the proximal end of each well unit 210. Overflow channel or
opening 239
facilitates the return flow of perfusion media out though the proximal side of
the scaffold
220.
Referring again to FIG. 17, an exemplary method of handling or manipulating a
scaffold or
scaffolds 220 according to the present invention is also shown. As shown with
respect to
well unit 211, as an initial step, a scaffold 220 or multiple scaffolds may be
loaded or
inserted into well units 210 of carrier 205. Once installed or loaded into
carrier 205, as
shown with respect to well unit 212, the scaffold(s) 220 may then be
manipulated such as by
being treated with chemicals, sterilized with ultraviolet radiation, seeded
with cells, or other
treatments. Similarly, as shown with respect to well unit 213, the scaffold
may be inserted
into a multi-well plate with cell culture media or biological agents to
conduct biological
experiments. As shown with respect to well unit 214 of FIG. 17, media can be
perfused
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through scaffold(s) 220. Also, if microscopy is necessary, carrier 205 can be
easily moved
to a separate or fresh dry plate for microscopy without the need to handle the
scaffolds
directly.
Exanzple 1
Referring to FIG. 8, one example of a cell biology experiment performed
according to the
invention is shown wherein primary rat hepatocytes were seeded onto alginate
scaffolds in
the perfusion chamber, and cultured witli Hepatostim media under perfusion
flow. The same
cells were also seeded onto matrigel substrates (typically known to maintain
basal CYP 3A1
activity for rat hepatocytes), and passive coated collagen type 1 substrate
were used as a
negative control (typically known to decrease basal CYP 3A1 activity for rat
hepatocytes).
After 48 hours, 100uM cortexolone was added to the media to induce CYP 3A1
expression.
At weekly time points, the basal 3A1 activity was monitored by testosterone
metabolism into
6B-hydroxytestosterone using HPLC analysis. The level of 6B-
hydroxytestosterone in the
culture is therefore indicative of CYP 3A1 expression and activity. The
collagen cultures
did not allow for CYP 3A1 expression, and the matrigel cultures helped the
hepatocytes
maintain CYP 3A1 expression for 3 weeks, at which point expression decreased.
Hepatocytes cultured under fluid flow on aligned scaffolds also maintained
elevated CYP
3A1 activity, but instead of decreasing at 4 weeks, the activity increased
dramatically
compared to the matrigel. This example demonstrates that cells may be grown in
this device
configuration and also suggests that the novel culture conditions allowed for
extended and
higher expression of differentiation-specific cell function for primary rat
cells. Under
perfusion flow, important p450 3A1 function is maintained for 4 weeks, longer
than industry
standard matrigel cultures.
Example 2
Referring to FIG. 14, one example of a cell biology experiment performed
according to the
invention is shown wherein mouse osteoblastic cells (MC3T3s) were seeded on
calcium
phosphate scaffolds, and cultured with Gibco Alpha media under perfusion flow.
The same
cells were also seeded on calcium phosphate scaffolds in a static condition.
The metabolic
activity of the cells was studied via absorbance under static and perfusion
conditions for a
period of 15 days. After 15 days, the cells under perfusion show a
statistically significant
increase of 34% in metabolic activity over the cells in the static condition.
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Example 3
Referring to FIG. 15, one example of a cell biology experiment performed
according to the
invention is shown wherein human mesenchymal stem cells (MSCs) were seeded on
calcium
phosphate scaffolds, and cultured with Osteogenesis media under perfusion
flow. The same
cells were also seeded on calcium phosphate scaffolds in a static condition.
The metabolic
activity of the cells was studied via absorbance under static and perfusion
conditions for a
period of 10 days. After 10 days, the cells under perfusion show a
statistically significant
increase of 42% in metabolic activity over the cells in the static condition.
Example 4
Referring to FIG. 16, one example of a cell biology experiment performed
according to the
invention is shown wherein rat liver slices were cultured with Gibco media
under perfusion
flow. The slices were also cultured in a static condition. The metabolic
activity of the cells
was studied via absorbance under static and perfusion conditions for a period
of 5 days.
After 5 days, the slices under perfusion show a statistically significant
increase of 136% in
metabolic activity over the slices in the static condition.
While the invention has been described in conjunction with specific
embodiments and
examples thereof, it is evident that many alternatives, modifications and
variations will be
apparent to those skilled in the art upon reading the present disclosure.
Accordingly, it is
intended to embrace all such alternatives, modifications and variations that
fall within the
spirit and broad scope of the appended claims.
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