U.S. patent application number 13/116174 was filed with the patent office on 2011-12-01 for bioreactors with multiple chambers.
This patent application is currently assigned to VANDERBILT UNIVERSITY. Invention is credited to Franz J. Baudenbacher, David Cliffel, Frederick R. Haselton, Eugene J. Leboeuf, Ales Prokop, Randall S. Reiserer, Mark A. Stremler, John P. Wikswo.
Application Number | 20110294202 13/116174 |
Document ID | / |
Family ID | 31978278 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110294202 |
Kind Code |
A1 |
Wikswo; John P. ; et
al. |
December 1, 2011 |
BIOREACTORS WITH MULTIPLE CHAMBERS
Abstract
A bioreactor for cultivating living cells in a liquid medium. In
one embodiment of the present invention, the bioreactor has a first
substrate having a first surface and an opposite second surface,
defining a chamber therebetween for receiving the cells and the
liquid medium. The bioreactor further has a barrier dividing the
chamber into a first subchamber and a second subchamber, wherein
the barrier has a porosity to allow the first subchamber and the
second subchamber in fluid communication and allow at least one
predetermined type of cells to permeate between the first
subchamber and the second subchamber.
Inventors: |
Wikswo; John P.; (Brentwood,
TN) ; Baudenbacher; Franz J.; (Franklin, TN) ;
Cliffel; David; (Nashville, TN) ; Haselton; Frederick
R.; (Nashville, TN) ; Leboeuf; Eugene J.;
(Franklin, TN) ; Prokop; Ales; (Franklin, TN)
; Reiserer; Randall S.; (Nashville, TN) ;
Stremler; Mark A.; (Franklin, TN) |
Assignee: |
VANDERBILT UNIVERSITY
Nashville
TN
|
Family ID: |
31978278 |
Appl. No.: |
13/116174 |
Filed: |
May 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10525549 |
Oct 24, 2005 |
7977089 |
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PCT/US03/26798 |
Aug 27, 2003 |
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13116174 |
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60406278 |
Aug 27, 2002 |
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Current U.S.
Class: |
435/287.9 ;
435/288.7 |
Current CPC
Class: |
B01L 2300/163 20130101;
B01L 2200/10 20130101; B01L 2300/0816 20130101; C12M 23/12
20130101; B01L 2300/0645 20130101; B01L 2300/0681 20130101; C12M
29/10 20130101; Y10S 435/808 20130101; B01L 2400/0472 20130101;
B01L 3/5027 20130101; C12M 41/00 20130101; C12M 23/16 20130101 |
Class at
Publication: |
435/287.9 ;
435/288.7 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Goverment Interests
STATEMENT OF FEDERALLY-SPONSORED RESEARCH
[0003] This invention was made with government support under
Contract No. N66001-01-C-8064 awarded by the Defense Advanced
Research Projects Administration and the Office of Naval Research.
The Government has certain rights in the invention.
Claims
1-32. (canceled)
33. A bioreactor for cultivating living cells in a liquid medium
comprising: (a) a substrate having a first surface and an opposite
second surface, defining a chamber therebetween for receiving the
cells and the liquid medium, wherein the chamber is formed with a
center and a boundary; (b) a first barrier enclosing the center and
a portion of the chamber to form a central chamber; (c) a second
barrier positioned between the first barrier and the boundary so as
to form an intermediate chamber and an outer chamber; and (d) means
for electrochemical measurements of the cells responsive to the
liquid medium in at least one of the outer chamber, the
intermediate chamber and the central chamber; (e) means for optical
measurements of the cells responsive to the liquid medium in at
least one of the outer chamber, the intermediate chamber, and the
central chamber, wherein the means for electrochemical measurements
is positioned between and in communication with the substrate and
the means for optical measurements, and wherein the first barrier
has a first porosity to allow the central chamber and the
intermediate chamber in fluid communication and allow at least a
first predetermined type of cells to permeate between the central
chamber and the intermediate chamber, and the second barrier has a
second porosity to allow the outer chamber and the intermediate
chamber in fluid communication and allow at least a second
predetermined type of cells to permeate between the outer chamber
and the intermediate chamber.
34. The bioreactor of claim 33, wherein the central chamber is
adapted for receiving a first type of material, the intermediate
chamber is adapted for receiving a second type of material, and the
outer chamber is adapted for receiving a third type of
material.
35. The bioreactor of claim 34, wherein each of the first type of
material, the second type of material and the third type of
material contains at least one selected from the group of cells,
chemicals, and fluids.
36. The bioreactor of claim 33, wherein the first predetermined
type of cells comprises tumor cells, which normally is received in
the central chamber corresponding to a tumor space.
37. The bioreactor of claim 36, wherein the second predetermined
type of cells comprises normal tissue cells, which normally is
received in the intermediate chamber corresponding to a tissue
space.
38. The bioreactor of claim 37, wherein the outer chamber is
corresponding to a vascular space adapted for receiving endothelial
cells, macrophage cells, neutophil cells, any combination of them,
or other immune cell type.
39. The bioreactor of claim 33, further comprising a biocompatible
coating layer applied to the chamber walls at the boundary.
40. The bioreactor of claim 39, wherein the biocompatible coating
layer comprises a material that may inhibit cell adhesion to the
biocompatible coating layer, enhance cell adhesion to the
biocompatible coating layer, or function as a fluorescent marker or
indicator of the state of cells.
41. The bioreactor of claim 33, further comprising at least one
inlet or outlet port and an input or output transfer channel in
fluid communication with the inlet or outlet port and the external
chamber for allowing delivery of the cells, fluids or chemicals to
the outer chamber.
42. The bioreactor of claim 41, further comprising at least one
inlet or outlet port and an input or output transfer channel in
fluid communication with the inlet or outlet port and the central
chamber for allowing delivery of the cells, fluids or chemicals to
the central chamber.
43. The bioreactor of claim 42, further comprising at least one
inlet or outlet port and an input or output transfer channel in
fluid communication with the inlet or outlet port and the
intermediate chamber for allowing delivery of the cells, fluids or
chemicals to the intermediate chamber.
44. The bioreactor of claim 33, wherein the substrate is fabricated
from glass, Mylar, PDMS, silicon, a polymer, a semiconductor, or
any combination of them.
45. The bioreactor of claim 33, wherein the first barrier comprises
a porous material.
46. The bioreactor of claim 45, wherein the first barrier is
microfabricated so as to form a first structure allowing the fluid
communication between the central chamber and the intermediate
chamber.
47. The bioreactor of claim 46, wherein the second barrier
comprises a porous material.
48. The bioreactor of claim 47, wherein the second barrier is
microfabricated so as to form a second structure allowing the fluid
communication between the outer chamber and the intermediate
chamber, and the second structure is different from the first
structure.
49. (canceled)
50. The bioreactor of claim 33, wherein the means for
electrochemical measurements comprises: (i) a reference electrode;
(ii) a counter electrode; and (iii) a plurality of individually
addressable working electrodes.
51. The bioreactor of claim 50, wherein the liquid medium comprises
at least one analyte, and wherein the plurality of individually
addressable working electrodes comprise a first group of
individually addressable working electrodes adapted for sensing the
concentration of a single analyte of the liquid medium at multiple
locations in the outer chamber or the concentrations of a plurality
of analytes of the liquid medium at multiple locations in the outer
chamber at a time period shorter than a characterization reaction
time related to at least one of cellular physiological activities
of the cells.
52. The bioreactor of claim 51, wherein the first group of
individually addressable working electrodes are further adapted for
measuring the metabolic variables related to the cells responsive
to the liquid medium at multiple locations in the outer chamber at
a time period shorter than a characterization reaction time related
to at least one of cellular physiological activities of the
cells.
53. The bioreactor of claim 52, wherein the plurality of
individually addressable working electrodes comprise a second group
of individually addressable working electrodes adapted for sensing
the concentration of a single analyte of the liquid medium at
multiple locations in the central chamber or the concentrations of
a plurality of analytes of the liquid medium at multiple locations
in the central chamber at a time period shorter than a
characterization reaction time related to at least one of cellular
physiological activities of the cells.
54. The bioreactor of claim 53, wherein the second group of
individually addressable working electrodes are further adapted for
measuring the metabolic variables related to the cells responsive
to the liquid medium at multiple locations in the central chamber
at a time period shorter than a characterization reaction time
related to at least one of cellular physiological activities of the
cells.
55. The bioreactor of claim 54, wherein the plurality of
individually addressable working electrodes comprise a third group
of individually addressable working electrodes adapted for sensing
the concentration of a single analyte of the liquid medium at
multiple locations in the intermediate chamber or the
concentrations of a plurality of analytes of the liquid medium at
multiple locations in the intermediate chamber at a time period
shorter than a characterization reaction time related to at least
one of cellular physiological activities of the cells.
56. The bioreactor of claim 55, wherein the third group of
individually addressable working electrodes are further adapted for
measuring the metabolic variables related to the cells responsive
to the liquid medium at multiple locations in the intermediate
chamber at a time period shorter than a characterization reaction
time related to at least one of cellular physiological activities
of the cells.
57. The bioreactor of claim 33, wherein the first barrier is
substantially circular.
58. The bioreactor of claim 33, wherein the second barrier is
substantially circular.
59. The bioreactor of claim 33, wherein the boundary is
substantially circular.
60. A bioreactor for cultivating living cells in a liquid medium
comprising: (a) a substrate having a first surface and an opposite
second surface, defining a chamber therebetween for receiving the
cells and the liquid medium with a boundary; (b) means for dividing
the chamber into plurality of subchambers; (c) means for
electrochemical measurements of the cells responsive to the liquid
medium in at least one of the plurality of subchambers; and (d)
means for optical measurements of the cells responsive to the
liquid medium in at least one of the plurality of subchambers,
wherein the means for electrochemical measurements is positioned
between and in communication with the substrate and the means for
optical measurements, and wherein each of the plurality of
subchambers is in fluid communication with at least another one of
the plurality of subchambers.
61. The bioreactor of claim 60, wherein the dividing means
comprises a barrier to divide the chamber into a first subchamber
and a second subchamber, and wherein the barrier has a porosity to
allow the first subchamber and the second subchamber in fluid
communication and allow at least one predetermined type of cells to
permeate between the first subchamber and the second
subchamber.
62. The bioreactor of claim 60, wherein the dividing means
comprises a first barrier and a second barrier to divide the
chamber into a first subchamber, a second subchamber and a third
subchamber, and wherein the first barrier has a first porosity to
allow the first subchamber and the intermediate subchamber in fluid
communication and at least a first predetermined type of cells to
permeate between the first subchamber and the second subchamber,
and the second barrier has a second porosity to allow the second
subchamber and the third subchamber in fluid communication and at
least a second predetermined type of cells to permeate between the
second subchamber and the third subchamber.
63. The bioreactor of claim 62, wherein the first porosity and the
second porosity can be same or different.
64. The bioreactor of claim 60, wherein the dividing means
comprises a plurality of n barriers, n being an integer greater
than zero, to divide the chamber into n+1 subchambers.
65. The bioreactor of claim 64, wherein each of n barriers has a
corresponding porosity that can be same or different from that of
other barriers.
Description
[0001] This application is a divisional patent application of U.S.
patent application Ser. No. 10/525,549, filed Oct. 24, 2005,
entitled "BIOREACTORS WITH MULTIPLE CHAMBERS", filed in the name of
John P. Wikswo et al., which is incorporated herein by reference in
its entirety and is a U.S. National Phase of PCT/US2003/26798,
filed Aug. 27, 2003, which itself claims the benefit, pursuant to
35 U.S.C. .sctn.119(e), of Provisional Application No. 60/406,278,
filed Aug. 27, 2002, which is incorporated herein by reference in
its entirety.
[0002] Some references, which may include patents, patent
applications and various publications, are cited in a reference
list and discussed in the description of this invention. The
citation and/or discussion of such references is provided merely to
clarify the description of the present invention and is not an
admission that any such reference is "prior art" to the invention
described herein. In terms of notation, hereinafter, "[n]"
represents the nth reference cited in the reference list. For
example, [.sup.11] represents the 11th reference cited in the
reference list, namely, Bu, W. S. and Aunins, J. G., Large-Scale
Mammalian Cell Culture, Curr. Opin. Biotechnol., 8, 148-153,
1997.
FIELD OF THE INVENTION
[0004] The present invention generally relates to an apparatus and
methods for growing and maintaining a living system. More
particularly, the present invention relates to an apparatus and
methods that have a channel configuration allowing perfusate flow
with diffusional exchange to tissue cells but no cell migration.
Additionally, the present invention relates to an apparatus and
methods that have capacity for growing and maintaining a living
microorganism such as protozoa.
[0005] The present invention also relates to an apparatus and
methods for dynamic analysis of a collection of cells such as a
biofilm. More particularly, the present invention relates to an
apparatus and methods for measuring response of a biofilm to one or
more dynamic streams of substance such as chemical stressors at
various depths of the biofilm.
[0006] Certain embodiments of the present invention comprise
apparatus and methods for growing and maintaining a living system
such as a cell or a collection of cells and monitoring the status
of such a living system that is metabolically active and responsive
to environmental change, wherein each metabolic activity of the
cell may be characterized by a characteristic time. More
particularly, the apparatus and methods comprise bioreactors with
multiple chambers and methods of using the same.
[0007] Certain other embodiments of the present invention comprise
apparatus and methods for growing and maintaining a living system
such as a cell or a collection of cells and monitoring the status
of such a living system that is metabolically active and responsive
to environmental change, wherein each metabolic activity of the
cell may be characterized by a characteristic time. More
particularly, the apparatus and methods comprise bioreactors with
an array of chambers with a common feed line and methods of using
the same.
[0008] Certain additional embodiments of the present invention
comprise apparatus and methods for growing and maintaining a living
system such as a cell or a collection of cells and monitoring the
status of such a living system that is metabolically active and
responsive to environmental change, wherein each metabolic activity
of the cell may be characterized by a characteristic time. More
particularly, the apparatus and methods comprise capillary perfused
bioreactors and methods of using the same.
[0009] Certain further embodiments of the present invention
comprise apparatus and methods for growing and maintaining a living
system such as a cell or a collection of cells and monitoring the
status of such a living system that is metabolically active and
responsive to environmental change, wherein each metabolic activity
of the cell may be characterized by a characteristic time. More
particularly, the apparatus and methods comprise bioreactors with
substance injection capability and methods of using the same.
BACKGROUND OF THE INVENTION
[0010] Bioreactor is a device that can be used for culturing living
cells. More particularly, bioreactors are vessels that provide a
proper physical and chemical environment as well as fast transport
of substrates and products to allow cellular biological reactions
to occur, ideally rapidly and efficiently. The simplest bioreactor
is a culture dish: fu conventional cell culture using well-plates,
culture-dishes, and flasks, the volume of the culture medium is
typically 200 to 1000 times the volume of the cells. This ratio,
when used in combination with buffering of the culture media,
allows the cells to grow for at least 24 hours without media
change. However, another consequence of this ratio is a
corresponding dilution of whatever extracellular factors are
produced by the cells and might otherwise provide paracrine
cell-to-cell communication, which is possible in tissue because the
extracellular volume might be only 10% of intracellular volume.
[0011] Much of the development of bioreactors was directed towards
either the functional tissues, or the generation of biochemicals
and pharmaceuticals. For example, over the last 20 years studies on
the generation of skin, pancreas, cartilage, liver, cornea and
bladder have taken particular importance.sup.1. In the United
States alone, there are more than 80,000 individuals waiting for an
organ transplant, and hence the need to develop improved bioreactor
technology is self-evident. There is also a growing recognition
that progress in understanding cell motility and chemotactic
signaling, as well as other complex cellular processes, is often
constrained by the laboratory techniques available for observing
and intervening at various points in the processes. Many of these
processes can be examined best in a properly instrumented
bioreactor.
[0012] There is a wide variety in bioreactors, including stirred
vessels, bubble column, packed beds.sup.2, air-lift reactors, and
membrane reactors.sup.3 that include plates, rotating plates,
spiral-wound and hollow fibres. Hollow-fiber reactors are of
special importance since (depending of their structure) they may
allow as much as 30,000 m.sup.2 of membrane area per m.sup.3 module
volume.sup.4-6. However, given that mammalian cells are very
sensitive to shearforces.sup.7-9 (which originate mainly from
agitation and aeration), it is important to reduce the forces as
much as possible in the reactor where the cells will be
grown.sup.9,10. Membranes have been used in bioreactors to increase
survival of cells. For instance, it has been known that liquid-gas
interface created in some models of reactors is particularly
damaging for mammalian cells. That potentially lethal interface can
be eliminated by the use of a hydrophobic membrane.sup.9.
[0013] Bioreactors may be also classified by means of their mode of
operation: batch, fed-batch and continuous cultivation (also called
perfused cultivation). In the first or batch mode, no substrate is
added, nor medium removed; in the case of the fed-batch mode there
is a continuous feeding, but nothing is removed until the reactions
are terminated and the reactor emptied. While these systems imply a
low effort for process control, the productivity is low compared to
that in perfused systems, the third mode, where a permanent inflow
of substrate and outflow of medium takes place. Besides the high
productivity, there is a better cell physiology control in this
kind of reactors.sup.11 and in the case of mammalian cell culture,
it has been shown to provide significant advantage over static
methods.sup.12,13.
[0014] One of the limitations when developing large
three-dimensional tissues is the lack of a proper vascular supply
for nutrient and metabolite transport. A number of studies have
analyzed the artificial vascular networks.sup.14-18, and there have
been a number of attempts to construct functional microfabricated
scaffolds.sup.3,16,19-21. The techniques by which these networks
have been produced include plasma etching, photolithography, soft
lithography, microcontact printing, microfluidic patterning using
microchannels, laminar flow patterning and stencil
patterning.sup.22-25. In the case of plasma etching technologies we
can consider the high aspect ratio micromachining (HARMS) as a very
powerful tool since it allows to etch channels of virtually
unlimited depth without increasing the width already achieved by
lithography. It is also possible to construct three dimensional
microchannel systems in PDMS with complex topologies and
geometries.sup.15.
[0015] Additionally, one needs to realize that the growth of
clinically-implantable tissue may require the ultimate
biodegradation and the mechanical properties of the tissue
scaffold.sup.16. These properties are directly related to the
crystallinity, molecular weight, glass transition temperature and
monomer hydrophobicity of the materials chosen to fabricate the
tissue.sup.19. Naturally derived materials such as collagen have
been employed.sup.26, as well as synthetic and semi synthetic ones.
Polyglycolic acid (PGA) possesses high porosity and it makes easy
the fabrication of devices, therefore, PGA fibre meshes have been
considered to transplant cells. However, they cannot resist
significant compressional forces. An alternative to solve this
problem is to use polymers of lactic and glycolic acid whose ratios
can be adjusted to control the crystallinity of the material and
hence the degradation rate and mechanical properties. Fibre-based
tubes have been fabricated from these polymers.sup.27.
[0016] It is important to compare the vascular nature of living
tissue with the capabilities provided by existing microfabricated
cell-perfusion bioreactor systems. In tissue, arteries divide into
progressively smaller vessels, eventually reaching arterioles and
then capillaries. The arterioles are important because they contain
the precapillary sphincters, which allow control of the perfusion
of individual capillary beds, but also provide the majority of the
peripheral resistance and hence the pressure drop associated with
the arterial supply. As a result, the pressure difference across
the capillary endothelium membrane is kept sufficiently low to
allow diffusional transport of nutrients and metabolites across the
membrane, as well as the trafficking of immune cells required for
tissue maintenance and infection control. Were the pressures in the
capillaries as high as those in the arterioles, the capillary wall
thickness would be too great to allow these critical transport
phenomena. The venous return system is in many ways a mirror of the
arterial system, albeit at lower pressures. Another feature of the
living vascular system is that the branching process described
above allows all cells to be within 50 to 200 microns of a
capillary, depending upon the specific tissue. As a result, the
arterial supply and venous return systems are intercalated in such
a manner that every capillary that perfuses a large group of cells
is connected to the larger supply and return systems with a
self-similarity that ensures uniform perfusion and transcapillary
pressures. It is this intercalation process that is so difficult to
replicate with microfabrication. For example, Borenstein et
al.,.sup.22 describe a process to build a two-dimensional vascular
system that could create a multi-scale perfusion system for
supporting endothelial cells, but there is no provision to
selectively limit diffusive transport across the smallest
capillaries to perfuse cells lying outside of the perfusion
network. More importantly, the networks they show have a large
region of the device that is covered with the larger vessels, and
the region of the bioreactor that is limited to capillary vessels
is in fact quite small.
[0017] Thus, there is a need for microfabricated migration
bioreactors that mimic in vitro the microenvironments of normal
tissue was well as that of tumors, infected tissue, and wounded
tissue, while providing independent control of chemokine and growth
factor gradients, shear forces, cellular perfusion, and the
permeability of physical barriers to cellular migration, thereby
allowing detailed optical and electrochemical observation of
normal, immune, and cancerous cells during cell migration,
intravasation, extravasation, and angiogenesis. Angiogenesis, tumor
metastasis, and leukocyte infiltration into tissue are complex
processes that are regulated not only by cellular responses to a
single chemokine, but also by external factors, such as multiple
competing chemokine and growth factor signals, autocrine feedback
loops, cell-cell interactions, and mechanical forces such as vessel
shear stress. Current approaches for assessing migration across
cellular barriers include Boyden and transwell chambers that
provide an integrated fluorescence assay of migration across
filters to allow quantitation of migration.sup.28-34, parallel
plate flow chambers.sup.35-38, in which adhesion and rolling on
endothelial cells in shear stress can be assessed.sup.35,39-44, and
in vivo intravital microscopy in which migration of cells in living
animals is visualized.sup.45-48. Each of these approaches has
limitations, including the inability to have sustained and
controlled chemotactic gradients (all systems), the inability to
visualize migration in real time or with physiologic shear stress
(Boyden and transwell chambers), the inability to observe
extravasation or angiogenesis into an underlying, deep cellular
matrix (parallel plate flow chambers) and the inability to control
all aspects of the experiments, e.g., having defined cell
populations and controlled microfluidics for independent control of
shear and tissue perfusion (all systems, especially intravital
microscopy). The development of a motility/metastasis model system
with independent control of endothelial shear stress, chemokine
gradients, tissue perfusion, and the ability to add different cell
types through different ports, combined with state-of the art
imaging techniques and sensor capabilities would represent a huge
advance over currently available systems.
[0018] Indeed, the need for such capabilities is quite urgent.
Angiogenesis is a dynamic process, influenced by the cellular
microenvironment and intricately linked to metastasis49,50. It has
been demonstrated that both VEGF and angiopoietin/tyrosine kinase
(Ang/Tie2) function are required for tumor angiogenesis.sup.51-53.
However, how signals from those two receptor systems are integrated
to mediate angiogenesis has not been determined, in part due to the
lack of good model systems. The next step would be to study the
coordination and integration of VEGF and Ang signaling in
endothelial cell migration, vascular sprouting and maturation, and
tumor transendothelium migration. As with angiogenesis, multiple
environmental inputs affect tumor metastasis and leukocyte
infiltration. Activation of one chemokine receptor in tumor cells
affects the induction of other ligands and receptors in tumor cells
as well as endothelial cells and leukocytes, but the mechanism is
poorly understood.sup.54. There is a need for an understanding of
how alteration of chemokine receptor internalization and/or changes
in receptor association with adaptor molecules such as AP-2 or
beta-arresting affect chemokine receptor activity as tumor cells
move through a complex matrix. How external factors such as
cell-cell adhesion, cell-matrix interactions, and vessel shear
stress affect cytoskeletal reorganization during migration through
tissues is also poorly understood. Cortactin over expression
increases the metastasis of breast cancer cells to bone.sup.55,
however the mechanism remains unclear. Likewise, lack of WASp
protein in humans leads to an X-linked immune disorder that may
result from signaling, proliferation or chemotaxis defects.sup.56.
There is a need to study the role of cortactin and WASp proteins in
chemotaxis of breast cancer and HL60 cells in a complex multicell
environment involving controllable shear, cell-cell interactions,
and chemokine gradients. As a final example, matrix
metalloproteinases (MMPs) are extracellularly expressed enzymes
found in many types of cancer and are thought to be important in
tumor development, growth, invasion and metastasis. It has recently
been discovered that skin tumors that develop in mice deficient for
MMP-3 (MMP-3 null mice) progress and grow much faster than skin
tumors from normal, wild-type mice. This difference is associated
with a reduced number of immune cells in the tumor and surrounding
tissue in the MMP-3 null mice. The logical progression of this
research is to determine how loss of an MMP affects the ability of
immune cells, namely monocytes and neutrophils, to infiltrate from
the peripheral blood circulation to the tumor site. The ability to
control the experimental environment, including multiple defined
cell populations, is critical to elucidate the relative importance
of tumor-host interactions in MMP-3 induced cellular
chemotaxis.
[0019] Despite the progress made over the years, however, currently
available bioreactors cannot provide a more physiologic environment
that would include a three-dimensional in vitro region with
multiple cell types, stimuli, and measurement capabilities and
allows study of molecular aspects of the chemotactic response.
Thus, bioreactors that mimic in vitro the microenvironments of
tumors and tissue while providing independent control of chemokine
and growth factor gradients, shear forces, cellular perfusion, and
the permeability of physical barriers to cellular migration,
thereby allowing detailed optical and electrochemical observation
of normal and cancerous cells during cell migration, intravasation,
extravasation, and angiogenesis need to be developed.
[0020] Therefore, a heretofore unaddressed need exists in the art
to address the aforementioned deficiencies and inadequacies.
SUMMARY OF THE INVENTION
[0021] In one aspect, the present invention relates to a bioreactor
for cultivating living cells in a liquid medium. In one embodiment,
the bioreactor has a first substrate having a first surface and an
opposite second surface, defining a chamber therebetween for
receiving the cells and the liquid medium. The bioreactor further
has a barrier dividing the chamber into a first subchamber and a
second subchamber, wherein the barrier has a porosity to allow the
first subchamber and the second subchamber in fluid communication
and allow at least one predetermined type of cells to permeate
between the first subchamber and the second subchamber.
[0022] As formed, the first subchamber is adapted for receiving a
first type of material and the second subchamber is adapted for
receiving a second type of material, wherein each of the first type
of material and the second type of material contains at least one
selected from the group of cells, chemicals, and fluids. The cells
can be any type of living cells, including, but not limited to,
bacteria, protozoa, or both, normal cells, tumor cells, or any
combination of them.
[0023] The bioreactor also includes a biocompatible coating layer
applied to the chamber walls, wherein the biocompatible coating
layer comprises a material that may inhibit cell adhesion to the
biocompatible coating layer, enhance cell adhesion to the
biocompatible coating layer, or function as a fluorescent marker or
indicator of the state of cells.
[0024] At least one inlet port and an input transfer channel are
formed in the first substrate, wherein the input transfer channel
is in fluid communication with the inlet port and one of the first
subchamber and the second subchamber for allowing delivery of the
cells, fluids or chemicals to the corresponding subchamber. At
least one outlet port and an outlet transfer channel are also
formed in the first substrate, wherein the outlet transfer channel
is in fluid communication with the outlet port and one of the first
subchamber and the second subchamber for allowing removal of the
cells, fluids or chemicals from the corresponding subchamber.
Additionally, at least one auxiliary port and an auxiliary channel
are formed in the first substrate, wherein the auxiliary channel is
in fluid communication with the auxiliary port and one of the input
transfer channel and the outlet transfer channel for flushing the
corresponding transfer channel. Furthermore, at least one access
port and an access channel are formed in the first substrate,
wherein the access channel is in fluid communication with the
access port and one of the first subchamber and the second
subchamber for allowing delivery or removal of the cells, fluids,
chemicals, coating material or sensing material to the
corresponding subchamber.
[0025] The bioreactor further includes a second substrate, wherein
the second substrate is positioned adjacent to the first surface of
the first substrate and defines a plurality of connection channels,
each of the connection channels being formed so as to be in fluid
communication with a corresponding one of the inlet port, the
outlet port, the auxiliary port, and the access port. The
bioreactor additionally has a plurality of connection ports, each
of the connection ports being formed with a channel and being
positioned to the second substrate such that each channel of the
connection ports is in fluid communication with a corresponding one
of the connection channels formed in the second substrate.
[0026] In one embodiment, the first substrate is fabricated from
glass, Mylar, PDMS, silicon, a polymer, a semiconductor, or any
combination of them. The barrier comprises a porous material,
wherein the barrier is microfabricated so as to form a structure
allowing the fluid communication between the first subchamber and
the second subchamber.
[0027] The bioreactor additionally includes a third substrate,
wherein the third substrate is positioned adjacent to the second
surface of the first substrate, and means positioned in the third
substrate and adapted for electrochemical measurements of the cells
responsive to the liquid medium in at least one of the first
subchamber and the second subchamber. The third substrate can be
formed with a semiconductor material such as silicon.
[0028] In one embodiment, the means for electrochemical
measurements includes a reference electrode, a counter electrode, a
plurality of edge connector pads, and a plurality of electrically
conductive leads, wherein a first electrically conductive lead
electrically couples the reference electrode to a corresponding
edge connector pad, and a second electrically conductive lead
electrically couples the counter electrode to a corresponding edge
connector pad. The means for electrochemical measurements further
includes a plurality of individually addressable working
electrodes, each being electrically coupled to a corresponding edge
connector pad through a corresponding electrically conductive lead,
wherein the liquid medium includes at least one or more analytes,
and wherein the plurality of individually addressable working
electrodes are adapted to be capable of sensing the concentration
of a single analyte of the liquid medium at multiple locations in
the chamber or the concentrations of a plurality of analytes of the
liquid medium at multiple locations in the chamber at a time period
shorter than a characteristic reaction time related to at least one
of cellular physiological activities of the cells. Additionally,
the plurality of individually addressable working electrodes are
further adapted to be capable of measuring the metabolic variables
related to the cells responsive to the liquid medium at multiple
locations in the chamber at a time period shorter than a
characteristic reaction time related to at least one of cellular
physiological activities of the cells.
[0029] The bioreactor further includes a fourth substrate, wherein
the fourth substrate is positioned above the second surface of the
first substrate, and means positioned in the fourth substrate and
adapted for optical measurements of the cells responsive to the
liquid medium in at least one of the first subchamber and the
second subchamber. The fourth substrate is at least partially
transparent. The fourth substrate can be formed with a
semiconductor material such as silicon.
[0030] In one embodiment, the means for optical measurements
includes a plurality of optical sensors, a plurality of edge
connector pads, and a plurality of leads, each optically coupling
an optical sensor to a corresponding edge connector pad, wherein
the plurality of optical sensors comprises at least one device
selected from the group of an LED and photodiode pair, a fiber
optic coupler, and an optical detecting head, wherein the liquid
medium includes at least one or more analytes, and wherein the
plurality of optical sensors are adapted to be capable of sensing
the concentration of a single analyte of the liquid medium at
multiple locations in the chamber or the concentrations of a
plurality of analytes of the liquid medium at multiple locations in
the chamber at a time period shorter than a characteristic reaction
time related to at least one of cellular physiological activities
of the cells. Additionally, the plurality of optical sensors are
further adapted to be capable of measuring the metabolic variables
related to the cells responsive to the liquid medium at multiple
locations in the chamber at a time period shorter than the
characteristic reaction time related to at least one of cellular
physiological activities of the cells.
[0031] In another aspect, the present invention relates to a
bioreactor for cultivating living cells in a liquid medium. In one
embodiment, the bioreactor has a substrate having a first surface
and an opposite second surface, defining a chamber therebetween for
receiving the cells and the liquid medium, wherein the chamber is
formed with a center and a boundary, a first barrier enclosing the
center and a portion of the chamber to form a central chamber, and
a second barrier positioned between the first barrier and the
boundary so as to form an intermediate chamber and an outer
chamber, wherein the first barrier has a first porosity to allow
the central chamber and the intermediate chamber in fluid
communication and allow at least a first predetermined type of
cells to permeate between the central chamber and the intermediate
chamber, and the second barrier has a second porosity to allow the
outer chamber and the intermediate chamber in fluid communication
and allow at least a second predetermined type of cells to permeate
between the outer chamber and the intermediate chamber.
[0032] As formed, the central chamber is adapted for receiving a
first type of material, the intermediate chamber is adapted for
receiving a second type of material, and the outer chamber is
adapted for receiving a third type of material, wherein each of the
first type of material, the second type of material and the third
type of material contains at least one selected from the group of
cells, chemicals, and fluids. The first predetermined type of cells
includes tumor cells, which normally is received in the central
chamber corresponding to a tumor space. The second predetermined
type of cells includes normal tissue cells, which normally is
received in the intermediate chamber corresponding to a tissue
space, wherein the outer chamber is corresponding to a vascular
space adapted for receiving endothelial cells, macrophage cells,
neutophil cells, any combination of them, or other immune cell
type.
[0033] The bioreactor further has a biocompatible coating layer
applied to the chamber walls at the boundary, wherein the
biocompatible coating layer includes a material that may inhibit
cell adhesion to the biocompatible coating layer, enhance cell
adhesion to the biocompatible coating layer, or function as a
fluorescent marker or indicator of the state of cells.
[0034] At least one additional inlet or outlet port and an input or
output transfer channel are formed in the substrate, wherein the
input or output transfer channel is in fluid communication with the
corresponding inlet or outlet port and the external chamber for
allowing delivery of the cells, fluids or chemicals to the outer
chamber. Additionally, at least one inlet or outlet port and an
input or output transfer channel are formed in the substrate,
wherein the input or output transfer channel is in fluid
communication with the inlet or outlet port and the central chamber
for allowing delivery of the cells, fluids or chemicals to the
central chamber.
[0035] Moreover, at least one another inlet or outlet port and an
input or output transfer channel are formed in the substrate,
wherein the input or output transfer channel is in fluid
communication with the inlet or outlet port and the intermediate
chamber for allowing delivery of the cells, fluids or chemicals to
the intermediate chamber.
[0036] In one embodiment, the substrate is fabricated from glass,
Mylar, PDMS, silicon, a polymer, a semiconductor, or any
combination of them. The first barrier includes a porous material,
wherein the first barrier is microfabricated so as to form a first
structure allowing the fluid communication between the central
chamber and the intermediate chamber. The second barrier includes a
porous material, wherein the second barrier is microfabricated so
as to form a second structure allowing the fluid communication
between the outer chamber and the intermediate chamber, and the
second structure is different from the first structure. In this
embodiment, the first barrier is substantially circular, the second
barrier is substantially circular, and the boundary is
substantially circular. Alternatively, each of the first barrier,
the second barrier, and the boundary can be formed with other
geometric shapes.
[0037] The bioreactor further includes means adapted for
electrochemical measurements of the cells responsive to the liquid
medium in at least one of the outer chamber, the intermediate
chamber and the central chamber, wherein the means for
electrochemical measurements includes a reference electrode, a
counter electrode, and a plurality of individually addressable
working electrodes, wherein the liquid medium includes at least one
or more analytes.
[0038] The plurality of individually addressable working electrodes
include a first group of individually addressable working
electrodes adapted to be capable of sensing the concentration of a
single analyte of the liquid medium at multiple locations in the
outer chamber or the concentrations of a plurality of analytes of
the liquid medium at multiple locations in the outer chamber at a
time period shorter than a characteristic reaction time related to
at least one of cellular physiological activities of the cells. The
first group of individually addressable working electrodes are
further adapted to be capable of measuring the metabolic variables
related to the cells responsive to the liquid medium at multiple
locations in the outer chamber at a time period shorter than a
characteristic reaction time related to at least one of cellular
physiological activities of the cells.
[0039] The plurality of individually addressable working electrodes
further include a second group of individually addressable working
electrodes adapted to be capable of sensing the concentration of a
single analyte of the liquid medium at multiple locations in the
central chamber or the concentrations of a plurality of analytes of
the liquid medium at multiple locations in the central chamber at a
time period shorter than a characteristic reaction time related to
at least one of cellular physiological activities of the cells. The
second group of individually addressable working electrodes are
further adapted to be capable of measuring the metabolic variables
related to the cells responsive to the liquid medium at multiple
locations in the central chamber at a time period shorter than a
characteristic reaction time related to at least one of cellular
physiological activities of the cells.
[0040] The plurality of individually addressable working electrodes
additionally has a third group of individually addressable working
electrodes adapted to be capable of sensing the concentration of a
single analyte of the liquid medium at multiple locations in the
intermediate chamber or the concentrations of a plurality of
analytes of the liquid medium at multiple locations in the
intermediate chamber at a time period shorter than a characteristic
reaction time related to at least one of cellular physiological
activities of the cells. The third group of individually
addressable working electrodes are further adapted to be capable of
measuring the metabolic variables related to the cells responsive
to the liquid medium at multiple locations in the intermediate
chamber at a time period shorter than a characteristic reaction
time related to at least one of cellular physiological activities
of the cells.
[0041] In yet another aspect, the present invention relates to a
bioreactor for cultivating living cells in a liquid medium. In one
embodiment, the bioreactor includes a substrate having a first
surface and an opposite second surface, defining a chamber
therebetween for receiving the cells and the liquid medium with a
boundary, and means for dividing the chamber into plurality of
chambers, wherein each of the plurality of subchambers is in fluid
communication with at least another one of the plurality of
subchambers.
[0042] The dividing means includes a barrier to divide the chamber
into a first subchamber and a second subchamber, and wherein the
barrier has a porosity to allow the first subchamber and the second
subchamber in fluid communication and allow at least one
predetermined type of cells to permeate between the first
subchamber and the second subchamber.
[0043] Alternatively, the dividing means includes a first barrier
and a second barrier to divide the chamber into a first subchamber,
a second subchamber and a third subchamber, and wherein the first
barrier has a first porosity to allow the first subchamber and the
intermediate subchamber in fluid communication and at least a first
predetermined type of cells to permeate between the first
subchamber and the second subchamber, and the second barrier has a
second porosity to allow the second subchamber and the third
subchamber in fluid communication and at least a second
predetermined type of cells to permeate between the second
subchamber and the third subchamber, wherein the first porosity and
the second porosity can be same or different.
[0044] Further alternatively, the dividing means includes a
plurality of n barriers, n being an integer greater than zero, to
divide the chamber into n+1 sub chambers, wherein each of n
barriers has a corresponding porosity that can be same or different
from that of other barriers.
[0045] These and other aspects of the present invention will become
apparent from the following description of the preferred embodiment
taken in conjunction with the following drawings, although
variations and modifications therein may be affected without
departing from the spirit and scope of the novel concepts of the
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1A schematically shows a top view of a bioreactor
according to one embodiment of the present invention.
[0047] FIG. 1B shows a perspective view of a bioreactor according
to another embodiment of the present invention.
[0048] FIG. 2 schematically shows a top view of a bioreactor
according to yet another embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0049] Various embodiments of the invention are now described in
detail. Referring to the drawings, like numbers indicate like parts
throughout the views unless the context clearly dictates otherwise.
As used in the description herein and throughout the claims that
follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise. Also, as
used in the description herein and throughout the claims that
follow, the meaning of "in" includes "in" and "on" unless the
context clearly dictates otherwise. Additionally, some terms used
in this specification are more specifically defined below.
DEFINITIONS
[0050] The terms used in this specification generally have their
ordinary meanings in the art, within the context of the invention,
and in the specific context where each term is used. For example,
conventional techniques of molecular biology, microbiology and
recombinant DNA techniques may be employed in accordance with the
present invention.
[0051] Such techniques and the meanings of terms associated
therewith are explained fully in the literature. See, for example,
Sambrook, Fitsch & Maniatis. Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N. Y. (referred to herein as "Sambrook et al.,
1989"); DNA Cloning: A Practical Approach, Volumes I and IT (D. N.
Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);
Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds.
1984); Animal Cell Culture (R. I. Freshney, ed. 1986); Immobilized
Cells and Enzymes (IRL Press, 1986); B. E. Perbal, A Practical
Guide to Molecular Cloning (1984); F. M. Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
(1994). See also, PCR Protocols: A Guide to Methods and
Applications, Innis et al., eds., Academic Press, Inc., New York
(1990); Saiki et al., Science 1988, 239:487; and PCR Technology:
Principles and Applications for DNA Amplification, H. Erlich, Ed.,
Stockton Press.
[0052] Certain terms that are used to describe the invention are
discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the devices
and methods of the invention and how to make and use them. For
convenience, certain terms are highlighted, for example using
italics and/or quotation marks. The use of highlighting has no
influence on the scope and meaning of a term; the scope and meaning
of a term is the same, in the same context, whether or not it is
highlighted. It will be appreciated that the same thing can be said
in more than one way. Consequently, alternative language and
synonyms may be used for anyone or more of the terms discussed
herein, nor is any special significance to be placed upon whether
or not a term is elaborated or discussed herein. Synonyms for
certain terms are provided. A recital of one or more synonyms does
not exclude the use of other synonyms. The use of examples anywhere
in this specification, including examples of any terms discussed
herein, is illustrative only, and in no way limits the scope and
meaning of the invention or of any exemplified term. Likewise, the
invention is not limited to various embodiments given in this
specification.
[0053] As used herein, "about" or "approximately" shall generally
mean within 20 percent, preferably within 10 percent, and more
preferably within 5 percent of a given value or range. Numerical
quantities given herein are approximate, meaning that the term
"about" or "approximately" can be inferred if not expressly
stated.
[0054] The term "molecule" means any distinct or distinguishable
structural unit of matter comprising one or more atoms, and
includes for example polypeptides and polynucleotides.
[0055] As used herein, "cell" means any cell or cells, as well as
viruses or any other particles having a microscopic size, e.g. a
size that is similar to that of a biological cell, and includes any
prokaryotic or eukaryotic cell, e.g., bacteria, fungi, plant and
animal cells. Cells are typically spherical, but can also be
elongated, flattened, deformable and asymmetrical, i.e.,
non-spherical. The size or diameter of a cell typically ranges from
about 0.1 to 120 microns, and typically is from about 1 to 50
microns. A cell may be living or dead. As used herein, a cell is
generally living unless otherwise indicated. As used herein, a cell
may be charged or uncharged. For example, charged beads may be used
to facilitate flow or detection, or as a reporter. Biological
cells, living or dead, may be charged for example by using a
surfactant, such as SDS (sodium dodecyl sulfate). Cell or a
plurality of cells can also comprise cell lines. Example of cell
lines include liver cell, macrophage cell, neuroblastoma cell,
endothelial cell, intestine cell, hybridoma, CHO, fibroblast cell
lines, red blood cells, electrically excitable cells, e.g. Cardiac
cell, myocytes (AT1 cells), cells grown in co-culture, NG108-15
cells (a widely used neuroblastoma X glioma hybrid cell line,
ATCC#HB-12317), primary neurons, a primary cardiac myocyte isolated
from either the ventricles or atria of an animal neonate, an AT-1
atrial tumor cardiac cell, Liver cells are also known as
Hepatocytes, Secretory cell (depolarize and it secretes things)
pancreatic beta cells secrete insulin, HELA cells (Helen Lane),
HEK293 Human Epithial Kidney c, Erythrocytes (primary red blood
cells), Lymphocytes and the like. Each cell line may include one or
more cells, same or different. For examples, the liver cell
comprises at least one of Human hepatocellular carcinoma ("HEPG2")
cell, CCL-13 cell, and H4IIE cell, the macrophage cells comprises
at least one of peripheral blood mononuclear cells ("PBMC"), and
skin fibroblast cells, the neuroblastoma cell comprises a U937
cell, the endothelial cell comprises a human umbilical
vein-endothelial cell ("Huv-ec-c"), and the intestine cell
comprises a CCL-6 cell.
[0056] "Culture" means a growth of living cells in a controlled
artificial environment. It may be a culture of microorganisms, such
as a bacterial culture, or one of animal or plant cells, such as a
tissue culture. The bioreactors according to the invention can do
both and more. Cultures require appropriate sources of food and
energy, provided by the culture medium, and a suitable physical
environment. Tissue cultures can themselves become a culture medium
for viruses, which grow only with live cells. Cultures of only one
kind of cells are known as pure cultures, as distinguished from
mixed or contaminated cultures.
[0057] "Tissue" means an aggregation of cells more or less similar
morphologically and functionally. The animal body is composed of
four primary tissues, namely, epithelium, connective tissue
(including bone, cartilage, and blood), muscle, and nervous tissue.
The process of differentiation and maturation of tissues is called
histogenesis.
[0058] A "sensor" is broadly defined as any device that can measure
a measurable quantity. For examples, a sensor can be a thermal
detector, an electrical detector, a chemical detector, an optical
detector, an ion detector, a biological detector, a radioisotope
detector, an electrochemical detector, a radiation detector, an
acoustic detector, a magnetic detector, a capacitive detector, a
pressure detector, an ultrasonic detector, an infrared detector, a
microwave motion detector, a radar detector, an electric eye, an
image sensor, any combination of them and the like. A variety of
sensors can be chosen to practice the present invention.
[0059] The term "analyte" means a material that can be consumed or
produced by a cell. Examples of analyte of interest include pH, K,
oxygen, lactate, glucose, ascorbate, serotonin, dopamine, ammonia,
glutamate, purine, calcium, sodium, potassium, NADH, protons,
insulin, NO (nitric oxide) and the like.
[0060] The term "flow" means any movement of fluid such as a liquid
or solid through a device or in a method of the invention, and
encompasses without limitation any fluid stream, and any material
moving with, within or against the stream, whether or not the
material is carried by the stream. For example, the movement of
molecules or cells through a device or in a method of the
invention, e.g. through channels of a substrate on microfluidic
chip of the invention, comprises a flow. This is so, according to
the invention, whether or not the molecules or cells are carried by
a stream of fluid also comprising a flow, or whether the molecules
or cells are caused to move by some other direct or indirect force
or motivation, and whether or not the nature of any motivating
force is known or understood. The application of any force may be
used to provide a flow, including without limitation, pressure,
capillary action, electroosmosis, electrophoresis,
dielectrophoresis, optical tweezers, and combinations thereof,
without regard for any particular theory or mechanism of action, so
long as molecules or cells are directed for detection, measurement
or sorting according to the invention.
[0061] A "liquid or medium" is a fluid that may contain one or more
substances that affecting growth of cells, one or more analytes, or
any combination of them. A medium can be provided with one or more
analytes to be consumed by one or more cells. A medium can have one
or more analytes generated by one or more cells. A medium can also
have at the same time one or more analytes to be consumed by one or
more cells and one or more analytes generated by one or more cells.
A medium may consist of natural materials, such as enzymatic
digests, extracts of yeast or beef, milk, potato slices, or chick
embryos. Artificial media are prepared by mixing various
ingredients according to particular formulas. A complex medium
contains at least one crude ingredient derived from a natural
material, hence of unknown chemical composition. A chemically
defined or synthetic medium is one in which the chemical structure
and amount of each component are known.
[0062] An "inlet region" is an area of a bioreactor that receives
molecules or cells or liquid. The inlet region may contain an inlet
port and channel, a well or reservoir, an opening, and other
features which facilitate the entry of molecules or cells into the
device. A bioreactor may contain more than one inlet region if
desired. The inlet region is in fluid communication with the
channel and is upstream therefrom.
[0063] An "outlet region" is an area of a bioreactor that collects
or dispenses molecules or cells or liquid. An outlet region is
downstream from a discrimination region, and may contain outlet
channels or ports. A bioreactor may contain more than one outlet
region if desired.
[0064] An "analysis unit" is a microfabricated substrate, e.g., a
microfabricated chip, having at least one inlet region, at least
one channel and chamber, at least one detection region and at least
one outlet region. A device of the invention may comprise a
plurality of analysis units.
[0065] A "channel" is a pathway of a bioreactor of the invention
that permits the flow of molecules or cells to pass a detection
region for detection (identification), or measurement. The
detection and discrimination regions can be placed or fabricated
into the channel. The channel is typically in fluid communication
with an inlet port or inlet region, which permits the flow of
molecules or cells or liquids into the channel. The channel is also
typically in fluid communication with an outlet region or outlet
port, which permits the flow of molecules or cells or liquid out of
the channel. The channel can also be used as a chamber to grown
cells, and vice versa.
[0066] A "detection region" or "sensing volume" or "chamber" is a
location within the bioreactor, typically in or coincident with the
channel (or a portion thereof) and/or in or coincident with a
detection loop, where molecules or cells to be grown, identified,
characterized, hybridized, measured, analyzed or maintained (etc.),
are examined on the basis of a predetermined characteristic. ill
one embodiment, molecules or cells are examined one at a time. ill
other embodiments, molecules, cells or samples are examined
together, for example in groups, in arrays, in rapid, simultaneous
or contemporaneous serial or parallel arrangements, or by affinity
chromatography.
[0067] "Reaction time" is the time that a system of interest
requires to respond to a change. For example, the reaction time of
a cell is the time required for at least one of the physiological
processes of a cell to adapt or respond to a change in its
environment. Each type of cell has its own characteristic reaction
time with respect to a particular change in its environment. The
reaction time of a sensor is the time required for the sensor to
respond to a change in the quantity that it is sensing. For
example, the reaction time of an electrochemical sensor is set by
the size of the sensor and the thickness and nature of protective
coatings on the activated surfaces of the sensor. The reaction time
of a microfluidic system is determined by, among other things, the
reaction time of the cell to changes in the environment, the time
required for chemical species to diffuse throughout the sensing
volume, the reaction time of the sensor(s) and the diffusion time
of the analyte being controlled by the actuators.
[0068] "Bacteria" are extremely small--usually 0.3-2.0 micrometers
in diameter--and relatively simple microorganisms possessing the
prokaryotic type of cell construction. Each bacterial cell arises
either by division of a preexisting cell with similar
characteristics, or through combination of elements from two such
cells in a sexual process.
[0069] "Protozoa" means a group of eukaryotic microorganisms
traditionally classified in the animal kingdom. Although the name
signifies primitive animals, some Protozoa (phytoflagellates and
slime molds) show enough plantlike characteristics to justify
claims that they are plants. Protozoa range in size from 1 to 106
micrometers. Colonies are known in flagellates, ciliates, and
Sarcodina. Although marked differentiation of the reproductive and
somatic zooids characterizes certain colonies, such as Volvox,
Protozoa have not developed tissues and organs.
[0070] Several embodiments are now described with reference to the
FIGS. 1-2, in which like numbers indicate like parts throughout the
FIGS. 1-2.
Overview of the Invention
[0071] The inventors of the present invention overcome the
disadvantages of the prior art and develop new bioreactors that
have, among other new and inventive features, the capability of
providing controlled chemokine gradients independent of the
perfusion flow and allow extravasation of a cellular matrix. Recent
advances in the fabrication ofnanofilters.sup.57-61 are used to
create perfused-membrane bioreactors according to the present
invention that allow the growth of mixed cultures of cells at
near-to-tissue densities in 1 mm.times.1 mm.times.100 micron
volumes, in the presence of controlled, stable chemokine or
growth-factor gradients within the device, to mimic the in vivo
tumor microenvironment.
[0072] One advantage of the present invention is that custom
devices can be constructed such that the isolated perfusion and
cell-delivery systems allow independent control of shear stress and
chemokine gradients during the course of an experiment. Moreover,
the optical and electrochemical metabolic microsensors can be
installed within these bioreactors to allow simultaneous
quantification of the local metabolic and chemical environment
(lactate, pH, O.sub.2, etc.) in selected regions within the
reactor, while cell migration or cell signaling events are imaged
by fluorescence microscopy. Hence, the bioreactors according to the
present invention can be considered as the next generation of
migration bioreactors that may move beyond a simple MicroTransWell
(MTW) system to one that more closely replicates in vitro the
microenvironment living tissue.
[0073] Moreover, the application of microfabrication techniques,
microfluidics, and microbiosensors with the bioreactors according
to the present invention offers an opportunity for study of the
molecular mechanism of tumor angiogenesis as well as leukocyte and
cancer cell extravasation. For example, the systematic examination
of the role of Tie2 and VEGF in vascular formation and remodeling
and may identify more specific molecular targets for
anti-angiogenic therapy. A similar microdevice model could be used
to examine leukocyte and cancer cell extravasation. These devices
will provide an appropriate cellular environment to host mouse
tumor explants, thereby potentially providing a metastasis assay
for tumor biopsy material. Metabolic sensing in these bioreactors
will help provide a clearer understanding of the tumor
microenvironment and confirm the validity of our in vitro
systems.sup.62-65.
[0074] Additionally, the limitation of the planar Borenstein design
that there is too little surface area of capillaries available to
support the growth of a substantial volume of cells is overcome by
the present invention, which remedies this problem by creating a
multi-layer intercalated supply and return bioreactor that allows
the full surface of a planar bioreactor to be covered with
capillaries, and hence capillary-perfused cells. More specifically,
in one aspect, the present invention relates to bioreactors.
[0075] These bioreactors are biomicroelectromechanical systems
(BioMEMS) that serve as migration microenvironments to study
molecular mechanisms of tumor angiogenesis, tumor metastasis and
leukocyte migration, but can also function as more general tissue
bioreactors and perfusion systems. Among other things, one unique
aspect of these microfluidic devices is their integration of
suitable cell culture and microfabrication techniques, which permit
cell growth in small, confined, well-perfused volumes at tissue
densities, provide independent control of multiple chemokines and
growth factor gradients, shear forces, tissue perfusion, and
permeability of physical barriers to cellular migration, and allow
detailed optical and electrochemical observation of normal and
cancerous cells during cell migration, intravasation,
extravasation, angiogenesis, and other cellular processes.
[0076] Recent advances in the fabrication of nanofilters.sup.57-61
can be used to practice the present invention to provide
perfused-membrane bioreactors that can allow the growth of mixed
cultures of cells at near-to-tissue densities in 1 mm.times.1
mm.times.100 micron volumes, in the presence of controlled, stable
chemokine or growth-factor gradients within the device, to mimic
the in vivo tumor microenvironment. One advantage of the present
invention is that custom devices can be constructed such that the
isolated perfusion and cell-delivery systems allow independent
control of shear stress and chemokine gradients during the course
of an experiment. Moreover, the optical and electrochemical
metabolic microsensors can be installed within these bioreactors to
allow simultaneous quantification of the local metabolic and
chemical environment (lactate, pH, O.sub.2, etc.) in selected
regions within the reactor, while cell migration or cell signaling
events are imaged by fluorescence microscopy. Hence the next
generation of migration bioreactors will eventually move beyond a
simple MicroTransWell (MTW) system to one that more closely
replicates in vitro the microenvironment living tissue.
[0077] The application of microfabrication techniques,
microfluidics, and microbiosensors offers an opportunity for study
of the molecular mechanism of tumor angiogenesis as well as
leukocyte and cancer cell extravasation. For example, the
systematic examination of the role of Tie2 and VEGF in vascular
formation and remodeling and may identify more specific molecular
targets for anti-angiogenic therapy. A similar microdevice model
could be used to examine leukocyte and cancer cell extravasation.
These bioreactors will provide an appropriate cellular environment
to host mouse tumor explants, thereby potentially providing a
metastasis assay for tumor biopsy material. Metabolic sensing in
these bioreactors will help provide a clearer understanding of the
tumor microenvironment and confirm the validity of our in vitro
systems.sup.62-65.
[0078] Without intent to limit the scope of the invention,
exemplary devices, application of them and related observations
according to the embodiments of the present invention are given
below. Note that titles or subtitles may be used in the examples
for convenience of a reader, which in no way should limit the scope
of the invention. Moreover, certain theories may have been proposed
and disclosed herein; however, in no way they, whether they are
right or wrong, should limit the scope of the invention so long as
the devices and applications of them are practiced according to the
invention without regard for any particular theory or scheme of
action.
EXAMPLES
Bioreactor with One Barrier
[0079] Referring now to FIGS. 1A and 1B, the present invention can
be practiced in association with an inventive bioreactor 100 as
shown in FIGS. 1A and 1B. In one embodiment, the bioreactor 100
includes a first substrate 140 having a first surface 140a and an
opposite second surface 104b, defining a chamber 101 therebetween
for receiving cells and a liquid medium. The bioreactor 100 has a
barrier 104 dividing the chamber 101 into a first subchamber 102
and a second subchamber 103, wherein the barrier 104 has a porosity
to allow the first subchamber 102 and the second subchamber 103 in
fluid communication and allow at least one predetermined type of
cells to permeate between the first subchamber 102 and the second
subchamber 103. The porosity of the barrier 104 can also be chosen
not to let any cells to permeate.
[0080] As formed, the first subchamber 102 is adapted for receiving
a first type of material such as cells 113 and the second
subchamber 103 is adapted for receiving a second type of material
such as cells 114, wherein each of the first type of material and
the second type of material contains at least one selected from the
group of cells, chemicals, and fluids. The cells can be any type of
living cells, including, but not limited to, bacteria, protozoa, or
both, normal cells, tumor cells, or any combination of them.
[0081] A biocompatible coating layer 116 can be applied to the
chamber walls of the bioreactor 100, wherein the biocompatible
coating layer 116 includes a material that may inhibit cell
adhesion to the biocompatible coating layer, enhance cell adhesion
to the biocompatible coating layer, or function as a fluorescent
marker or indicator of the state of cells.
[0082] The bioreactor 100 further includes at least one or more
inlet ports 105, 106 and one or more corresponding input transfer
channel 112, 107. As formed, the input transfer channel 112 is in
fluid communication with the corresponding inlet port 105 and the
first subchamber 102, and the input transfer channel 107 is in
fluid communication with the corresponding inlet port 106 and the
second subchamber 103 for allowing delivery of the cells, fluids or
chemicals to the corresponding subchamber 102 or 103, respectively.
For example, a fluid can be introduced from an external device (not
shown) into the first subchamber 102 through the inlet port 105 and
the corresponding input transfer channel 112. Inlet ports 105, 106
each can be in fluid communication with an external device or port
(not shown).
[0083] The bioreactor 100 additionally includes at least one or
more outlet ports 111, 109 and one or more corresponding outlet
transfer channel 110, 108. As formed, the outlet transfer channel
110 is in fluid communication with the corresponding outlet port
111 and the first subchamber 102, and the outlet transfer channel
108 is in fluid communication with the corresponding outlet port
109 and second subchamber 103 for allowing removal of the cells,
fluids or chemicals from the corresponding subchamber 102 or 103,
respectively. For example, a fluid can be introduced away from the
first subchamber 102 through the outlet transfer channel 110 and
the corresponding outlet port 111. Outlet ports 111, 109 each can
be in fluid communication with an external device or port (not
shown).
[0084] The bioreactor 100 further includes at least one or more
auxiliary ports 115 and one or more auxiliary channels 115a. As
formed, each auxiliary channel 115a is in fluid communication with
a corresponding auxiliary port 115 and a corresponding one of the
input transfer channels 112, 107 and the outlet transfer channels
110, 108 for flushing the corresponding transfer channel. Auxiliary
ports 115 each can be in fluid communication with an external
device or port (not shown). Auxiliary ports 115 and auxiliary
channels 115a can be utilized to prevent clogging by cells or
cellular debris in the bioreactor 100. They can also be utilized to
selectively apply coatings to the channels to which they are in
fluid communication.
[0085] The bioreactor 100 additionally includes one or more access
ports 117 and one or more access channels 117a. As formed, each
access channel 117a is in fluid communication with a corresponding
access port 117 and a corresponding one of the first subchamber 102
and the second subchamber 103 for allowing delivery or removal of
the cells, fluids, chemicals, coating material or sensing material
to the corresponding subchamber. The access ports 117 and
corresponding access channels 117a are strategically positioned so
as to provide direct access to the first subchamber 102 and the
second subchamber 103. For example, a fluid can be introduced into
the first subchamber 102 through an access channels 117a and the
corresponding access port 117 fast because the distance between the
access port 117 and the first subchamber 102 is the shortest for
this embodiment. Each access port 117 can be in fluid communication
with an external device or port (not shown).
[0086] Moreover, the bioreactor 100 has a second substrate 150,
wherein the second substrate 150 is positioned adjacent to the
first surface 140a of the first substrate 140 and defines a
plurality of connection channels 155. Each of the connection
channels 155 is formed so as to be in fluid communication with a
corresponding one of the inlet ports 105, 106, the outlet ports
111, 109, the auxiliary ports 115, and the access ports 117 as set
forth above.
[0087] The bioreactor 100 further includes a plurality of
connection ports 151 corresponding to the plurality of connection
channels 155. Each of the connection ports 151 is formed with a
channel 153 and is strategically positioned to the second substrate
150 such that each channel 153 of the connection ports 151 is in
fluid communication with a corresponding one of the connection
channels 155 formed in the second substrate 150 as shown in FIG.
1B.
[0088] The first substrate 140 can be fabricated from glass, Mylar,
PDMS, silicon, a polymer, a semiconductor, or any combination of
them. The barrier 104 is formed with a porous material. The barrier
104 can be microfabricated so as to form a structure allowing the
fluid communication between the first subchamber 102 and the second
subchamber 103, which may allow permeation of the barrier 104 by
certain predetermined types of cells but not by other types of
cells. For example, in the embodiment shown in FIGS. 1A and 1B, the
barrier 104 is formed with a plurality of posts spaced from each
other so as to allow bacteria to cross over but not protozoa.
[0089] The bioreactor 100 further has a third substrate 160, which
is positioned adjacent to the first surface of the first substrate
140, and means strategically positioned in the third substrate 160
and adapted for electrochemical measurements of the cells
responsive to the liquid medium in one or both of the first
subchamber 102 and the second subchamber 103. The third substrate
160 can be formed with a semiconductor material such as
silicon.
[0090] In one embodiment as shown in FIG. 1B, the means for
electrochemical measurements includes a reference electrode 161, a
counter electrode 162, a plurality of edge connector pads 164, and
a plurality of electrically conductive leads 163. A first
electrically conductive lead 163 electrically couples the reference
electrode 161 to a corresponding edge connector pad 164, and a
second electrically conductive lead 163 electrically couples the
counter electrode 162 to a corresponding edge connector pad 164.
The means for electrochemical measurements further includes a
plurality of individually addressable working electrodes 165. Each
of the plurality of individually addressable working electrodes 165
is electrically coupled to a corresponding edge connector pad 164
through a corresponding electrically conductive lead 163. The
sensing heads of the plurality of individually addressable working
electrodes 165 are strategically positioned in a region shown by
outline 166 in FIG. 1B.
[0091] In operation, the liquid medium being introduced into one or
both of the first subchamber 102 and the second subchamber 103 may
include one or more analytes, and the plurality of individually
addressable working electrodes are adapted for sensing the
concentration of a single analyte of the liquid medium at multiple
locations in the chamber 101 or the concentrations of a plurality
of analytes of the liquid medium at multiple locations in the
chamber 101 at a time period shorter than a characteristic reaction
time related to at least one of cellular physiological activities
of the cells. The plurality of individually address able working
electrode scan be further adapted to be capable of measuring the
metabolic variables related to the cells responsive to the liquid
medium at multiple locations in the chamber 101 at a time period
shorter than a characteristic reaction time related to at least one
of cellular physiological activities of the cells. The sensing
heads of the plurality of individually addressable working
electrodes 165 are strategically positioned in a region shown by
outline 166 corresponding to that of the chamber 101 to perform
such tasks.
[0092] The bioreactor 100 further includes a fourth substrate 170,
wherein the fourth substrate 170 is positioned above the second
surface 140b of the first substrate 140, and means strategically
positioned in the fourth substrate 170 and adapted for optical
measurements of the cells responsive to the liquid medium in at
least one of the first subchamber 102 and the second subchamber
103. The fourth substrate 170 is at least partially transparent.
For examples, it can be formed with a semiconductor material or a
glass or both.
[0093] In one embodiment as shown in FIG. 1B, the means for optical
measurements includes a plurality of optical sensors 171, a
plurality of edge connector pads 173, and a plurality of leads 172,
each coupling an optical sensor 171 to a corresponding edge
connector pad 173. The plurality of optical sensors 171 may include
at least one device selected from the group of an LED and photo
diode pair, a fiberoptic coupler, and an optical detecting
head.
[0094] In operation, the liquid medium being introduced into one or
both of the first subchamber 102 and the second subchamber 103 may
include one or more analytes, and the plurality of optical sensors
171 are adapted to be capable of sensing the concentration of a
single analyte of the liquid medium at multiple locations in the
chamber 101 or the concentrations of a plurality of analytes of the
liquid medium at multiple locations in the chamber 101 at a time
period shorter than a characteristic reaction time related to at
least one of cellular physiological activities of the cells. The
plurality of optical sensors 171 can be further adapted for
measuring the metabolic variables related to the cells responsive
to the liquid medium at multiple locations in the chamber 101 at a
time period shorter than the characteristic reaction time related
to at least one of cellular physiological activities of the cells.
The sensing heads of the plurality of optical sensors 171 are
strategically positioned in a region shown by outline 174
corresponding to that of the chamber 101 to perform such tasks.
Bioreactor with Multiple Barriers
[0095] Referring now to FIG. 2, the present invention can also be
practiced in association with an inventive bioreactor 700 as shown
in FIG. 2. In one embodiment, the bioreactor 700 includes a
substrate 730 having a first surface and an opposite second
surface, defining a chamber 732 therebetween for receiving cells
and a liquid medium, wherein the chamber 732 is formed with a
center 734 and a boundary 736. The bioreactor 700 also has a first
barrier 738, which encloses the center 734 and a portion of the
chamber 732 to form a central chamber 706, and a second barrier
740, which is positioned between the first barrier 738 and the
boundary 736 so as to form an intermediate chamber 705 and an outer
chamber 704.
[0096] In one embodiment, the first barrier 738 has a first
porosity to allow the central chamber 706 and the intermediate
chamber 705 in fluid communication and allow at least a first
predetermined type of cells to permeate between the central chamber
706 and the intermediate chamber 705, and the second barrier 740
has a second porosity to allow the outer chamber 704 and the
intermediate chamber 705 in fluid communication and allow at least
a second predetermined type of cells to permeate between the outer:
chamber 704 and the intermediate chamber 705.
[0097] Moreover, the central chamber 706 is adapted for receiving a
first type of material such as tumor cells 714, the intermediate
chamber 705 is adapted for receiving a second type of material such
as normal tissue cells 713, and the outer chamber 704 is adapted
for receiving a third type of material such as endothelial cells
712. Each of the first type of material, the second type of
material and the third type of material contains at least one
selected from the group of cells, chemicals, and fluids.
[0098] The first predetermined type of cells includes tumor cells
714, which normally is received in the central chamber 706 that is
formed to simulate a tumor space. The second predetermined type of
cells includes normal tissue cells 713, which normally is received
in the intermediate chamber 705 that is formed to simulate a tissue
space. Furthermore, the outer chamber 704 is formed to simulate a
vascular space adapted for receiving endothelial cells, macrophage
cells, neutophil cells, any combination of them, or other immune
cell type.
[0099] A biocompatible coating layer 742 can be applied to the
chamber walls at the boundary 736, wherein the biocompatible
coating layer 742 includes a material that may inhibit cell
adhesion to the biocompatible coating layer, enhance cell adhesion
to the biocompatible coating layer, or function as a fluorescent
marker or indicator of the state of cells.
[0100] The bioreactor 700 further includes one or more inlet or
outlet ports 701 and corresponding one or more input or output
transfer channels 751, where each of the input or output transfer
channel 751 is in fluid communication with a corresponding inlet or
outlet port 701 and the outer chamber 704 for allowing delivery of
cells, fluids or chemicals to the outer chamber 704.
[0101] The bioreactor 700 additionally may include one or more
inlet or outlet ports 702 and corresponding one or more input or
output transfer channels 752, where each of the input or output
transfer channels 752 is in fluid communication with a
corresponding inlet or outlet port 702 and the central chamber 706
for allowing delivery of the cells, fluids or chemicals to the
central chamber 706.
[0102] The bioreactor 700 may further include one or more inlet or
outlet ports 703 and corresponding one or more input or output
transfer channels 753, where each of the input or output transfer
channels 753 is in fluid communication with a corresponding inlet
or outlet port 703 and the intermediate chamber 705 for allowing
delivery of the cells, fluids or chemicals to the intermediate
chamber 705.
[0103] The substrate 730 can be fabricated from glass, Mylar, PDMS,
silicon, a polymer, a semiconductor, or any combination of them.
The first barrier 738 is formed with a porous material. The first
barrier 738 can be microfabricated so as to form a first structure
allowing the fluid communication between the central chamber 706
and the intermediate chamber 705. The second barrier 740 is formed
with a porous material. The second barrier 740 can be
microfabricated so as to form a second structure allowing the fluid
communication between the outer chamber 704 and the intermediate
chamber 705. The first barrier 738 and the second barrier 740 can
be formed with same or different porous materials. And the second
structure can be same or different from the first structure. For
example, in the embodiment shown in FIG. 2, the first barrier 738
is formed with a plurality of posts spaced from each other more
condensed than the second barrier 740. The first barrier 738 and
the second barrier 740 can also be formed into same or different
shapes. For example, in the embodiment shown in FIG. 2, the first
barrier 738 and the second barrier 740 are substantially circular.
The boundary 736 can take various geometric shapes as well. For
example, in the embodiment shown in FIG. 2, the boundary 736 is
substantially circular.
[0104] The bioreactor 700 further includes means strategically
positioned and adapted for electrochemical measurements of the
cells responsive to the liquid medium in one or more of the outer
chamber 704, the intermediate chamber 705 and the central chamber
706.
[0105] In one embodiment as shown in FIG. 2, the means for
electrochemical measurements includes a reference electrode 707, a
counter electrode 708, and a 20 plurality of individually
addressable working electrodes.
[0106] In operation, the liquid medium being introduced into one or
more of the outer chamber 704, the intermediate chamber 705 and the
central chamber 706 may include one or more analytes, and the
plurality of individually addressable working electrodes include a
first group of individually addressable working electrodes 709, a
second group of individually addressable working electrodes 710 and
a third group of individually addressable working electrodes 711,
respectively.
[0107] For the embodiment shown in FIG. 2, the first group of
individually addressable working electrodes 709 are adapted to be
capable of sensing the concentration of a single analyte of the
liquid medium at multiple locations in the outer chamber 704 or the
concentrations of a plurality of analytes of the liquid medium at
multiple locations in the outer chamber 704 at a time period
shorter than a characteristic reaction time related to at least one
of cellular physiological activities of the cells. The first group
of individually addressable working electrodes 709 are further
adapted to be capable of measuring the metabolic variables related
to the cells responsive to the liquid medium at multiple locations
in the outer chamber 704 at a time period shorter than a
characteristic reaction time related to at least one of cellular
physiological activities of the cells.
[0108] The second group of individually addressable working
electrodes 710 adapted to be capable of sensing the concentration
of a single analyte of the liquid medium at multiple locations in
the central chamber 706 or the concentrations of a plurality of
analytes of the liquid medium at multiple locations in the central
chamber 706 at a time period shorter than a characteristic reaction
time related to at least one of cellular physiological activities
of the cells. The second group of individually addressable working
electrodes 710 are further adapted to be capable of measuring the
metabolic variables related to the cells responsive to the liquid
medium at multiple locations in the central chamber 706 at a time
period shorter than a characteristic reaction time related to at
least one of cellular physiological activities of the cells.
[0109] Similarly, the third group of individually addressable
working electrodes 711 are adapted to be capable of sensing the
concentration of a single analyte of the liquid medium at multiple
locations in the intermediate chamber 705 or the concentrations of
a plurality of analytes of the liquid medium at multiple locations
in the intermediate chamber 705 at a time period shorter than a
characteristic reaction time related to at least one of cellular
physiological activities of the cells. The third group of
individually addressable working electrodes 711 are further adapted
to be capable of measuring the metabolic variables related to the
cells responsive to the liquid medium at multiple locations in the
intermediate chamber 705 at a time period shorter than a
characteristic reaction time related to at least one of cellular
physiological activities of the cells.
[0110] As such formed, among other things, bioreactor 700 can be
utilized to nurture, culture, observe, detect and explore cells,
collection of cells, biofilm formed by cells and related cell
activities. For examples, as shown in FIG. 2, bioreactor 700 allows
a spectrum of cell activities to take place, including: a cell 715,
which can be an immune type of cell such as a macrophage or
neutophil, undergoing extravasation across the second barrier 740
from the outer chamber 704 into the intermediate chamber 705, a
cell 716, which can be a tumor cell metastasizing from the central
chamber 706 through the surrounding tissue into the vascular space,
undergoing intravasation across the second barrier 740 from the
intermediate chamber 705 into the outer chamber 704, and a cell
717, for example, an endothelial cell, undergoing tube formation
across the second barrier 740 that may eventually lead to
vascularization of the tumor, respectively.
[0111] While there has been shown various embodiments of the
present invention, it is to be understood that certain changes can
be made in the form and arrangement of the elements of the
apparatus and steps of the methods to practice the present
invention as would be known to one skilled in the art without
departing from the underlying scope of the invention as is
particularly set forth in the Claims. Furthermore, the embodiments
described above are only intended to illustrate the principles of
the present invention and are not intended to limit the claims to
the disclosed elements. Indeed, since many embodiments of the
invention can be made without departing from the spirit and scope
of the invention, the invention resides in the claims hereinafter
appended.
LIST OF REFERENCES
[0112] 1. Godbey, W. T. and Atala, A., In Vitro Systems for Tissue
Engineering, Ann. 10 N. Y., Acad. Sci., 961, 10-26, 2002. [0113] 2.
Murdin, A. D., Thorpe, J. S., Kirkby, N., Groves, D. J., Spier, R.
E., Immobilisation and Growth of Hybridomas in Packed Beds, In:
Bioreactors and biotransformations, Moody, G. W. and Baker, P. R,
eds. Elsevier Applied Science Publishers, London, N.Y., 99-110,
1987. [0114] 3. De Bartolo, L., Jarosch-Von Schweder, G., Haverich,
A., Bader, A., A Novel Full-Scale Flat Membrane Bioreactor
Utilizing Porcine Hepatocytes: Cell Viability and Tissue-Specific
Functions, Biotechnol. Prog., 16, 102-108, 2000. [0115] 4.
McDuffie, N. G., Cell Culture Bioreactors. In: Bioreactor Design 20
Fundamentals, Butterworth-Heinemann, Boston, 93-119, 1991. [0116]
5. Drioli, E, et al., Biocatalytic Membrane Reactors, Applications
in Biotechnology and the Pharmaceutical Industry, Taylor &
Francis, London, Philadelphia, 1999. [0117] 6. Labecki, M., Bowen,
B. D., Piret, J. M., Protein Transport in Ultrafiltration
Hollow-Fiber Bioreactors for Mammalian Cell Culture, In: Membrane
Separations in Biotechnology, Wang, W. K., ed., M. Dekker, New
York, 1-62, 2001. [0118] 7. Nollert, M. U., Diamond, S. L.,
McIntire, L. V., Hydrodynamic Shear-Stress and Mass-Transport
Modulation of Endothelial-Cell Metabolism, Biotechnol. Bioeng., 38,
588-602, 1991. [0119] 8. Augenstein, D. C., Sinskey, A. l, Wang, D.
I. C., Effect of Shear on Death of Two Strains of Mammalian Tissue
Cells, Biotechnol. Bioeng., 13,409-418, 1971. [0120] 9. Millward,
H. R., Bellhouse, B. l, Sobey, I. J., The Vortex Wave Membrane
Bioreactor: Hydrodynamics and Mass Transfer, Chemical Engineering
Journal and the Biochemical Engineering Journal, 62, 175-181, 1996.
[0121] 10. Beeton, S., Bellhouse, B. J., Knowles, C. J., Millward,
H. R., Nicholson, A. M., Wyatt, J. R., A Novel Membrane Bioreactor
for Microbial-Growth, Appl. Microbiol. Biotechnol., 40, 812-817,
1994. [0122] 11. Hu, W. S, and Aunins, J. G., Large-Scale Mammalian
Cell Culture, Curr. Opin. Biotechnol., 8, 148-153, 1997. [0123] 12.
Tobert, W. R., Lewis, C. Jr., White, P. l, Feder, l, Perfusion
Culture Systems for Production of Mammalian Cell Biomolecules, In:
Large-Scale Mammalian cell culture, Feder, J. and Tolbert, W. R.,
eds., Academic Press, Orlando, 97-123, 1985. [0124] 13. Voisard,
D., Meuwly, F., Ruffieux, P. A., Baer, G., Kadouri, A., Potential
of Cell Retention Techniques for Large-Scale High-Density Perfusion
Culture of Suspended Mammalian Cells, Biotechnol. Bioeng., 82,
751-765, 2003. [0125] 14. MacNeill, B. D., Pomerantseva, I., Lowe,
H. C., Oesterle, S. N., Vacanti, J. P., Toward a New Blood Vessel,
Vase. Med., 7, 241-246, 2002. [0126] 15. Wu, R. K, Odom, T. W.,
Chiu, D. T., Whitesides, G. M., Fabrication of Complex
Three-Dimensional Microchannel Systems in PDMS, l Am. Chem. Soc.,
125, 554-559, 2003. [0127] 16. Griffith, L. G., Emerging Design
Principles in Biomaterials and Scaffolds for Tissue Engineering,
Reparative Medicine: Growing Tissues and Organs, 961, 83-95, 2002.
[0128] 17. Snyder, J. D. and Desai, T. A., Fabrication of Multiple
Microscale Features on Polymer Surfaces for Applications in Tissue
Engineering, Biomedical Microdevices, 3, 293-300, 2001. [0129] 18.
Solan, A, Prabhakar, V., Niklason, L., Engineered Vessels:
Importance of the Extracellular Matrix, Transplant. Proc., 33,
66-68, 2001. [0130] 19. Griffith, L. G. and Naughton, G., Tissue
Engineering-Current Challenges and Expanding Opportunities,
Science, 295, 1009-2002. [0131] 20. Powers, M. l, Domansky, K,
Kaazempur-Mofrad, M. R, Kalezi, A., Capitano, A., Upadhyaya, A,
Kurzawski, P., Wack, K E., Stolz, D. B., Karnm, R, Griffith, L. G.,
A Microfabricated Array Bioreactor for Perfused 3D Liver Culture,
Biotechnol. Bioeng., 78, 257-269, 2002. [0132] 21. Park, T. R. and
Shuler, M. L, Integration of Cell Culture and Microfabrication
Technology, Biotechnol. Prog., 19,243-253, 2003. [0133] 22.
Borenstein, I T., Terai, R., King, K R, Weinberg, E. J.,
Kaazempur-Mofrad, M. R, Vacanti, J. P., Microfabrication Technology
for Vascularized Tissue Engineering, Biomedical Microdevices, 4,
167-175, 2002. [0134] 23. Kaihara, S., Borenstein, J., Koka, R.,
Lalan, S., Ochoa, E. R, Ravens, M., Pien, R., Cunningham, B.,
Vacanti, J. P., Silicon Micromachining to Tissue Engineer Branched
Vascular Channels for Liver Fabrication, Tissue Eng., 6, 105-117,
2000. [0135] 24. Allen, J. W. and Bhatia, S. N., Improving the Next
Generation of Bioartificial Liver Devices, Seminars in Cell &
Developmental Biology, 13, 447-454, 2002. [0136] 25. Passeraub, P.
A, Almeida, A C., Thakor, N. V., Design, Microfabrication and
Analysis of a Microfluidic Chamber for the Perfusion of Brain
Tissue Slices, Biomedical Microdevices, 5, 147-155, 2003. [0137]
26. Fink, C., Ergun, S., Kralisch, D., Remmers, U., Weil, J.,
Eschenhagen, T., Chronic Stretch of Engineered Heart Tissue fuduces
Hypertrophy and Functional Improvement, FASEB J., 14, 669-679,
2000. [0138] 27. Mooney, D. T., Mazzoni, C. L., Breuer, C.,
McNamara, K., Hem, D., Vacanti,. J. P., Langer, R, Stabilized
Polyglycolic Acid Fibre Based Tubes for Tissue Engineering,
Biomaterials, 17, 115-124, 1996. [0139] 28. Boyden, S., The
Chemotactic Effect of Mixtures of Antibody and Antigen on
Polymorphonuclear Leucocytes, J. Exp. Med., 115,453-466, 1962.
[0140] 29. Harvath, L., Falk, W., Leonard, E. J., Rapid
Quantitation of Neutrophil Chemotaxis--Use of A
Polyvinylpyrrolidone--Free Polycarbonate Membrane in A Multiwell
Assembly, J. Immunol. Methods, 37, 39-45, 1980. [0141] 30. Falk,
W., Goodwin, R H., Leonard, E. J., A 48-Well Micro Chemotaxis
Assembly for Rapid and Accurate Measurement of Leukocyte Migration,
J. Immunol. Methods, 33, 239-247, 1980. [0142] 31. Yao, J.,
Harvath, L., Gilbert, D. L., Colton, C. A., Chemotaxis by A Cns
Macrophage, the Microglia, J. Neurosci. Res., 27, 36-42, 1990.
[0143] 32. Roth, S J., Can, M. W., Rose, S. S., Springer, T. A.,
Characterization of Transendothelial Chemotaxis of T Lymphocytes,
J. Immunol. Methods, 188, 97-116, 1995. [0144] 33. Klemke, R L.,
Leng, J., Molander, R, Brooks, P. C., Vuori, K., Cheresh, D. A.,
CAS/Crk Coupling Serves As a "Molecular Switch" for Induction of
Cell Migration, Journal of Cell Biology, 140,961-972, 1998. [0145]
34. Ding, Z., Xiong, K, Issekutz, T. B., Chemokines Stimulate Human
T Lymphocyte Transendothelial Migration to Utilize VLA-4 in
Addition to LFA-1, J. Leukoc. Biol., 69, 458-466, 2001. [0146] 35.
Jones, D. A., Abbassi, O., McIntire, L. V., McEver, R P., Smith, C.
W., P-Selectin Mediates Neutrophil Rolling on Histamine-Stimulated
Endothelial Cells, Biophys. J., 65, 1560-1569, 1993. [0147] 36.
Brown, D. and Larson, R., Improvements to Parallel Plate Flow
Chambers to Reduce Reagent and Cellular Requirements, BMC
Immunology, 2, 9-16, 2001. [0148] 37. Cinamon, G. and Alon, R, A
Real Time in Vitro Assay for Studying Leukocyte Transendothelial
Migration Under Physiological Flow Conditions, J. Immunol. Methods,
273, 53-62, 2003. [0149] 38. Renard, M., Heutte, F.,
Boutherin-Falson, O., Finet, M., Boisseau, M. R., Induced Changes
of Leukocyte Slow Rolling in an in Flow Pharmacological Model of
Adhesion to Endothelial Cells, Biorheology, 40, 173-178, 2003.
[0150] 39. Munn, L. L., Melder, R. J., Jain, R. K., Analysis of
Cell Flux in the Parallel-Plate Flow Chamber-Implications for Cell
Capture Studies, Biophys. J., 67, 889-895, 1994. [0151] 40. Ley,
K., The Selectins As Rolling Receptors. In: The selectins:
initiators of leukocyte endothelial adhesion, Vestweber, D, ed.
Harwood Academic Publishers, Australia, 63-104, 1997. [0152] 41.
Papadaki, M. and McIntire, L. V., Quantitative Measurement of
Shear-Stress Effects on Endothelial Cells. In: Tissue engineering
methods and protocols, Morgan, J. R. and Yarmush, M. L, eds. Humana
Press, Totowa, N. J., 577-593, 1999. [0153] 42. Ramos, C. L. and
Lawrence, M. B., Quantitative Measurement of Cell-Cell Adhesion
Under Flow Conditions, In: Tissue engineering methods and
protocols, Morgan, J. R. and Yarmush, M. L., eds. Humana Press,
Totowa, N. J., 507-519, 1999. [0154] 43. Hannner, D. A. and Brunk,
D. K., Measuring Receptor-Mediated Cell Adhesion Under Flow:
Cell-Free Systems. In: Tissue engineering methods and protocols,
Morgan, J. R and Yarmush, M. L., eds. Humana Press, Totowa, N. J.,
543-552, 1999. [0155] 44. Jain, R. K., Munn, L. L., Fukumura, D.,
Melder, R J., In Vitro and In Vivo Quantification of Adhesion
Between Leukocytes and Vascular Endothelium. In: Tissue engineering
methods and protocols, Morgan, J. R. and Yarmush, M. L., eds.
Humana Press, Totowa, N. J., 553-575, 1999. [0156] 45. Li, C. Y.,
Shan, S., Huang, Q., Braun, R D., Lanzen, J., Hu, K., Lin, P.,
Dewhirst, M. W., Initial Stages of Tumor Cell-Induced Angiogenesis:
Evaluation Via Skin Window Chambers in Rodent Models, J Nat! Cancer
Inst, 92, 143-7, 2000. [0157] 46. Jain, R K, Munn, L. L., Fukumura,
D., Dissecting Tumour Pathophysiology Using Intravital Microscopy.
Nat. Rev Cancer, 2, 266-76, 2002. [0158] 47. Jain, R K, Munn, L. L,
Fukumura, D., Dissecting Tumour Pathophysiology Using Intravital
Microscopy. Nature Reviews Cancer, 2, 266-276, 2002. [0159] 48.
Jain, R K., Angiogenesis and Lymphangiogenesis in Tumors: Insights
From Intravital Microscopy, Cold Spring Harb. Symp. Quant. Biol.,
67, 239-248, 2002. [0160] 49. Folkman, J., Bach, M., Rowe, J. W.,
Davidoff, F., Lambert, P., Hirsch, C., Goldberg, A., Hiatt, H. H.,
Glass, J., Henshaw, B., Tumor Angiogenesis-10 Therapeutic
Implications, N. Engl. J. Med., 285, 1182-1186, 1971. [0161] 50.
Weidner, N., Semple, J. P., Welch, W. R, Folkman, J., Tumor
Angiogenesis and Metastasis--Correlation in Invasive
Breast--Carcinoma, N. Engl. J. Med., 324, 1-8, 1991. [0162] 51.
Lin, P., Buxton, l A, Acheson, A, Radziejewski, C, Maisonpierre, P.
C., Yancopoulos, G. D., Channon, K. M., Hale, L. P., Dewhirst, M.
W., George, S. B., Peters, K G., Antiangiogenic Gene Therapy
Targeting the Endothelium-Specific Receptor Tyrosine Kinase Tie2,
Proc. Natl. Acad Sci USA, 95, 8829-34, 1998. [0163] 52. Lin, P.,
Polyerini, P., Dewhirst, M., Shan, S., Rao, P. S., Peters, K.,
Inhibition of Tumor Angiogenesis Using a Soluble Receptor
Establishes a Role for Tie2, in Pathologic Vascular Growth, J Clin
Invest, 100, 2072-8, 1997. [0164] 53. Lin, P., Sankar, S., Shan,
S., Dewhirst, M. W., Polyerini, P. J., Quinn, T. Q., Peters, K. G.,
Inhibition of Tumor Growth by Targeting Tumor Endothelium Using a
Soluble Vascular Endothelial Growth Factor Receptor, Cell Growth
Differ, 9, 49-58, 1998. [0165] 54. Heidemann, J., Ogawa, H.,
Dwinell, M. B., Rafiee, P., Maaser, C., Gockel, H. R, Otterson, M.
F., Ota, D. M., Lugering, N., Domschke, W., Binion, D. G.,
Angiogenic Effects of Interleukin 8 (CXCL8) in Human Intestinal
Microvascular Endothelial Cells Are Mediated by CXCR2, J. Biol.
Chem., 278, 8508-8515, 2003. [0166] 55. Li, Y., Tondravi, M., Liu,
l, Smith, E., Haudenschild, C. C., Kaczmarek, M., Zhan, X.,
Cortactin Potentiates Bone Metastasis of Breast Cancer Cells,
Cancer Res, 61, 6906-11, 2001. [0167] 56. Higgs, R. N. and Pollard,
T. D., Regulation of Actin Filament Network Formation Through
Arp2/3 Complex: Activation by a Diverse Array of Proteins, Annu
Rev. Biochem., 70, 649-676, 2001. [0168] 57. Li, F. Y., Zhang, L.,
Metzger, R M., On the Growth of Highly Ordered Pores in Anodized
Aluminum Oxide, Chem. Mater., 10, 2470-2480, 1998. [0169] 58. Li,
A. P., Muller, F., Birner, A., Nielsch, K, Gosele, D., Hexagonal
Pore Arrays With a 50-420 Nm Interpore Distance Formed by
Self-Organization in Anodic Alumina, J. Appl. Phys., 84, 6023-6026,
1998. [0170] 59. Black, C. T., Guarini, K W., Milkove, K. R, Baker,
S. M., Russell, T. P., Tuominen, M. T., Integration of
Self-Assembled Diblock Copolymers for Semiconductor Capacitor
Fabrication, Appl. Phys. Lett., 79, 409-411, 2001. [0171] 60.
Black, C. T. and Guarini, K W., Diblock Copolymers: Self-Assembly
for Applications in Microelectronics, In: Encyclopedia of
Materials: Science and Technology, Buschow, KHJ, ed. Elsevier,
N.Y., 1-6, 2002. [0172] 61. Guarini, K W., Black, C. T., Zhang, Y,
Kim, H., Sikorski, E. M., Babich, I V., Process Integration of
Self-Assembled Polymer Templates into Silicon Nanofabrication,
Journal of Vacuum Science & TechnologyB, 20, 2788-2792, 2002.
[0173] 62. MartinezZaguilan, R, Sefior, B. A., Sefior, R E. B.,
Chu, Y. W., Gillies, R J., Hendrix, M. J. C., Acidic PH Enhances
the Invasive Behavior of Human Melanoma Cells, Clinical &
Experimental Metastasis, 14, 176-186, 1996. [0174] 63. Gillies, R
l, Raghunand, N., Karczmar, G. S., Bhujwalla, Z. M., MRI of the
Tumor Microenvironment, J. Magn. Reson. Imaging, 16,430-450, 2002.
[0175] 64. Bhujwalla, Z. M., Artemov, D., Ballesteros, P., Cerdan,
S., Gillies, R J., Solaiyappan, M, Combined Vascular and
Extracellular PH Imaging of Solid Tumors, NMR Biomed., 15, 114-119,
2002. [0176] 65. Helmlinger, G., Schell, A., Dellian, M., Forbes,
N. S., Jain, R K, Acid Production in Glycolysis-hnpaired Tumors
Provides New Insights into Tumor Metabolism, Clin. Cancer Res., 8,
1284-1291, 2002.
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