U.S. patent application number 11/901277 was filed with the patent office on 2008-06-19 for selective deposition of materials on contoured surfaces.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Christopher S. Chen, Donald E. Ingber, Emanuele Ostuni, George M. Whitesides.
Application Number | 20080145908 11/901277 |
Document ID | / |
Family ID | 23063650 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080145908 |
Kind Code |
A1 |
Ostuni; Emanuele ; et
al. |
June 19, 2008 |
Selective deposition of materials on contoured surfaces
Abstract
The present invention is directed to a method of patterning
materials, such as proteins, on a contoured surface by depositing
them onto protrusions on the surface and to a cell containment
device that may be constructed by this method. In one embodiment,
the present invention is directed to a method of selectively
depositing a material on a substrate including a contoured surface
including a protrusion and a recess. The method includes applying a
first fluid to the contoured surface of the substrate and allowing
the first fluid to distribute across a portion of the contoured
surface such that the first fluid contacts the protrusion and not
the recess. The method also includes allowing a first material to
deposit on the substrate where the substrate is in contact with the
first fluid. Optionally, this method may further include applying a
second fluid to the contoured surface of the substrate, allowing
the second fluid to distribute across a portion of the contoured
surface such that the second fluid contacts the recess, and
allowing a second material to deposit on the substrate where the
substrate is in contact with the second fluid. Optionally, the
method may still further include applying a third fluid to the
contoured surface of the substrate, allowing the third fluid to
distribute across a portion of the contoured surface, and allowing
a third material with an affinity for one of the first material and
the second material to deposit on the substrate only where the one
of the first material and the second material is deposited. In one
embodiment of this method the first material is a cytophobic
material, the second material is a cytophilic material and the
third material is a cell. In another embodiment, the present
invention is directed to a cell containment device, including a
substrate having a contoured surface, a recess in the surface of
the substrate, a cytophobic material connected to the surface of
the substrate outside the recess and a cytophilic material
connected to the recess.
Inventors: |
Ostuni; Emanuele;
(Watertown, MA) ; Chen; Christopher S.;
(Baltimore, MD) ; Ingber; Donald E.; (Boston,
MA) ; Whitesides; George M.; (Newton, MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
23063650 |
Appl. No.: |
11/901277 |
Filed: |
September 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10668679 |
Sep 23, 2003 |
7288394 |
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11901277 |
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PCT/US02/09149 |
Mar 25, 2002 |
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10668679 |
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60278092 |
Mar 23, 2001 |
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Current U.S.
Class: |
435/180 |
Current CPC
Class: |
B01J 2219/00605
20130101; C12N 11/08 20130101; Y10S 530/812 20130101; Y10S 530/815
20130101; B01J 2219/00637 20130101; B01L 3/5085 20130101; B01J
2219/00659 20130101; B01J 2219/00317 20130101; B01J 2219/00743
20130101; B01J 2219/00619 20130101; B01J 2219/00621 20130101; B01L
3/5088 20130101; B01J 2219/00612 20130101 |
Class at
Publication: |
435/180 |
International
Class: |
C12N 11/08 20060101
C12N011/08 |
Goverment Interests
GOVERNMENT FUNDING
[0002] Funding for research leading to the invention(s) described
and claimed herein was provided in part by Federal grants NSF
ECS-9729405 and ONR N65236-97-1-5814. The government has certain
rights in the invention.
Claims
1-39. (canceled)
40. A patterned substrate comprising: a contoured surface having at
least one recess therein, the recess being contiguous with and
forming a pattern in the contoured surface, wherein the pattern has
a lateral dimension of about 500 .mu.m or less; and a fluid
comprising a material chosen from: a polymer, a polymer precursor,
a metal, an organic electroluminescent material, a protein, a
cytophobic material, a cell-adhesion inhibiting agent, a
cell-adhesion promoting agent, and combinations thereof covering at
least a portion of the contoured surface and the at least one
recess therein, wherein the fluid forms an advancing angle of about
90.degree. or greater on areas of the contoured surface surrounding
the at least one recess, and wherein air is trapped within the at
least one recess that is covered by the fluid such that the fluid
does not contact the surface of the at least one recess.
41. The patterned substrate of claim 1, wherein the fluid comprises
a polymer chosen from: a thermoset polymer, a photolytically
polymerized polymer, a convectively polymerized polymer, a
radiatively polymerized polymer, a free-radical polymerized
polymer, and combinations thereof.
42. The patterned substrate of claim 1, wherein the fluid comprises
a polymer chosen from: a polyurethane, a polyamide, a polyamine, a
polycarbonate, a polyacetylene, a polydiacetylene, a
polyphosphazine, a polysiloxane, a polyolefin, a polyester, a
polyether, a poly(ether ketone), a poly(alkylene oxide), a
poly(ethylene terephthalate), a poly(methyl methacrylate), a
polystyrene, an alkyl acrylate, an alkyl methacrylate, an
.alpha.-methylstyrene, a vinyl chloride, a maleic anhydride, an
acrylic acid, an acrylonitrile, a hexafluoroisopropyl methacrylate,
a bromophenyl acrylate, a bromophenyl methacrylate, and
combinations thereof
43. The patterned substrate of claim 1, wherein the fluid comprises
a material that forms a covalent bond with the contoured surface
surrounding the at least one recess.
44. The patterned substrate of claim 1, wherein the at least one
recess has a lateral dimension of about 100 .mu.m or less.
45. The patterned substrate of claim 1, wherein the at least one
recess has a depth of about 50 .mu.m or less.
46. The patterned substrate of claim 1, wherein the fluid further
comprises a solvent.
47. The patterned substrate of claim 1, wherein the fluid is
hydrophobic.
48. A patterned substrate comprising: a contoured surface having at
least one recess therein, the recess being contiguous with and
forming a pattern in the contoured surface, wherein the pattern has
a lateral dimension of about 500 .mu.m or less; a coating
comprising a component chosen from a polymer, a polymer precursor,
a metal, an organic electroluminescent material, a protein, a
cytophobic material, a cell-adhesion inhibiting agent, a
cell-adhesion promoting agent, and combinations thereof covering at
least a portion of the contoured surface surrounding the at least
one recess; and a fluid comprising a material chosen from a second
polymer, a second polymer precursor, a second metal, a second
organic electroluminescent material, a second protein, a second
cytophobic material, a second cell-adhesion inhibiting agent, a
second cell-adhesion promoting agent, an antibody, and combinations
thereof, wherein the fluid fills the at least one recess, and
wherein the areas of the contoured surface surrounding the at least
one recess are free from the fluid.
49. The patterned substrate of claim 9, wherein the fluid comprises
a material that forms a covalent bond with a surface of the at
least one recess.
50. The patterned substrate of claim 9, wherein the coating is
hydrophobic.
51. The patterned substrate of claim 9, wherein the fluid is
hydrophilic.
52. The patterned substrate of claim 9, wherein the fluid further
comprises a solvent.
53. The patterned substrate of claim 9, wherein the fluid forms an
advancing angle of about 90.degree. or less on areas of the
contoured surface surrounding the at least one recess.
54. The patterned substrate of claim 9, further comprising a second
coating deposited on the fluid.
55. The patterned substrate of claim 15 wherein the second coating
comprises a component that forms a covalent bond with a material
present in the fluid.
56. The patterned substrate of claim 15, wherein the second coating
comprises a component chosen from: a third polymer, a third polymer
precursor, a third protein, a cell, and combinations thereof
57. The patterned substrate of claim 15, wherein the second coating
is hydrophilic.
58. The patterned substrate of claim 9, wherein the at least one
recess has a lateral dimension of about 100 .mu.m or less.
59. A method to pattern a contoured surface having at least one
recess therein, the recess being contiguous with and forming a
pattern in the contoured surface, wherein the pattern has a lateral
dimension of about 500 .mu.m or less, the method comprising:
applying a fluid comprising a component chosen from a polymer, a
polymer precursor a metal, an organic electroluminescent material,
a protein, a cytophobic material, a cell-adhesion inhibiting agent,
a cell-adhesion promoting agent, and combinations thereof to the
contoured surface; allowing the fluid to distribute across the
contoured surface such that the first fluid contacts an area of the
surface surrounding the recess, but does not contact the surface of
the recess; and depositing the component from the fluid onto the
area of the surface surrounding the recess.
60. The method of claim 20, wherein the component comprises a
polymer chosen from: a thermoset polymer, a photolytically
polymerized polymer, a convectively polymerized polymer, a
radiatively polymerized polymer, a free-radical polymerized
polymer, and combinations thereof.
61. The method of claim 20, wherein the component comprises a
polymer chosen from: a polyurethane, a polyamide, a polyamine, a
polycarbonate, a polyacetylene, a polydiacetylene, a
polyphosphazine, a polysiloxane, a polyolefin, a polyester, a
polyether, a poly(ether ketone), a poly(alkylene oxide), a
poly(ethylene terephthalate), a poly(methyl methacrylate), a
polystyrene, an alkyl acrylate, an alkyl methacrylate, an
.alpha.-methylstyrene, a vinyl chloride, a maleic anhydride, an
acrylic acid, an acrylonitrile, a hexafluoroisopropyl methacrylate,
a bromophenyl acrylate, a bromophenyl methacrylate, and
combinations thereof.
62. The method of claim 20, wherein the at least one recess has a
lateral dimension of about 100 .mu.m or less.
63. The method of claim 20, wherein the at least one recess has a
depth of about 50 .mu.m or less.
64. The method of claim 20, further comprising: applying a second
fluid comprising a second component chosen from a second polymer, a
second polymer precursor a second metal, a second organic
electroluminescent material, a second protein, a second cytophobic
material, a second cell-adhesion inhibiting agent, a second
cell-adhesion promoting agent, and combinations thereof to the
contoured surface; allowing the second fluid to distribute across
the contoured surface such that the second fluid contacts a surface
of the recess, but does not contact the areas of the surface
surrounding the recess; and depositing the second component from
the second fluid onto the surface of the recess.
65. A product prepared by the process of claim 20.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/668,679, filed Sep. 23, 2003, which is a
continuation of International Application No. PCT/US02/09149 filed
Mar. 25, 2002, which was published under PCT Article 21(2) in
English, and claims priority to U.S. provisional application Ser.
No. 60/278,092, filed Mar. 23, 2001, each of which is incorporated
by reference herein.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention is directed to a method of patterning
materials, such as proteins, on a contoured surface by depositing
them onto protrusions on the surface and to a cell containment
device that may be constructed by this method.
[0005] 2. Description of the Related Art
[0006] A number of methods in biochemistry require a technique for
patterning single cells with a high degree of spatial selectivity.
Many laboratories are trying to take advantage of the high
sensitivity of living cells in sensing units for biosensor
applications; in these systems, the cells must be located precisely
on the circuitry of the device. In tissue engineering, it may be
useful to pattern different kinds of cells on different areas of a
substrate in order to build defined architecture into
multifunctional tissues. Automated technologies for high-throughput
screening require the placement of cells in well-ordered arrays
that can be addressed individually. Basic studies of cellular
function and metabolism will also benefit from the ability to
control the microenvironment of patterned cells, and to perturb
them individually. Assays aimed at identifying the phenotype of a
cell in a population of heterogeneously transfected cells might be
simplified if individual cells were localized; the production of a
fluorescently labeled gene product could be detected at the
location, rather than remaining unidentified in solution.
[0007] Although cell-based assays are commonly available,
applications involving patterned single cells have been limited by
technological problems: e.g., the selective delivery of small
volumes of liquid to a well with a 50 .mu.m diameter, the placement
of cells on a defined grid, and the prevention of non-selective
adhesion and cell migration. Improving the technology to generate
regular arrays of cells would make it possible to develop: i)
analytical systems based on single cells for the detection of toxic
agents; ii) systems for high-throughput screening of combinatorial
libraries and gene products; iii) research tools to study the
effect of the adhesive environment on the behavior of a cell; iv)
new methods for the study of cellular function and metabolism at
the level of single cells and individually isolated groups of
cells.
[0008] To pattern single mammalian cells onto a substrate, the best
strategy is often to pattern adhesive extracellular matrix (ECM)
proteins onto that substrate. Current methods for patterning ECM
proteins use self-assembled monolayers (SAMs) of alkanethiolates on
gold, or of alkyltrichlorosiloxanes on silicon. Previously, it has
been shown that mammalian cells can adhere to flat or contoured
gold surfaces patterned with SAMs. Earlier work on patterning cells
focused on their interaction with SAMs of alkylsiloxanes.
Photolithographic patterning of siloxanes allowed the definition of
patterns of functional groups that were recognized nonspecifically
by various types of cells; complex biological ligands, however, are
not compatible with these photolithographic methods. Microcontact
printing has also been used to directly print patterns of proteins
onto surfaces. (See U.S. Pat. Nos. 5,776,748 and 5,976,826, which
are hereby incorporated by reference in their entirety.)
[0009] You et al. coated arrays of large wells (1-mm diameter)
non-selectively with ECM proteins; mink lung cells were forced into
the wells from a suspension by dragging a flat piece of PDMS across
the array of wells. (You, A. J.; Jackman, R. J.; Whitesides, G. M.;
Schreiber, S. L. Chem. & Biol. 1997, 4, 969-975.) Parce et al.
used gravitational sedimentation to deposit cells in arrays of 50
.mu.m wells that were fabricated using silicon micromachining.
(Parce, J. W.; Owicki, J. C.; Kercso, K. M.; Sigal, G. B.; Wada, H.
G.; Muir, V. C.; Bousse, L. J.; Ross, K. L.; Sikic, B. I.;
McConnell, H. M. Science 1989, 246, 243-247.) Methods have also
been developed for patterning cells onto surfaces using elastomeric
membranes as resists against the adsorption of proteins and the
adhesion of cells. (Ostuni, E; Kane, R.; Chen, C. S.; Ingber, D.
E.; Whitesides, G. M. Langmuir, 2000, 16, 7811-7819.)
SUMMARY OF THE INVENTION
[0010] In one embodiment, the present invention is directed to a
method of selectively depositing a material on a substrate
including a contoured surface including a protrusion and a recess.
The method includes applying a first fluid to the contoured surface
of the substrate and allowing the first fluid to distribute across
a portion of the contoured surface such that the first fluid
contacts the protrusion and not the recess. The method also
includes allowing a first material to deposit on the substrate
where the substrate is in contact with the first fluid. Optionally,
this method may further include applying a second fluid to the
contoured surface of the substrate, allowing the second fluid to
distribute across a portion of the contoured surface such that the
second fluid contacts the recess, and allowing a second material to
deposit on the substrate where the substrate is in contact with the
second fluid. Optionally, the method may still further include
applying a third fluid to the contoured surface of the substrate,
allowing the third fluid to distribute across a portion of the
contoured surface, and allowing a third material with an affinity
for one of the first material and the second material to deposit on
the substrate only where the one of the first material and the
second material is deposited. In one embodiment of this method the
first material is a cytophobic material, the second material is a
cytophilic material and the third material is a cell.
[0011] In another embodiment, the present invention is directed to
a method including selectively depositing a protein on an
outward-facing portion of a protrusion of a contoured surface
including a protrusion and a recess, at least one of the protrusion
and recess having a maximum lateral dimension of no more than about
1 mm, while leaving the recess free of the protein.
[0012] In another embodiment, the present invention is directed to
method of selectively depositing a material on a substrate having a
contoured surface including a protrusion and a recess. The method
includes applying a fluid to the contoured surface without urging
the fluid against the surface mechanically, and allowing the fluid
to contact the protrusion and not the recess. The method further
includes allowing a first material to be deposited from the fluid
onto the protrusion but not the recess.
[0013] In another embodiment, the present invention is directed to
a cell containment device including a substrate including a
contoured surface including a protrusion and a recess. The cell
containment device further includes a cytophobic material connected
to the protrusion and a cytophilic material connected to the
recess.
[0014] In another embodiment, the present invention is directed to
an article including a contoured surface including at least one
protrusion and at least one recess, and a cytophilic agent on the
surface within the recess. In this embodiment, the surface at the
protrusion being free of the cytophilic agent.
[0015] In another embodiment, the present invention is directed to
an article including a contoured surface including a plurality of
protrusions and recesses, and at least one cell in at least one
recess, wherein the at least one recess has a maximum lateral
dimension of 500 .mu.m.
[0016] In another embodiment, the present invention is directed to
an article including a contoured surface including a plurality of
protrusions and recesses, and a single cell in at least one
recess.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Preferred, non-limiting embodiments of the present invention
will be described by way of example with reference to the
accompanying drawings, in which:
[0018] FIGS. 1-5 are a schematic of one embodiment of a substrate
according to the present invention at different stages of one
embodiment of the method of present invention;
[0019] FIG. 6 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0020] FIG. 7 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0021] FIG. 8 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0022] FIG. 9 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0023] FIG. 10 is a schematic of another embodiment of a substrate
according to the present invention;
[0024] FIG. 11 is an expanded view of a portion of the embodiment
of FIG. 10;
[0025] FIG. 12 is another expanded view of a portion of the
embodiment of FIG. 10;
[0026] FIG. 13 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0027] FIG. 14 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0028] FIG. 15 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0029] FIG. 16 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0030] FIG. 17 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0031] FIG. 18 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0032] FIG. 19 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0033] FIG. 20 is photocopy of an image from a microscope of a
substrate according to another aspect of the present invention;
[0034] FIGS. 21-24 are a schematic of another embodiment of a
substrate according to the present invention at different stages of
another embodiment of the method of present invention; and
[0035] FIG. 25 is a photocopy of an image from a microscope of a
substrate according to another aspect of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] In one embodiment, the present invention is directed to a
method of selectively depositing a material on a substrate
including a contoured surface including a protrusion and a recess.
As used herein, a substrate is any material having a surface
capable of having a contoured surface including a protrusion and a
recess formed therein. Also as used herein, a protrusion may
include a microprotrusion (a protrusion having a height and
diameter of less than 1 mm), ridge, mesa, projection, or the like
and a recess may include a microwell (a well having a depth and
diameter of less than 1 mm), cavity, divot, hole, nook, or the
like. Each of the protrusions and/or recesses may be of essentially
any shape and size, as will be discussed below. A protrusion may
also be defined by surrounding recesses, such as the area between
microwells, and a recess may also be defined by surrounding
protrusions, such as the area between microprotrusions.
[0037] The method includes applying a first fluid to the contoured
surface of the substrate and allowing the first fluid to distribute
across a portion of the contoured surface such that the first fluid
contacts the protrusion and not the recess. The method also
includes allowing a first material to deposit on the substrate
where the substrate is in contact with the first fluid. Fluid is
used herein to describe both traditional solutions and other
liquids that may deposit a material on a surface, such as
suspensions or pre-polymers that polymerize where in contact with
the substrate. Deposit is used herein to broadly indicate that a
material is left on a surface when fluid is removed and may refer
to physical deposition, such as sedimentation, chemical and
electrochemical deposition, electroless plating, biological
attachment, and the like.
[0038] Optionally, this method may further include applying a
second fluid to the contoured surface of the substrate, allowing
the second fluid to distribute across a portion of the contoured
surface such that the second fluid contacts the recess, and
allowing a second material to deposit on the substrate where the
substrate is in contact with the second fluid. Optionally, the
method may still further include applying a third fluid to the
contoured surface of the substrate, allowing the third fluid to
distribute across a portion of the contoured surface, and allowing
a third material with an affinity for one of the first material and
the second material to deposit on the substrate only where the one
of the first material and the second material is deposited. In one
embodiment of this method the first material is a cytophobic
material, the second material is a cytophilic material and the
third material is a cell. As used herein, a cytophobic material is
one which has no affinity for, or is repellant or toxic to, cells
and a cytophilic material is one which has an affinity for cells.
In some embodiments cells may be deposited in recesses. In these
embodiments, it may be preferred to deposit a single cell within a
recess, meaning that the recess is devoid of other cells.
[0039] In another embodiment, the present invention is directed to
a cell containment device, including a substrate having a contoured
surface including a protrusion and a recess, a cytophobic material
connected to the protrusion and a cytophilic material connected to
the recess.
[0040] The present invention exploits the observation that certain
fluids do not enter recesses in a substrate when the fluid is
applied to the substrate. This phenomenon is used to selectively
deposit a material on a substrate. As the surface of the recess may
then remain free for deposit of a second material, in some
embodiments a fluid that does enter the recesses is applied to the
substrate, allowing a second material to be deposited within the
recesses. One example embodiment of a structure that may be made
according to the present invention is a cell containment device
including a cytophobic material deposited on the surface of a
substrate and a cytophilic material deposited in recesses. Such a
containment device may allow, for example, the study of individual
cells and improved automation of certain processes involving
cells.
[0041] The phenomenon by which certain liquids trap air in recesses
on a surface can be explained using the Laplace-Young equation to
define an advancing angle (.theta..sub.a) (eq. 1);
cos .theta. a = .gamma. sv - .gamma. sl .gamma. lv ( 1 )
##EQU00001##
.gamma. is the interfacial tension of the solid-vapor (sv),
solid-liquid (sl), and liquid-vapor (lv) interfaces. The
solid-vapor interface of some materials, such as
polydimethylsiloxane (PDMS), for example, has lower energy
(.gamma..sub.SV.about.21 dyn/cm) than the liquid-vapor interface of
drops of some liquids, such as water, (.gamma..sub.LV of
water.about.73 dyn/cm). Fluids having values of
.theta..sub.a>90.degree. imply that the values of .gamma..sub.sl
are high. Under such conditions, it is thermodynamically favorable
for the area of all interfaces with the liquid to be minimized.
Hence, a drop of liquid that contacts the flat regions of a
substrate, but does not fill recesses, forms a system of lower free
energy than one in which liquid contacts the entire surface of the
substrate, including the recesses.
[0042] Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1944, 40,
546-551, which is hereby incorporated by reference in its entirety,
discloses an equation (eq. 2) to describe the contact angle of a
liquid on a contoured surface by taking into account the areas of
the solid-liquid and liquid-vapor interfaces.
cos .theta..sub.b=f.sub.1 cos .theta..sub.a-f.sub.2 (2)
In eq. 2, .theta..sub.b is the apparent contact angle,
.theta..sub.a is the contact angle measured on the flat surface,
f.sub.1 is the area of the solid-liquid interface, and f.sub.2 is
the area of the liquid-vapor interface. Porosity causes the surface
of a material on which a liquid has .theta..sub.a>90.degree.
(<90.degree.) to appear more hydrophobic (hydrophilic) than a
flat surface of the same material. Cassie and Shuttleworth used
equation 2 to predict that a droplet of liquid with
.theta..sub.a>90.degree. would trap air inside recesses in a
surface. (Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1944,
40, 546-551 and Shuttleworth, R.; Bailey, G. L. J. Discuss. Faraday
Soc. 1948, 3, 16-22, which is hereby incorporated by reference in
its entirety.) This prediction has been confirmed experimentally.
The derivations assume that the contribution of gravitational force
to the wetting behavior is minor; this assumption appears valid
since the measured values of contact angles on porous surfaces are
in agreement with the predicted ones.
[0043] Referring now to the figures, and in particular to FIGS.
10-12, a drop of fluid, namely liquid 100, with a high value of
.theta..sub.a pins at the edge of a recess 20 until its contact
angle on the wall of recess 20 reaches the advancing value. Liquids
with a value of .theta..sub.a>90.degree. reach the other edge of
recess 20 before the angle between the liquid and the vertical wall
reaches .theta..sub.a; the edge of the liquid, therefore, advances
over the face of recess 20 and traps air inside recess 20. Liquids
with values of the receding contact angle
(.theta..sub.r)<90.degree. fill recesses as drops of the liquids
recede on the substrate. Accordingly, the portions of the method
described herein where the liquid is not desired to enter the
recess are preferably used with liquids that advance on the
substrate (.theta..sub.a>90.degree.).
[0044] Referring now to FIGS. 1-5 and 21-24, one example embodiment
of the method of the present invention will be described. The
method includes providing a substrate 10 including recesses 20 and
protrusions 22 on a contoured surface 12. Substrate 10 may be
constructed in any manner and of any material(s) that allow a
recess 20 or protrusion 22 to be formed therein. As recess 20 or
protrusion 22 may be less than 100 .mu.m across, substrate 10 may
be constructed of a material that is able to be shaped to include
an appropriately sized recess 20 or protrusion 22. Furthermore, as
the geometry of recess 20 or protrusion 22 may impact whether a
first fluid 30 enters recess 20, the material of substrate 10 may
be capable of being accurately constructed in a desired geometry.
Surface 12 of substrate 10 need not be flat, nor have any other
particular geometry, as long as it allows first fluid 30 to contact
it without entering recess 20. Substrate 10 may include any number
of recesses 20 or protrusions 22 arranged in any desired pattern
upon surface 12 of substrate 10. For example, substrate 10 may
include evenly spaced recesses 20 or protrusions 22 arranged in a
grid. By way of illustration, FIGS. 1-5 correspond to FIGS. 21-24,
except that in FIGS. 1-5 recesses 20 are microwells and protrusion
22 is the space therebetween, while in FIGS. 21-24 protrusions 22
are microprotrusions and recesses 20 are the spaces
therebetween.
[0045] Substrate 10 may have a surface that interacts with first
fluid 30 such that fluid 30 does not enter recesses 20. For
example, substrate 10 may be constructed out of a material upon
which first fluid 30 has an advancing angle of about 90 degrees or
greater. Substrate 10 may also have a surface that favors
deposition of materials, such as a first material 32 from first
fluid 30 or a second material 42 from a second fluid 40. Substrate
10 may also be constructed of a durable, relatively inexpensive
material that is easy to construct and work with. For example,
substrate 10 may be constructed of a material that may be easily
cast or molded, such as many polymeric materials. As an alternate
example, substrate 10 may be constructed of a material that is
easily constructed with etching, photolithography or machining,
such as a silicon-based material or a metal. A preferred polymeric
substance for the construction of substrate 10 is an elastomer such
as polydimethylsiloxane (PDMS), which is an easily molded,
inexpensive, durable, transparent and relatively inert elastomer.
PDMS is also hydrophobic, making it easier to bind proteins. The
elastomeric nature of PDMS allows it to be stretched or deformed,
and fitted to non-planar surfaces. PDMS is also permeable to O and
CO.sub.2. Other suitable elastomers are described in U.S. Pat. No.
5,512,131 and PCT publication nos. WO 96/29629 and WO 99/54786,
which are hereby incorporated by reference in their entirety.
[0046] Substrate 10 constructed from PDMS or other moldable
materials may be fabricated from reusable masters by replica
molding. Where substrate 10 is constructed by replica molding, the
size and shape of recess 20 may be defined using standard
photolithography, e-beam writing or rapid prototyping as described
in Xia, Y.; Whitesides, G. M. Angew. Chem. Int. Ed. Engl. 1998, 37,
4000-4025 and Qin, D.; Xia, Y.; Whitesides, G. M. Adv. Mater. 1996,
8, 917-919, which are hereby incorporated by reference in their
entirety. Rapid prototyping is preferred for microwells greater
than about 30 .mu.m in diameter and standard photolithography is
preferred for microwells between about 1-30 .mu.m in diameter.
[0047] Recess 20 or protrusion 22 may be constructed in any manner
that allows recess 20 to inhibit entry of particular fluids, such
as first fluid 30. In some embodiments, recess 20 or protrusion 22
may also be constructed to promote entry of other fluids, such as a
second fluid 40. For example, recess 20 or protrusion 22 may be
symmetrical or irregular, and may have any shape or dimension that
allows recess 20 to selectively inhibit the entry of particular
fluids. The walls of recess 20 or protrusion 22 may be sloped
inwardly or outwardly, or may be straight. By way of illustration,
rather than limitation, recess 20 is assumed herein to be
cylindrical and protrusion 22 is assumed to be defined by the area
surrounding recess 20. Recess 20 may also have any dimensions that
allow it to inhibit entry of particular fluids, such as first fluid
30, and, in some embodiments, to promote entry of other fluids,
such as a second fluid 40. The dimensions of recess 20 may also be
selected based upon a material to be contained within recess 20.
For example, where recess 20 is to be constructed to contain cells,
it may be sized to contain, for example, a single cell. Typically,
recess 20 is less than about 100 .mu.m in diameter and 50 .mu.m in
depth, however, it should be appreciated that these dimensions are
dependent, for example, upon the material of substrate 10, the
fluids used and the application.
[0048] The embodiment of the method illustrated in FIGS. 1-5 and
21-24 also includes applying first fluid 30 containing first
material 32 to surface 12 of substrate 10, such that a bubble 14 is
formed in recess 20 and substantially none of first fluid 30 enters
recess 20. FIG. 1 illustrates substrate 10 with a drop of first
fluid 30 thereon. First fluid 30 may be delivered to surface 12 by
any conventional manner of transferring fluid, such as with a
pipette or micropipette. In some instances, and particularly where
the advancing angle between first fluid 30 and substrate 10 is
about 90 degrees, it may be desired to apply first fluid 30 to
substrate 10 in a controlled manner and to protect substrate 10
from shock, vibration, and the like while first fluid 30 is in
contact with substrate 10 so that bubble 14 is not mechanically
dislodged from recess 20.
[0049] First material 32 may be any material(s) desired to be
deposited onto substrate 10. For example, first material 32 may be
a material that may impart a desired surface activity to substrate
10. Accordingly, first material 32 may be any of a variety of
materials, such as proteins, polymers, other chemicals or
combinations thereof. By way of illustration, where substrate 10 is
to become part of a cell containment device, first material 32 may
be a cytophobic material intended to prevent cells from attaching
to substrate 10 except at recesses 20. Cell-adhesion inhibiting
agents are well known. Examples of cell-adhesion inhibiting agents
include polyethylene glycol-based agents and bovine serum albumin
(BSA). Those of ordinary skill in the art can easily screen agents
for cell-adhesion promotion or inhibition as follows. Various
agents can be applied to surfaces, cells can be applied to those
surfaces, and the ability of the cells to adhere to the surface can
be studied via morphology or other characteristics. This is routine
for those of ordinary skill in the art.
[0050] The embodiment of the method illustrated in FIGS. 1-5
further includes allowing first material 32 to deposit on substrate
10 where substrate 10 is in contact with first fluid 30. How the
deposit of first material 32 is accomplished may vary, for example,
with the material of substrate 10 and the nature of first material
32 and first fluid 30. For example, the amount of time required for
a desired amount of first material 32 to deposit may vary with
these and other factors. By way of illustration, it has been found
that 1 mg/ml BSA in PBS deposits satisfactorily on PDMS in
approximately one hour at room temperature.
[0051] Other types of selective deposition of the materials may be
accomplished in accordance with the principles of the invention in
other manners. For example, metal deposition, such as "wet"
electrochemical deposition or electroless deposition, can be
carried out from fluid precursors defining electrochemical or
electroless plating baths. Alternatively, a prepolymeric fluid
precursor can be used, such as a fluid containing species that can
react to form a thermoset polymer on projection 22, or a fluid
pre-polymer that can be polymerized on protrusion 22 via
photolysis, convective or radiative heat, free-radical
polymerization, and the like. Additionally, a relatively
low-melting polymer can be applied in a molten form and allowed to
solidify on projection 22. These and other forms of polymerization
are known to those of ordinary skill in the art and can be applied
to the techniques of the present invention without undue
experimentation. All types of polymerization, including cationic,
anionic, copolymerization, chain copolymerization, cross-linking,
and the like can be employed, and essentially any type of polymer
or copolymer formable from a fluid precursor can be patterned in
accordance with the invention. An example, non-limiting list of
polymers that are suitable include polyurethane, polyamides,
polyamines, polycarbonates, polyacetylenes and polydiacetylenes,
polyphosphazenes, polysiloxanes, polyolefins, polyesters,
polyethers, poly(ether ketones), poly(alkylene oxides),
poly(ethylene terephthalate), poly(methyl methacrylate),
polystyrene, and derivatives and block, random, radial, linear, or
teleblock copolymers, cross-linkable materials such as
proteinaceous material and/or blends of the above. Gels may be
suitable. Also suitable are polymers formed from monomeric alkyl
acrylates, alkyl methacrylates, alpha-methylstyrene, vinyl chloride
and other halogen-containing monomers, maleic anhydride, acrylic
acid, acrylonitrile, specifically, methyl methacrylate, imides,
carbonates, hexafluoroisopropyl methacrylate, acrylonitrile,
bromophenyl acrylates or bromophenyl methacrylates, and the like.
Monomers can be used alone, or mixtures of different monomers can
be used to form homopolymers and copolymers. Non-linear and
ferroelectric polymers can be advantageous. The particular polymer,
copolymer, blend, gel, protein or other material selected is not
critical to the invention, and those of skill in the art can tailor
a particular material for any of a wide variety of
applications.
[0052] As yet another example, a variety of organic
electroluminescent materials may also be used, including those
described in the following articles, each incorporated herein by
reference: Renak, et al., "Microlithographic Process for Patterning
Conjugated Emissive Polymers", Adv. Mater., 1997, 9, 5, 392-394;
Yam, "Plastics Get Wired", Scientific American, Jul. 1995, 83-87;
Kijima, et al., "RGB Luminescence from Passive-Matrix Organic
LED's", IEEE Transactions on Electron Devices, 44, 8, Aug. 1997;
Shen, et al., "Three-Color, Tunable, Organic Light-Emitting
Devices", Science, 6, Jun. 27, 1997; Burrows, et al., "Achieving
Full-Color Organic Light-Emitting Devices for Lightweight
Flat-Panel Displays", IEEE Transactions on Electron Devices, 44, 8,
Aug. 1992.
[0053] By way of another example, according to one embodiment, a
polymerizable or cross-linkable species (optionally in a fluid
carrier) including an admixed biochemically active agent such as a
protein can be made to form a pattern on a substrate surface
according to the described technique. For example, carboxylated
Dextran.TM. can carry admixed protein, be applied to surface 12 of
substrate 10, and hardened. Where the Dextran.TM. carries admixed
biologically active agent, the substrate can be exposed to a medium
suspected of containing a biological binding partner of the
biochemical agent, and any biochemical binding or other interaction
detected via, for example, diffraction.
[0054] In some embodiments, first material 32 may be combined with
a compatible carrier to form first fluid 30. The carrier may be
selected such that first fluid 30 has an advancing angle of more
than about 90 degrees with substrate 10 and promotes, or does not
inhibit, the deposit of first material 32 on substrate 10. An
acceptable carrier for many biological materials, such as proteins,
is phosphate buffered saline (PBS).
[0055] As illustrated in FIG. 2, first fluid 30 may be removed from
substrate 10, leaving first material 32 deposited thereon. Where
first fluid 30 it to be removed from substrate 10, it may be
removed in any manner that will remove first fluid 30 without
removing first material 32 deposited on substrate 10. For example,
first fluid 30 may be removed from substrate 10 by vacuum,
absorption, drying, and the like.
[0056] As illustrated in FIG. 3, in some embodiments of the
invention, the method may further include applying second fluid 40
containing second material 42 to surface 12 of substrate 10, such
that second fluid 40 enters recess 20. Second fluid 40 may be
delivered to surface 12 by any conventional manner of transferring
fluid, such as with a pipette or micropipette. In some instances,
and particularly where the contact angle between second fluid 40
and substrate 10 is about 90 degrees, it may be desired to apply
shock, vibration, and the like to, substrate 10 while second fluid
40 is in contact with substrate 10 so that any bubble 14 is
mechanically dislodged from recess 20. Alternatively, a vacuum may
be applied to the environment of second fluid 40 and substrate 10
to promote the release of any bubble 14. For example, a vacuum of
about 400 mm Hg was found to loosen bubbles in some
applications.
[0057] Second material 42 may be any material(s) desired to be
deposited within recesses 20. In some embodiments, second material
42 is preferred not to deposit where first material 32 has
deposited. Second material 42 may be a material that may impart a
desired surface activity to substrate 10. Accordingly, second
material 42 may be a variety of materials, such as proteins,
polymers, other chemicals or combinations thereof. By way of
illustration, where substrate 10 is to become part of a cell
containment device, second material 42 may be a cytophilic material
intended to promote the attachment of cells to substrate 10 within
recess 20.
[0058] In one embodiment, second material 42 can be a cell-adhesion
promoter, i.e. second material 42 can have physical (e.g., "sticky"
materials) and/or chemical properties that allow cell adherence to
second material 42 while maintaining the integrity of the cell.
Cell adhesion can be achieved by specific or non-specific
interactions. Surfaces which promote non-specific interactions of
proteins adhere most cells. Examples of such surfaces include ionic
or charged surfaces. Hydrophilic surfaces also promote non-specific
protein absorption and cell adhesion. An example of a surface
involved in non-specific interactions includes polymer surfaces
used in biomaterials such as polylysine or plasma-treated
polystyrene. Cell-specific interactions generally result when a
cell has a receptor which recognizes certain surfaces. For example,
mammalian cells have receptors which recognize extracellular matrix
proteins. Examples of cell-adhesion promoting agents include
extracellular matrix proteins such as vitronectin, laminin,
fibronectin (FN), collagens and gelatins. Alternatively, a surface
can be modified with antibodies which recognize certain cellular
receptors.
[0059] Many of the fluids and materials described for first fluid
30 and first material 32 may also find use as second fluid 40 and
second material 42 if their surface interaction with the substrate
is modified, or a vacuum or other technique used to remove bubbles
14 from substrate 10.
[0060] Second material 42 may be combined with a compatible carrier
to form second fluid 40. The carrier may be selected such that
second fluid 40 has a contact angle of less than about 90 degrees
with the substrate and promotes, or does inhibit, the deposit of
second material 42 on substrate 10.
[0061] The embodiment of the method illustrated in FIGS. 1-5
further includes allowing second material 42 to deposit on
substrate 10 where substrate 10 is in contact with second fluid 40.
How the deposit of second material 42 is accomplished may vary, for
example, with the material of substrate 10 and the nature of second
material 42 and second fluid 40. For example, the amount of time
required for a desired amount of second material 42 to deposit may
vary with these and other factors. For example, it has been found
that 50 .mu.g/ml FN in PBS deposits satisfactorily on PDMS in
approximately one hour at room temperature.
[0062] As illustrated in FIG. 4, second fluid 40 may be removed
from substrate 10, leaving second material 42 deposited thereon.
Where second fluid 40 is to be removed from substrate 10, it may be
removed in any manner that will remove second fluid 40 without
removing second material 42 deposited on substrate 10. For example,
second fluid 40 may be removed from substrate 10 by vacuum,
absorption, drying, and the like.
[0063] As illustrated in FIG. 5, the method may still further
include applying a third fluid 50 containing a third material 52
with an affinity for one of first material 32 and second material
42 to upper surface 12 of substrate 10 and allowing third material
52 to deposit on substrate 10 where the one of the first material
32 and second material 42 is deposited. Third fluid 50 may be
delivered to surface 12 by any conventional manner of transferring
fluid, such as with a pipette or micropipette. Alternatively,
substrate 10 may be immersed in third fluid 50 such that third
fluid 50 contacts surface 12. In some instances, and particularly
where the contact angle between third fluid 50 and substrate 10 is
about 90 degrees, it may be desired to apply shock, vibration, and
the like to substrate 10 while third fluid 50 is in contact with
substrate 10 so that any bubble 14 is mechanically dislodged from
recess 20. Alternatively, a vacuum may be applied to the
environment of third fluid 50 and substrate 10 to promote the
release of any bubble 14.
[0064] Third material 52 may be any material(s) desired to be
deposited within recesses 20. In some embodiments, third material
52 is preferred not to deposit where first material 32 has
deposited. Third material 52 may be a material that is desired to
be isolated, for example for study. Accordingly, third material 52
may be a variety of materials, such as proteins, polymers, other
chemicals, cells or combinations thereof. By way of illustration,
where substrate 10 is part of a cell containment device, third
material 52 may be a cell of interest. Any cell of interest to
those skilled in the art may be patterned this way. An example of a
cell that may be contained within a recess 20 is a bovine adrenal
capillary endothelial (BCE) cell.
[0065] Third material 52 may be combined with any compatible
carrier to form third fluid 50. The carrier may be selected such
that third fluid 50 has a contact angle of less than about 90
degrees with the substrate and promotes, or does not inhibit, the
deposit of third material 52 on substrate 10.
[0066] The embodiment of the method illustrated in FIGS. 1-5
further includes allowing third material 52 to deposit on substrate
10 where second material 42 is deposited. How the deposit of third
material 52 is accomplished may vary, for example, with the
material of substrate 10 and the nature of third material 52 and
third fluid 40. For example, the amount of time required for a
desired amount of third material 52 to deposit may vary with these
and other factors. Where third material 52 includes living cells,
the deposition process may include incubation. For example, it has
been found that BCE cells in a chemically defined medium (10
.mu.g/mL high density lipoprotein, 5 .mu.g/mL transferrin, 5 ng/mL
bFGF in BSA/DMEM), incubated in 10% CO.sub.2 at 37.degree. C. for 4
hours in recesses 20 deposited satisfactorily.
EXAMPLES
Example 1
[0067] An experiment was performed to demonstrate the feasibility
of production of substrates including arrays of microwells. PDMS
substrates were prepared as described in Jackman, R. J.; Duffy, D.
C.; Ostuni, E.; Willmore, N. D.; Whitesides, G. M. Anal. Chem.
1998, 11, 2280-2287, which is incorporated herein by reference in
its entirety. To summarize, a master consisting of posts of
photoresist supported on a silicon wafer was prepared
photolithographically; the height of the posts, which corresponds
to the depth of the microwells, was controlled by the choice of the
photoresist and the spinning rate. The chrome masks used for
photolithography had circular features with diameters between 25
.mu.m and 50 .mu.m and spacing between 50 .mu.m and 75 .mu.m; masks
were prepared by Advanced Reproductions, North Andover, Mass. The
PDMS (Dow Corning, Sylgard 184) was cured against these masters at
60.degree. C. for two hours and peeled away from the silicon wafers
to give an array of microwells molded into the surface of the
polymer. Substrates were then cut to the desired size (typically
1-2 cm.sup.2) and washed with ethanol and distilled water before
use. These experiments demonstrate that production of substrates
having arrays of microwells is feasible and relatively
straightforward.
Example 2
[0068] Experiments were performed to demonstrate that fluids having
an advancing angle of greater than 90 degrees do not enter
microwells and fluids having advancing angles of less that 90
degrees do enter microwells. First, the contact angles of various
fluids on PDMS were measured using a contact angle goniometer
(Rame-Hart, Mountain Lakes, N.J.). Advancing contact angles were
measured for three separate drops on each substrate by
delivering/withdrawing aliquots (3.times.5 .mu.l) of fluid with a
micropipette (Matrix Technologies, MicroElectrapette). The results
are reported in Table I. Each entry in Table I is the average of
the three measurements. Occasionally, the drops pinned on
heterogeneities on the surface of the polymer; in such cases, the
values of the contact angles deviated from the average by more than
50% and new measurements were taken on another region of the
surface.
[0069] Next, the fluids for which the advancing angle was known
were applied to a substrate including an array of microwells to
determine if the fluid entered the microwells. The results of this
experiment are reported in Table I.
Table I. Advancing contact angles of various fluids on flat PDMS
substrates. The columns under "filling" report whether the tested
liquids filled wells with the specified dimensions (reported in the
format "diameter (depth)," both in micrometers). A "+" indicates
wells that are filled by the liquid and a "-" indicates cases when
filling was not observed.
TABLE-US-00001 .theta..sub.a(PDMS) Filling (.+-.5 deg) 25(5)
50(1.3) 50(5) Distilled Water 111 - - - Fibronectin/PBS (50
.mu.g/mL) 109 - - - BSA/PBS (1 mg/mL) 100 - - - Ethylene glycol
(EG) 92.sup.a +/-.sup.b +/-.sup.b +/-.sup.b Tri(ethylene glycol)
76.sup.a + + + Butan-1-ol 36.sup.a + + + Ethanol 31.sup.a + + +
.sup.aObtained from You, A. J.; Jackman, R. J.; Whitesides, G. M.;
Schreiber, S. L. Chem. & Biol. 1997, 4, 969-975. .sup.bEG does
not fill the wells immediately; over minutes, the trapped bubbles
escape.
[0070] When drops of aqueous fluids with values of
.theta..sub.a>90.degree. were placed on top of a substrate that
presented microwells and allowed to spread, air was trapped in the
microwells. FIG. 6 shows the appearance of an array of microwells
covered with a drop of solution of BSA (1 mg/mL) in PBS buffer
(.theta..sub.a=100.degree.). Microwells filled with air were much
brighter than the rest of the substrate when imaging the system in
reflection mode on an inverted microscope (light coming from the
"bottom" of the sample) using light of high intensity (100 W Hg arc
lamp). The difference between the index of refraction of PDMS and
air (ca. 0.4) is higher than the difference between PDMS and buffer
(ca. 0.05); hence, the microwells that were filled with air
reflected more light and appeared brighter than the rest of the
sample.
[0071] Liquids with values of .theta..sub.a>90.degree. were
allowed to cover the entire surface of the substrates by detaching
the air from the microwells with a brief exposure of the system to
house vacuum for 30 sec; the bubbles in the microwells expanded and
detached when vacuum was applied, and the microwells filled when
vacuum was released. Microwells that were filled with liquid were
not brighter than the rest of the surface, as illustrated in FIG.
7.
[0072] The results obtained with optical microscopy were confirmed
using confocal microscopy. FIG. 8 shows a confocal image of a well
filled with air; the image was obtained in reflection mode (light
coming from the "top" of the sample). A complex optical diffraction
pattern produced by the light reflected from the air-filled cavity
was observed, as illustrated in FIG. 9. Indistinguishable
observations were made with microwells 50 .mu.m wide and 1.3 .mu.m
deep, 25 .mu.m wide and 5 .mu.m deep and 50 .mu.m wide and 50 .mu.m
deep when distilled water or PBS buffer were used to cover the
substrate. Air was also trapped in the wells when the substrates
were placed upside-down, on top of a drop of liquid.
[0073] Liquids such as ethanol and butan-1-ol having values of
.theta..sub.a<90.degree. on PDMS filled the microwells. When
ethylene glycol (EG, .theta..sub.a=92.+-.5.degree.) covered the
microwells, air was trapped in the wells, but the bubbles escaped
within minutes.
[0074] These experiments demonstrate that fluids having an
advancing angle of greater than about 90 degrees do not enter
microwells, while those having an advancing angle of less than
about 90 degrees do enter microwells.
Example 3
[0075] Experiments were performed to demonstrate that materials
could be selectively deposited on a substrate including microwells.
The surface between the microwells was first coated with BSA by
placing a solution of the protein in PBS on the substrate for 1
hour at room temperature. In this interval, air trapped in the
microwells protected their surfaces from contact with the solution
of BSA (FIG. 6); the trapped air effectively acted as a resist
against the adsorption of protein. This result was confirmed by
performing the same incubation with a solution of fluorescently
labeled BSA (FIG. 15).
[0076] Following the adsorption of BSA on the regions between the
microwells, the substrate was rinsed with PBS and exposed to house
vacuum for 30 seconds to remove the air bubbles from the microwells
and ensure that liquid filled them. The PBS was then exchanged with
a solution of FN (50 .mu.g/mL) in buffer. FN adsorbed on those
hydrophobic regions of the surface not already covered with BSA,
that is the surface of the microwells; the adsorbed BSA protected
the regions between the microwells from the adsorption of FN.
[0077] Immunofluorescence staining of FN confirmed that this
protein was delivered and adsorbed to the microwells selectively
(FIG. 16). A drop of FN left on top of an uncoated PDMS substrate,
allowed the protein to adsorb only to the regions of the array
between the wells (FIG. 17).
[0078] These experiments demonstrate that it is possible to
selectively deposit material on a substrate including microwells
according to one embodiment of the method of the present invention.
A similar experiment was performed on a substrate including a
plurality of microprotrusions. Immunofluorescence staining of FN
confirmed that this protein was delivered and adsorbed to the
microprotrusions selectively (FIG. 25).
Example 4
[0079] An experiment was performed to demonstrate that the method
of the present invention is capable of producing a working cell
containment device. Initially, PDMS substrates were placed inside
sterile Petri dishes. A drop of BSA (Intergen Company, Purchase,
N.Y.; 1 mg/mL) in PBS buffer was placed on top of the substrate for
1 hour to allow the protein to deposit on the surface between
wells. The liquid trapped air bubbles inside the microwells; these
air bubbles protected the interior surface of the wells from
contact with the solution containing BSA. Shaking or vibrating the
substrate was avoided to prevent accidental dislodgment of the
bubbles. The substrate was then washed gently with PBS three times.
Brief exposure (30 sec) of the substrates to house vacuum (ca. 400
mm Hg) ensured that all the bubbles escaped from the microwells
during the washing process.
[0080] The PBS wash was exchanged with a solution of FN
(Collaborative Biomedical; 50 .mu.g/mL) in PBS by rapidly
aspirating the buffer and placing the solution of FN on the surface
of the substrate; during this procedure, drying the sample was
avoided to prevent the formation of bubbles and damage to the
deposited BSA. The deposit of FN on the PDMS was then allowed to
proceed at room temperature for 1 hr.
[0081] Bovine adrenal capillary endothelial (BCE) cells were
cultured under 10% CO.sub.2 on petri dishes (Falcon) coated with
gelatin in Dulbecco's Modified Eagle's Medium (DMEM) containing 10%
calf serum, 2 mM glutamine, 100 .mu.g/mL streptomycin, 100 .mu.g/mL
penicillin, and 1 ng/mL basic fibroblast growth factor (bFGF).
Prior to incubation with the polymeric substrate, cells were
dissociated from culture plates with
* * * * *