Title: Method for cell patterning
Abstract: The present invention provides a masking system for selectively applying cells to predetermined regions of a surface. A mask is positioned adjacent to a surface to cover some portions of the surface while allowing other portions of the surface to remain uncovered. Cells then are applied to uncovered portions of the surface and the mask removed. Alternatively, a cell-adhesion promoter is applied to uncovered portions of the surface, and then cells are applied to the surface before or after removal of the mask from the surface. The masking system can be pre-coated, at least on those surfaces which will come into contact with cells, with a cell-adhesion inhibitor to resist absorption of cells and thereby avoid cell damage when the mask is removed (if cells are deposited prior to removal of the mask). A polymeric elastomeric mask that comes into cohesive-conformal contact with a surface to be patterned can be used.
Patent Number: 6,893,850 Issued on 05/17/2005 to Ostuni, et al.
| Inventors:
|
Ostuni; Emanuele (Cambridge, MA);
Kane; Ravi (Troy, NY);
Whitesides; George M. (Newton, MA);
Jackman; Rebecca J. (Boston, MA);
Duffy; David C. (Cambridge, MA)
|
| Assignee:
|
President and Fellows of Harvard College (Cambridge, MA)
|
| Appl. No.:
|
808745 |
| Filed:
|
March 15, 2001 |
| Current U.S. Class: |
435/174; 435/176; 435/177; 435/178; 435/179; 435/180; 435/182; 435/243; 435/325; 435/395 |
| Intern'l Class: |
B32B 031/00; C12N011/08; C12B011/14 |
| Field of Search: |
435/2521,174,176,177,178,179,180,182,243,325,395,4,173.9,173.4,173.5,173.8
|
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|
Primary Examiner: Haff; David M.
Assistant Examiner: Ware; Deborah K.
Attorney, Agent or Firm: Wolf, Greenfield & Sacks, P.C.
Goverment Interests
This invention was sponsored by NSF Grant Nos. PHY-9312572, DMR-9809363, ECS-9729405
and AFOSR/SPAWAR Grant No. N66001-98-1-8915. The government has certain rights
in the invention.
Parent Case Text
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application Ser.
No. 60/190,399, filed Mar. 17, 2000, entitled CELL PATTERNING VIA AN ELASTOMERIC MASK.
Claims
1. A method for patterning cells, comprising:
providing a masking system, the masking system comprising a flexible cohesive
mask, and the masking system including a first surface, an opposing second surface,
and a plurality of channels passing through the masking system connecting the first
surface with the second surface;
pre-coating a second agent onto the second surface and the plurality of channels
of the masking system, wherein the first surface of the masking system is free
of the second agent;
thereafter, shielding a first portion of a surface of an article with the masking
system by contacting the first surface of the masking system with the article,
wherein the flexible cohesive mask is in conformal contact with the surface of
the article;
applying a first agent through a channel within the masking system to a second
portion of the surface of the article while preventing application of the first
agent to the first portion of the surface of the article, the channel being one
of the plurality of channels;
thereafter, applying cells through the channel within the masking system to the
second portion of the surface of the article so the cells contact the first agent
while preventing application of the cells to the first portion of the surface of
the article; and
removing the masking system from the first portion of the surface of the article.
2. The method of claim 1, wherein the first portion of the surface of the article
is contiguous with the second portion.
3. The method of claim 1, wherein the channel has a dimension for controlling
the growth of a single cell.
4. The method of claim 1, wherein the first agent is a cell-adhesion promoter.
5. The method of claim 4, wherein the first agent is a protein.
6. The method of claim 5, wherein the protein is fibronectin.
7. The method of claim 4, wherein the second agent is a cell-adhesion inhibitor.
8. The method of claim 7, further comprising adding a third agent to the first
portion of the surface of the article.
9. The method of claim 8, further comprising allowing the cells applied to the
first agent to spread onto the third agent.
10. The method of claim 8, wherein the first agent is a first cell-adhesion promoter
and the third agent is a second cell-adhesion promoter.
11. The method of claim 10, further comprising adding cells of a second type
to the third agent.
Description
FIELD OF INVENTION
The present invention relates to methods for patterning cells on substrate surfaces
via an elastomeric mask. These methods allow for the study of cell migration and growth.
BACKGROUND OF THE INVENTION
Cell adherence on substrate surfaces, particularly surfaces used for cell-culture
such as glass or plastic, is necessary in many instances for the study of cells
in furthering applications such as tissue engineering, biosensors, etc. Cell patterning,
i.e. placing cells in discrete portions of a surface, has been provided by photolithography.
Although the technology of photolithography is very highly developed, it presents
several disadvantages. Photolithography presents harsh conditions which can destroy
the cells themselves. Clean-room facilities and other complex equipment are also
required and such facilities and equipment are not readily accessible to most biologists.
Photolithography is not amenable to controlling the molecular properties of a surface
required for many sophisticated cell-biological experiments. In addition, photolithography
modifies a surface only at the beginning of an experiment. Once cells are deposited,
photolithography cannot be used to make further surface modifications.
Laminar flow (FLO) patterning involves surface modification via laminar flow
of adjacent fluid streams with low Reynolds numbers. FLO patterning is restricted
to simple patterning and thus is useful for patterning the environment of a cell
and for cell labeling. This technique, however, is not suited for patterning the
shape and size of the cells.
Accordingly, there is a need to pattern cells in a facile manner while
subjecting the cells to relatively mild conditions.
SUMMARY OF THE INVENTION
One aspect of the present invention provides a method for patterning cells. The
method involves shielding a first portion of a surface of an article with a masking
system. The masking system comprises a cohesive mask in conformal contact with
a surface of the article. The method also involves applying an agent to a channel
within the masking system to a second portion of the surface of the article while
preventing application of the agent to the first portion of the surface of the
article. The method also involves applying cells onto the agent.
Another aspect of the invention provides a method for patterning cells comprising
shielding a first portion of a surface of an article with a masking system. The
masking system comprises a cohesive mask in conformal contact with the surface
of the article. The method further involves applying a cell-adhesion inhibitor
through a channel within the masking system to a second portion of the surface
of the article while preventing application of the cell-adhesion inhibitor to the
first portion of the surface of the article.
Another aspect of the present invention provides a method for patterning
cells comprising shielding a first portion of a surface of an article with a masking
system. The masking system comprises a cohesive mask in conformal contact with
the surface of the article. The method further involves applying a cell-adhesion
promoter through a channel within the masking system to a second portion of the
surface of the article while preventing application of the cell-adhesion promoter
to the first portion of the surface of the article.
Another aspect of the present invention provides a method for patterning
cells comprising providing an article having a first pattern of cells of a first
type. The method also involves applying an agent to a portion of a surface of the
article, the portion being contiguous with the first pattern.
Another aspect of the present invention provides an article comprising a
first pattern of cells of a first type contiguous with a second pattern of cells
of a second type.
Another aspect of the present invention provides a method comprising shielding
a first portion of a surface of an article with a masking system. The method involves
allowing a cell-adhesion promoter to be applied to a second, unshielded portion
of the surface of the article while preventing application of the cell-adhesion
promoter to the first portion of the surface of the article with the masking system.
The method further involves applying a cell to the second portion of the surface.
Another aspect of the present invention provides a method for patterning
cells comprising shielding a first portion of a surface of an article with a polymeric
masking system. The method involves applying an agent to a channel within the masking
system to a second portion of the surface of the article while preventing application
of the agent to a first portion of the surface of the article. The method further
involves applying cells onto the agent.
Other advantages, novel features, and objects of the invention will become
apparent from the following detailed description of the invention when considered
in conjunction with the accompanying drawings, which are schematic and which are
not intended to be drawn to scale. In the figures, each identical or nearly identical
component that is illustrated in various figures is represented by a single numeral.
For purposes of clarity, not every component is labeled in every figure, nor is
every component of each embodiment of the invention shown where illustration is
not necessary to allow those of ordinary skill in the art to understand the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic diagram of lift-off membrane (masking system) patterning
to pattern cells onto a surface of an article according to the invention;
FIG. 2 shows a schematic diagram for lift-off membrane patterning involving
a pre-coated masking system according to the invention;
FIG. 3 shows a schematic diagram for the fabrication of a masking system for
use in the invention;
FIG. 4 shows a photocopy of a scanning electron micrograph of a masking system
for use in the invention having channels shaped as holes having a diameter of about
100 μm;
FIG. 5A shows a photocopy of a fluorescence micrograph displaying comparative
results of completely coating a substrate with a cell-adhesion protein followed
by the addition of cells over the entire assembly;
FIG. 5B shows a photocopy of a fluorescence micrograph of the cells adhered
selectively to the surface of the substrate;
FIG. 6A shows a photocopy of a fluorescence micrograph displaying a pattern
of fibronectin after peeling the masking system in a process of the invention;
FIG. 6B shows a photocopy of a fluorescence micrograph displaying a pattern
of cells adhered to circular islands of fibronectin of FIG. 6A.
FIG. 7A shows a photocopy of an optical micrograph of cells patterned on circular
islands having a diameter of about 100 μm according to the invention;
FIG. 7B shows a photocopy of an optical micrograph of cells patterned on square
islands having a sides of a length of about 100 μm according to the invention;
FIG. 8A shows a photocopy of a phase-contrast micrograph and a fluorescence
micrograph of cells patterned with a BSA pre-coated membrane for features having
a diameter of 250 μm in a process of the invention;
FIG. 8B shows a photocopy of a phase-contrast micrograph and a fluorescence
micrograph of cells patterned without a BSA pre-coated membrane for features having
a diameter of 250 μm;
FIG. 8C shows a phase-contrast micrograph and a fluorescence micrograph of cells
patterned with a BSA pre-coated membrane for features having a diameter of 100 μm;
FIG. 8D shows a phase-contrast micrograph and a fluorescence micrograph of cells
patterned without a BSA pre-coated membrane for features having a diameter of 100 μm;
FIG. 8E shows a phase-contrast micrograph of a surface of the membrane removed
from the process of FIG. 8B, showing attached cells; and
FIGS. 9A-D show photocopies of scanning electron micrographs displaying the
results of cell spreading after (a) 7 h, (b) 8.2 h, (c) 9.5 h, and (d) 11 h.
DETAILED DESCRIPTION
The present invention provides methods for patterning cells involving a masking
system, and surfaces modified optionally using the system. The methods are particularly
advantageous in that various cell patterns can be provided without the aid of photolithographic
steps and thus patterns can be achieved in a relatively simple and inexpensive
manner. The present invention is applicable for patterning cells on a broad range
of substrates, which include most materials routinely used in cell culture. The
masking system has flexibility for patterning on substrates of essentially any
shape, and has rigidity to be reused a number of times.
A resulting pattern of cells can be used for a variety of applications including
observing cell growth and spreading, chemotaxis, haptotaxis, morphogenesis, and
the patterning of multiple cell types. In addition, cell patterning can have long
range applications in the study of regeneration, partial regeneration or healing
of human organs and wounds, i.e. tissue engineering. Other applications involve biosensors.
One aspect of the present invention provides a method for patterning cells. One
method involves shielding a first portion of a surface of an article with a masking
system. Subsequently, a second, unshielded portion of the surface of the article
is exposed to an agent such as a cell-adhesion promoter, a cell-adhesion inhibitor,
or a cell, before or after removal of the masking system. The masking system can
be polymeric and, in one embodiment, the masking system comprises a mask having
a flexible surface which allows the mask to conform to the surface. By "conform"
it is meant to define essentially continuous contact between the masking system
and the portions of the article to be patterned. This embodiment is to be distinguished
from, for example, a metal screen or a rigid polymer, each of which can contact
a surface to be masked but which may not be flexible enough to conformally contact
the surface. The flexibility of the mask can be provided by the use of an elastomeric
mask. The mask can be made of a polymeric material such as polydimethylsiloxane
(PDMS), or the like. In one embodiment, the mask can shield selected portions of
the surface by being brought into contact with those portions. Due to the flexibility
of the mask, the surface can be either a planar or non-planar surface.
In one embodiment, there is a channel within the masking system, and preferably
a plurality of channels within the masking system. The masking system can comprise
first and second opposing surfaces where the channel passes through the mask, connecting
the first surface with the second surface. The channel can function to expose certain
portions (a second portion) of the surface of the article, whereas a first portion
of the article is shielded due to conformal contact of the article with the masking
system. In one embodiment, the first portion is contiguous with the second portion.
In one embodiment, a channel within the mask is a hole through the mask, and by
placing one surface of the mask onto a substrate, wells are formed as defined by
the walls of the channel and the substrate surface (second portion of the surface).
The mask can contain a variety of liquid or solid agents within these wells.
In one embodiment, the mask is a polymer. A preferred polymer is a polymeric
elastomer
that can form a seal against the surface of the article. "Seal" in this context
means that when the mask is sealingly engaged with a surface and a fluid is applied
to the masked surface, the fluid is allowed to contact only those portions of the
masked surface in register with channels of the mask and the fluid does not pass
under the mask and contact shielded portions of the article surface covered by
solid portions of the mask, so long as the fluid does not degrade the mask or the
surface to be patterned (in which case fluid could pass under the mask due to degradation
of the mask and/or surface). For example, the seal can prevent a protein solution
from seeping under the mask. "Sealing" in this context is to be distinguished from
the operation of other rigid or flexible masks that may be brought into conformal
contact with a surface, but that can not seal against the surface. It is a feature
of the invention that masks of the invention can form a seal against a substrate
surface in the absence of any clamping apparatus or other apparatus used to apply
a force against the mask in a direction of the substrate surface. Where elastomeric
surfaces are used, and the elastomeric surface and substrate surface to be masked
are clean, sealing can occur essentially instantaneously upon contact without application
of significant pressure, and sealing can be maintained without maintenance of any
pressure. This sealing is reversible, that is, the mask can be removed from the
substrate surface by being peeled off, and can be reused on the same or a different
substrate surface. Reusability of a particular mask increases with the thickness
of the mask.
Exemplary techniques for fabricating a mask are described in PCT publication
WO 99/54786, entitled "ELASTOMERIC MASK AND USE IN FABRICATION OF DEVICES, INCLUDING
PIXELATED ELECTROLUMINESCENT DISPLAYS," by Jackman et al., published Oct. 28, 1999,
and which is incorporated herein by reference. For example, a flexible mask can
be created by a number of polymerization methods. One method, described in PCT
publication WO 99/54786, involves spin-coating a pre-polymer layer onto a substrate
surface having an array of cylindrical posts.
In one embodiment, the method involves applying an agent through the channel.
The method allows the agent to contact the exposed (second) portion of the surface
of the article while preventing application of the agent to the shielded (first)
portion of the article. The agent can be applied via deposition, chemical reaction,
or the like. For example, if the agent is provided as a solution, the deposition
can involve spraying or dripping the solution onto the mask and through the channel,
or dipping the entire substrate and masking system assembly into the solution.
In one embodiment, a vacuum may be applied to remove any air bubbles within the
solution in the channel to ensure optimal surface coverage.
In one embodiment, the agent has physical and/or chemical properties that allow
its adherence to the surface of the article via adsorption. Application of agent
can result in chemical reaction resulting in covalent or ionic interactions between
the surface of the article and the agent.
In one embodiment, the agent can be a cell-adhesion promoter, i.e. the agent
can
have physical (e.g., "sticky" materials) and/or chemical properties that allow
cell adherence to the agent while maintaining the integrity of the cell, and the
method involves applying cells onto the agent. Cell adhesion can be achieved by
specific or non-specific interactions. Surfaces which promote non-specific interactions
adhere most cells. Examples of such surfaces include ionic or charged surfaces.
Hydrophilic surfaces also promote non-specific cell adhesion. An example of a surface
involved in non-specific interactions include 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. Thus,
cells can be patterned onto surfaces using masking systems of the invention by
first applying a cell-adhesion promoter agent to the surface, preferably using
a masking system, or applying a masking system to a surface which already is cell-adhesion
promoting. Both cell-adhesion promoting agents and cell-adhesion promoting surfaces
are well-known in the art (some of which are described immediately above). Examples
of cell-adhesion promoting agents include extracellular matrix proteins such as
vitronectin, laminin, fibronectin, collagens and gelatins. Alternatively, a surface
can be modified with antibodies which recognize certain cellular receptors. Cell-adhesion
inhibiting surfaces and cell-inhibiting agents also are well-known. Examples of
cell-adhesion inhibiting agents include polyethylene glycol-based agents. Those
of ordinary skill in the art can easily screen surfaces for their natural cell-adhesion
promoting or inhibiting characteristics, or agents for cell-adhesion promotion
or inhibition as follows. Various untreated surfaces can be studied, or 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.
In one embodiment, the article or surface of the article can be a metal oxide
such as silica, alumina, quartz, glass, and the like or derivatives thereof, or
metals such as gold, silver and copper. The surface can be derivatized with functional
groups including amides, carboxylic acids, phosphoryl groups, hydroxyl groups,
amino acid groups, amines, sulfonyl groups. Oxy compounds or plastics can also
be used in accordance with the present invention. Additional materials and functional
groups can be found in U.S. Pat. No. 5,512,131, issued Apr. 30, 1996 and incorporated
herein by reference. In one embodiment, the surface can be that of an article typically
used to study cells, such as a microscope slide, petri dish, test tube or other
articles. Typically, these articles are made of polystyrene, glass or polycarbonate.
Functional groups discussed above, and other functionality can be provided on the
surface by coating the surface with a self-assembled monolayer as described in
U.S. Pat. No. 5,512,131. Self-assembled monolayers are well-known and typically
involve molecules each including a group that adheres to a surface and a spacer
moiety that can assemble, or pack with other spacer moieties such that when a plurality
of the molecules are exposed to a surface they orient themselves in an ordered
manner with the groups that adhere to the surface against the surface and the spacer
moieties packed relative to each other and extending from the surface. At the other
end of each, or selected of these molecules can be provided functional groups providing
the exposed portion of the self-assembled monolayer with a desired chemical functionality.
FIG. 1 shows a schematic diagram of one example for patterning agents associated
with cell deposition, according to the present invention. FIG.
1(
a)
shows an article
10 having a surface
11 with a first portion
12
and a second portion
14. A masking system can comprise a mask
16.
Mask
16 (shown in cross section) is brought into conformal contact with
the surface of article
10 such that the first portion
12 of surface
11 is shielded. FIG.
1(
b) shows the results of applying an
agent
20 through channel
18 of masking channel
16. Because
mask
16 shields first portion
12, agent
20 is applied only
to second portion
14 and is prevented from being applied to first portion
12. Agent
20 can be a cell-adhesion promoter (e.g., fibronectin).
Cells can be applied onto agent
20 on second portion
14 at this stage.
Cells, however, will also adhere to all surfaces coated by agent
20 (e.g.,
see FIG.
5A). Alternatively, mask
16 can be removed prior to applying
cells onto agent
20, as shown in FIG.
1(
c). In FIG.
1(
c),
substrate
10 has a pattern of agent
20 on second portion
14,
whereas first portion
12 comprises a surface free of agent
20 (e.g.,
see FIG.
6A).
FIG.
1(
d) shows the results of adding a second agent
22
to the exposed first portion
12. Second agent
22 can be a cell-adhesion
inhibitor, such as bovine serum albumin. Due to the inability of cells to adhere
to a cell-adhesion inhibitor, a cell-adhesion inhibitor functions to localize the
deposition of cells to a confined area, specifically portion
14. Generally,
cells will not grow or spread onto a surface that comprises a cell-adhesion inhibitor.
By adding cells
24 to the surface of FIG.
1(
d), a pattern
of cells can be established in register with portions
14, as shown in FIG.
1(
e) (e.g., see FIG.
6B).
FIG. 1 demonstrates that with techniques of the invention material can be patterned
through the holes against the substrate, and the mask removed, leaving an array
of pixels, without the requirement of steps and apparatus involved in laser ablation,
photolithography, and shadow mask procedures.
As discussed more fully below, in one embodiment cells can be deposited onto
portion
14 while mask
16 is on surface
11 providing that the cells
do not adhere to the mask or have been subjected to a pre-coating treatment (e.g.,
see FIG.
2 and discussion). The amount of cells deposited in each channel
can depend on factors such as the diameter and height of the channels or the density
of cells in a fluid suspension. Each channel may contain one cell or several tens
of cells. An advantageous feature of the invention is that cell(s) are confined
to the space defined by the channel until mask
16 is removed from article
10. Depending on the number of cells to be deposited, the channels or holes
of the mask can have a diameter of less than about 1 mm, less than about 500 μm,
less than about 250 μm, less than about 100 μm, less than about 50
μm, less than about 25 μm, less than about 10 μm, less than about
5 μm, down to less than about 1.5 micron.
Any pattern of channels
18 in the mask, for example a pattern defined
by a single channel or many channels that can be circular, oval, square, rectangular,
and the like, and arranged in a grid-like array (as illustrated) or a non-array
(for example random pattern) can be used.
The mask and channels can be of a variety of dimensions. In one embodiment, the
mask has a thickness of no more than about 1 mm, preferably no more than about
500 μm, more preferably no more than about 200 μm, preferably no more
than about 100 μm, more preferably still no more than about 25 μm.
In one embodiment, channel
18 has a preferred cross-sectional dimension
19 that corresponds to a thickness
17 of the mask
20 to create
a length to diameter ratio of channels of no more than about 5 to 1, and preferably
no more than about 2 to 1. Of course, the number of channels and the shape of channels
can be varied by any method known to one of ordinary skill in the art.
The conformal contact of mask
16 with article
10 should be strong
enough to prevent slippage of the mask on the article surface yet capable of being
removed by a peeling process. In one embodiment, the mask has a thickness of at
least about 50 μm. This thickness helps ensure the integrity of the mask
through several peeling processes. Preferably, the peeling should not disturb the
integrity of the pattern. It is a feature of the invention that the mask is cohesive
and can be removed from a surface as a single unit and re-used, i.e., the mask
facilitates a "dry lift-off" procedure. The mask is cohesive in that attractive
forces within the mask that hold the mask together are stronger than forces typically
required to remove the mask from a surface. That is, the mask can be used to seal
a surface during a deposition process, then can be removed by lifting a portion
of the mask which draws the entire mask away from the surface, and the mask then
can be reused. This is to be distinguished from a lithographically-created mask
such as a photoresist mask. Use of a cohesive mask of the invention allows formation
of the mask on the surface to be masked without degrading, at the surface, portions
defining channels
32 (such as are degraded in creation of a lithographically-created mask).
Alternatively, agent
20 is a cell-adhesion inhibitor, and upon
removal of mask
16, a cell-adhesion promoter can be applied to the bare
surface to achieve the arrangement of FIG.
1(
d).
To describe FIG. 1 with a specific example, a PDMS mask (i.e., masking system
16, alternatively referred to as a membrane) is used as a resist against
the adsorption of the cell-adhesion promoter fibronectin (FN, an extracellular
matrix protein) to the surface of the substrate. FN adsorbs only to the surface
of the substrate that is exposed by the pores of the membranes (see FIG.
6).
Removal of the mask from the surface generates a pattern of FN. The substrate is
then exposed to bovine serum albumin (BSA)-containing culture media to ensure that
the remainder of the surface is coated by a protein that resists cell attachment.
Cells from a suspension adhered to this substrate only in the pattern defined by
the pores of the membrane (see FIG.
7). FIG. 1 is not drawn to scale and
FIG. 1 does not imply that layers of BSA and FN have the same thickness. The mask
features may be curved at the top as a result of menisci formation during spin
coating or other processes.
For certain cell types, it may be preferable to pattern the cells by confining
the cells within mask channels. The mask channels provide a physical barrier to
contain and thus maintain control of cell size and shape, or the size and shape
of a layer of cells. In this embodiment, ideally, a mask is positioned on the surface
to create certain wells as defined by mask channels and the surface, and cells
are deposited into these wells. Because the mask itself may have cell-adherent
properties, however, removal of the mask may result in tearing of cell walls in
some instances, particularly where cells are adhered simultaneously to the mask
and the substrate. Accordingly, to ensure that contacting the cells with the mask
will not damage cell walls upon mask removal, in one embodiment, a cell pattern
is provided by use of a pre-coated mask. At least a portion of the mask, preferably
those portions that could contact cells in the process, can be pre-coated with
an agent that is a cell-adhesion inhibitor, such as bovine serum albumin. Thus,
a cell does not have a tendency to adhere to the mask and peeling off the mask
does not damage the cells.
FIG. 2 shows an example for patterning cells involving a pre-coating treatment
of the masking system. FIG.
2(
c) shows an article
30 having
a surface
31 comprising a first portion
32 and a second portion
34.
Mask
36 (shown in cross section) shields first portion
32 by being
in conformal contact with first portion
32 whereas channels
38 expose
second portion
34. FIG.
2(
c) also shows a coating of a first
agent, a cell-adhesion inhibitor agent
40 which has been applied only to
exposed surfaces of mask
36 and not on exposed surfaces of second portion
34 of article
30. Addition of a second agent
42, such as a
cell-adhesion promoter, provides a coating over second portions
34 of article
30, as shown in FIG.
2(
d). Preferably, agent
40 is
selected to resist adsorbtion of agent
42. The addition of cells
43
results in cell adhesion on agent
42 only, and cell adhesion is inhibited
on surfaces covered by agent
40, as shown in FIG.
2(
e) (e.g.,
see FIG.
5B). The arrangement shown in FIG.
2(
e) provides
the advantage that upon peeling the masking system
36 from article
30,
the cell-adhesion inhibitor nature of the surface of article
36 coated with
agent
40 will reduce any friction between masking system
36 and the
cells, thus promoting cell integrity (e.g., see FIG.
8).
In this embodiment, article
36 can be pre-coated with a cell-adhesion
inhibitor
42 as shown in FIGS.
2(
a) and (
b). In FIG.
2(
a),
a first surface
37 of mask
36 is contacted with a surface
46
of substrate
44. Preferably, first surface
37 is brought into conformal
contact with surface
46. FIG.
2(
b) shows the results of coating
agent
40 (a cell-adhesion inhibitor) onto mask
36 and substrate
44.
Surface
37 of mask
36 is free of the agent
40. Removal of
mask
36 from substrate
44 followed by placement of mask
36
on surface
31 of article
30 results in unexposed surfaces (second
portions
34) of article
30 free of agent
40. Subsequently,
mask
36 can be used to shield portions of article
30, as shown in
FIG.
2(
c).
FIG.
2(
f) shows the results of removing mask
36 from article
30, exposing first portions
32, followed by application of agent
50 (either a cell-adhesion inhibitor or promoter) to first portions
32.
Where agent
50 is a cell-adhesion promoter, the cells applied onto agent
42 can be allowed to spread onto agent
50, the results of which are
shown schematically in FIG.
2(
h). Thus, the invention provides a
novel medium to study the effects of cell spreading, or other cellular phenomena,
such as chemotaxis, haptotaxis or morphogenesis from a predetermined area (i.e.,
shape and size) of an individual cell to groups of cells.
It can be seen that in this aspect of the invention, a method is provided for
a simple and inexpensive method to grow attached cells within patterned constraints.
In one embodiment, the constraints can be released to allow the cells to spread.
Most current techniques for patterning cells are not directly compatible with a
process that requires the cells to be grown within patterned constraints, and then
releasing those constraints and allowing the cells to migrate. Patterning of cells
is an experimental tool that can be useful, for example, for studying and controlling
the behavior of anchorage-dependent cells. Patterning of cells has been previously
achieved with microcontact printing (see for example, C. S. Chen et al.
Science
1997, 276, 1425-1428; R. Singhvi et al.
Science 1994, 264, 696-698;
Prog. 1998, 14, 378-387; G. P. Lopez et al.
J. Am. Chem. Soc. 1993,
115, 5877-5878; A. Kumar et al.
Appl. Phys. Lett. 1993, 63, 2002-2004; M.
Mrksich et al.
Trends Biotech. 1995, 13, 228-235). Although microcontact
printing is an experimentally convenient technique that has sufficient resolution
to allow the patterning of single cells, in its simplest configuration it does
not allow the cells to be "released" from the pattern; that is, once a pattern
of SAMs has been formed, fibronectin adsorbed, and cells attached, there is no
practical way of changing the pattern or allowing the cells to spread beyond the
boundaries of this pattern. Other patterning methods involve more complex processes.
In another embodiment, agent
42 is a first cell-adhesion promoter and
agent
50 is a second cell-adhesion promoter. This embodiment provides a method
for patterning multiple cell types onto a single substrate in which cells of a
second type can be applied onto agent
50. In one embodiment, the first cell-adhesion
promoter is specific for cells
43 of a first type and the second cell-adhesion
promoter is specific for cells
45 of a second type (FIG.
2(
g)).
FIG. 2 can be described with reference to a specific example. The mask
36
is pre-coated with agent
40, namely BSA, selectively on one of its sides
and in interior channel surfaces and the pre-coated mask is used during the adsorption
of FN to a clean substrate surface (FIG. 2
d). Cells adhere to the surface
of the substrate that is coated with FN while being prevented from adhering to
the walls of the membrane channels or the top of the mask, coated with BSA. Accordingly,
upon peeling, the mask does not damage the cells that remain attached to the surface
