WO2011028579A2 - Aligning cells on wrinkled surface - Google Patents

Aligning cells on wrinkled surface Download PDF

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WO2011028579A2
WO2011028579A2 PCT/US2010/046670 US2010046670W WO2011028579A2 WO 2011028579 A2 WO2011028579 A2 WO 2011028579A2 US 2010046670 W US2010046670 W US 2010046670W WO 2011028579 A2 WO2011028579 A2 WO 2011028579A2
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method
cell
nanometers
surface
cells
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PCT/US2010/046670
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French (fr)
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WO2011028579A3 (en
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Mark L. Baum
James B. Panther Ii
Michelle Khine
Kara Mccloskey
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The Regents Of The University Of California
Shrink Technologies, Inc.
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Publication of WO2011028579A2 publication Critical patent/WO2011028579A2/en
Publication of WO2011028579A3 publication Critical patent/WO2011028579A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells being immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells being immobilised on or in an organic carrier carrier being a synthetic polymer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068General culture methods using substrates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0603Embryonic cells ; Embryoid bodies
    • C12N5/0606Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657Cardiomyocytes; Heart cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00Supports or coatings for cell culture, characterised by material
    • C12N2533/30Synthetic polymers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00Supports or coatings for cell culture characterised by topography
    • C12N2535/10Patterned coating

Abstract

A method is provided for preparing a tissue patch comprising the steps of placing one or more cells on a surface having a texture, which texture has an average height of from about 100 nanometers to about 5 micrometers; allowing the cells to migrate or divide on the surface; and removing the cell population from the textured surface, thereby forming tissue patch. Also provided a method to prepare the surface which method comprises the steps of: a) depositing a metal onto a pre-stressed thermoplastic material; b) reducing the surface area of the receptive material by at least about 60%; and c) preparing the surface via lithography.

Description

ALIGNING CELLS ON WRINKLED SURFACE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional patent application serial number 61/237,245, filed August 26, 2009, the contents of which is hereby incorporated by reference in its entirety into the present disclosure.

BACKGROUND OF THE INVENTION

[0002] Throughout this disclosure, various technical and patent publications are referenced to more fully describe the state of the art to which this invention pertains. These publications are incorporated by reference, in their entirety, into this application.

[0003] Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. Engineered tissues can be used to repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, etc.). Often, the tissues involved require certain mechanical and structural properties for proper functioning. Efforts have been made to make tissues that perform specific biochemical functions using cells within an artificially-created support system (e.g., an artificial pancreas, or a bioartificial liver).

[0004] Examples of engineered tissues include bioartificial liver device, artificial pancreas, artificial bladders, cartilage, Doris Taylor's heart in ajar, tissue-engineered airway, artificial skin constructed from human skin cells embedded in collagen, and artificial bone marrow.

[0005] Advances in stem cell biology made it possible for myocardial regeneration after infarction. Myocardium may be formed in infarcted rodent hearts using human embryonic stem cell (hESC)-derived cardiomyocytes. However, small and highly variable cell to graft size limits its application. In some methods, dispersed cells are injected enzymatically or mechanically directly into the injured left ventricular wall. Advances in cardiac tissue engineering, which seek to generate myocardium-like tissue in vitro and then implant the tissue in vivo, provide better ways to control cell seeding efficiency and graft size.

[0006] In tissue engineering, cells are often implanted or 'seeded' into an artificial structure capable of supporting three-dimensional tissue formation. These structures, typically called scaffolds, are often critical, both ex vivo as well as in vivo, to recapitulating the in vivo milieu and allowing cells to influence their own microenvironments. Scaffolds usually serve at least one of the following purposes: allow cell attachment and migration; deliver and retain cells and biochemical factors; enable diffusion of vital cell nutrients and expressed products; and exert certain mechanical and biological influences to modify the behavior of the cell phase.

[0007] Substrate topographical features of the scaffolds have a great impact on cell attachment and migration and morphology. Surface roughness can affect biocompatibility. Optimal micro-roughness depends on cell type. Nano scale roughness is reported to promote cell attachment, migration and proliferation. This may be partly due to a similarity between the nano scale roughness of the scaffold surface and nanostructures found in natural extra-cellular matrix such as nano-stripes in collagen.

[0008] A number of technologies have been introduced to make rough surface structures, including nano imprint lithography (Charest et al. (2005) J. Vac. Sci. Technol. B 23:3011- 4), laser holography (Clark et al. (2002) Int. J. Biochem. Cell Biol. 34:816e25; Clark et al . (1991) J. Cell Sci. 99:73-7), nano-lithography (Arnold et al. (2004) ChemPhysChem 5:383- 8), nano imprinting methods (Lenherta et al. (2005) Biomaterials 26:563-70), laser machining (Lu et al. (2003) Mater. Lett. 58:29-32; Mirzadeh (2003) Radiat. Phys. Chem. 67:381-5; Zhu et al. (2004) J. Biomed. Mater. Res. B 70B:43-8; Wang et al. (2006) J. Biomed. Mater. Res. A 78:746-54), electrospinning (Schindler et al. (2005) Biomaterials 26:5624-31) and polymer demixing (Dalby et al. (2004) Cell Biol. Int. 28:229-36).

However, these methods require costly manufacture of devices. There is a need to develop a cost effective method to make rough surface structures suitable for tissue engineering and to develop methods for removal of the tissue for use without a scaffold.

SUMMARY OF THE INVENTION

[0009] This invention provides a new method to align or grow cells on a textured, or alternatively termed "wrinkle", surface. The invention also provides methods to make the textured surface conveniently at a very low cost. The invention is useful for tissue engineering, such as generating a cardiac patch.

[0010] Thus in one aspect, this invention provides a method for preparing an aligned cell population comprising the steps of: 1) placing one or more cells on a surface having a texture, which texture has an average height of from about 100 nanometers to about 5 micrometers; and 2) allowing the cells to migrate or divide on the surface, thereby forming an aligned cell population on the textured surface.

[0011] In one aspect, preparation of the textured surface comprises the steps of: a) depositing a metal onto a pre-stressed thermoplastic material; b) reducing the surface area of the receptive material by at least about 60%; and c) preparing the surface via lithography.

[0012] In some embodiments, the cell placed on the textured surface is an isolated stem cell. In one aspect, the isolated stem cell is an embryonic stem cell, a pluriopotent stem cell, a somatic stem cell and or iPS stem cell. In another aspect, the cell is a fetal or neonatal cardiac cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows alignment of cardiac cells. 1 A shows that cardiac cells placed on a flat surface of a polydimethylsiloxane (PDMS) base did not align after 24 hours culturing. IB shows that cardiac cells placed on a textured surface of a PDMS base, as prepared by methods of this invention, align into clusters of cells.

[0014] Figure 2 shows cell alignment after different period of time on surfaces without texture or with different sizes of texture.

[0015] Figure 3 shows the formation of embryoid bodies (EBs). 3A shows the unique high-throughput approach to generating large arrays of EBs by printing honeycombed microwells onto thermoplastics and molding with PDMS. 3B shows EBs generated from mouse ESC aggregates, plated on gelatin on day 8. On Day 14, 17% of the EBs were spontaneously beating (left). Cells were analyzed on day 17 for Flk-1 (middle) and cardiac troponin T (right).

[0016] Figure 4 shows the process flow to fabricate a cardiac waveguide.

[0017] Figure 5A shows a SEM of a wrinkle waveguiding structure; Figure 5B shows aligned CM stained for Cardiac Troponin I, Phalloidin, and DAPI, nucleus; Figure 5C shows distribution of wrinkle wavelengths as a function of metal thickness layer.

[0018] Figure 6 shows fluorescent micrographs over time of cardiac cells cultured on waveguiding substrates. The first column is Actin, the second is Cardiac Troponin and the nuclear stain is also shown. The third column is an overlay of the first 2 columns and the final column is an overlay including the brightfield showing the substrate as well. DETAILED DESCRIPTION OF THE INVENTION

[0019] As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a microfluidic channel" includes a plurality of microfluidic channels.

[0020] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for preparing the microfluidic device. Embodiments defined by each of these transition terms are within the scope of this invention.

[0021] A "thermoplastic material" is intended to mean a plastic material which shrinks upon heating. In one aspect, the thermoplastic materials are those which shrink uniformly without distortion. The shrinking can be either bi-axially (isotropic) or uni-axial

(anisotropic). Suitable thermoplastic materials for inclusion in the methods of this invention include, for example, high molecular weight polymers such as acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVC alloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal), polyacrylates (Acrylic), Poly(methyl methacrylate) (PMMA),

polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), Polycyclohexylene Dimethylene Terephthalate (PCT), polycarbonate (PC), polyhydroxyal kanoates (PHAs), polyketone (PK), polyester polyethylene (PE), polyetheretherketone (PEEK),

polyetherimide (PEI), polyethersulfone (PES), polysulfone polyethylenechlormates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), polyolefm, and spectralon. A "Shrinky-Dink" is a commercial thermoplastic which is used as a children's toy. As used herein, the terms "thermoplastic base" and "thermoplastic cover" refer to thermoplastic material having been subjected to both the etching process as well as heating process. The "thermoplastic base" would be located at the bottom or within the device, and the "thermoplastic cover" is the last layer of one or more layers of thermoplastic base.

[0022] A "solution" is intended to refer to a substantially homogeneous mixture of a solute, such as a solid, liquid, or gaseous substance, with a solvent, which is typically a liquid. The solution can be either aqueous or non-aqueous. Examples of suitable solutes in solutions include fluorescent dyes, biological compounds, such as proteins, DNA and plasma, and soluble chemical compounds. Examples of suitable solids include beads, such as polystyrene beads, and powders, such as a metal powder. A "suspension" is intended to refer to a substantially heterogeneous fluid containing a solid, wherein the solid is dispersed throughout the liquid, but does not substantially dissolve. The solid particles in a suspension will typically settle as the particle size is large, compared to a colloid, where the particle size is small such that the suspension does not settle. Examples of suitable suspensions include biological suspensions such as whole blood, cell compositions, or other cell containing mixtures. It is contemplated that any solution, solid or suspension can be mixed using the mixers disclosed herein, provided that the solid has a particle size sufficiently small to move throughout the channels in the mixer. [0023] Disposed on the thermoplastic material may be an image-forming material is one which is compressed upon heating, and bonds to the plastic and is durable (can be used as a mold for multiple iterations). For example, an "image-forming material" is, in one aspect, intended to mean a composition, typically a liquid, containing various pigments and/or dyes used for coloring a surface to produce an image or text such as ink and printer toner. In addition to an ink, the image forming material can be a metal, such as gold, titanium, silver, a protein, a colloid, a dielectric substance, a paste or any other suitable metal or

combination thereof. Examples of suitable proteins include biotin, fibronectin and collagen. Examples of suitable colloids include pigmented ink, paints and other systems involving small particles of one substance suspended in another. Examples of suitable dielectric substances include metal oxides, such as aluminum oxide, titanium dioxide and silicon dioxide. Examples of suitable pastes include conductive pastes such as silver pastes. [0024] The image forming material can be applied to the thermoplastic material by a variety of methods known to one skilled in the art, such as printing, sputtering and evaporating. The term "evaporating" is intended to mean thermal evaporation, which is a physical vapor deposition method to deposit a thin film of metal on the surface of a substrate. By heating a metal in a vacuum chamber to a hot enough temperature, the vapor pressure of the metal becomes significant and the metal evaporated. It recondenses on the target substrate. As used herein, the term "sputtering" is intended to mean a physical vapor deposition method where atoms in the target material are ejected into the gas phase by high- energy ions and then land on the substrate to create the thin film of metal. Such methods are well known in the art (Bowden et al. (1998) Nature (London) 393: 146-149; Bowden et al. (1999) Appl. Phys. Lett. 75: 2557-2559; Yoo et al. (2002) Adv. Mater. 14: 1383-1387; Huck et al. (2000) Langmuir 16: 3497-3501; Watanabe et al. (2004) J. Polym. Sci. Part 6: Polym. Phys. 42: 2460-2466; Volynskii et al. (2000) J. Mater. Sci. 35: 547-554; Stafford et al. (2004) Nature Mater. 3:545-550; Watanabe et al. (2005) J. Polym. Sci. Part 6: Polym. Phys. 43: 1532-1 537; Lacour et al. (2003) Appl. Phys. Lett. 82: 2404-2406).

[0025] In addition, the image forming material can be applied to the thermoplastic material using "pattern transfer." The term "pattern transfer" refers to the process of contacting an image-forming device, such as a mold or stamp, containing the desired pattern with an image-forming material to the thermoplastic material. After releasing the mold, the pattern is transferred to the thermoplastic material. In general, high aspect ratio pattern and sub-nanometer patterns have been demonstrated. Such methods are well known in the art (Sakurai, et al, US Patent 7,412,926; Peterman, et al, US Patent 7,382,449; Nakamura, et al, US Patent 7,362,524; Tamada, US Patent 6,869,735).

[0026] Another method for applying the image forming material includes, for example "micro-contact printing." The term "micro -contact printing" refers to the use of the relief patterns on a PDMS stamp to form patterns of self-assembled monolayers (SAMs) of an image-forming material on the surface of a thermoplastic material through conformal contact. Micro-contact printing differs from other printing methods, like inkjet printing or 3D printing, in the use of self-assembly (especially, the use of SAMs) to form micro patterns and microstructures of various image-forming materials. Such methods are well known in the art (Cracauer et al, US Patent 6,981 ,445; Fujihira et al, US Patent 6,868,786; Hall et al, US Patent 6,792,856; Maracas et al, US Patent 5,937,758). [0027] "Soft-lithography" is intended to refer to a technique commonly known in the art. Soft-lithography uses a patterning device, such as a stamp, a mold or mask, having a transfer surface comprising a well defined pattern in conjunction with a receptive or conformable material to receive the transferred pattern. Microsized and nanosized structures are formed by material processing involving conformal contact on a molecular scale between the substrate and the transfer surface of the patterning device.

[0028] The term "receptive material" is intended to refer to a material which is capable of receiving a transferred pattern. In certain embodiments, the receptive material is a conformable material such as those typically used in soft lithography comprise of elastomeric materials, such as polydimethylsiloxane (PDMS), gelatin, agarose, polyethylene glycol, cellulose nitrate, polyacrylamide or chitosan. The thermoplastic receptive material, or thermoplastic material, is also a receptive material as it can be etched, for example.

[0029] "Imprint lithography" is intended to refer to a technique commonly known in the art. "Imprint lithography" typically refers to a three-dimensional patterning method which utilizes a patterning device, such as a stamp, a mold or mask.

[0030] A "mold" is intended to mean an imprint lithographic mold.

[0031] A "patterning device" is intended to be broadly interpreted as referring to a device that can be used to convey a patterned cross-section, corresponding to a pattern that is to be created in a target portion of the substrate.

[0032] A "pattern" is intended to mean a mark or design.

[0033] "Bonded" is intended to mean a fabrication process that joins materials, usually metals or thermoplastics, by causing coalescence. This is often done by melting the materials to form a pool of molten material that cools to become a strong joint, with pressure sometimes used in conjunction with heat, or by itself, to produce the bond.

[0034] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about." It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. METHODS FOR PREPARING WRINKLES AND ALIGNING CELLS

[0035] In one aspect, the present invention discloses a method for preparing an aligned cell population comprising the steps of 1) placing one or more cells on a surface having a texture, which texture has an average height of from about 100 nanometers to about 5 micrometers; and 2) allowing the cells to migrate or divide on the surface, thereby forming an aligned cell population on the textured surface.

[0036] In one aspect, the texture has an average height selected from the group consisting of about 200 nanometers, about 300 nanometers, about 500 nanometers, about 700 nanometers, about 1 micrometer, about 2 micrometers, about 3 micrometers, about 4 micrometers, and about 5 micrometers.

[0037] In one aspect, the texture has a periodicity in the range of from about 10 nanometers to about 600 nanometers. In another aspect, the texture has a periodicity selected from the group consisting of about 15 nanometers, about 30 nanometers, about 60 nanometers, and about 600 nanometers.

[0038] The optimal height and periodicity of the texture are cell type dependent. Referring to Fig. 2, the optimal height and periodicity of the texture for a cell type can be

experimentally determined.

[0039] In one aspect, the textured surface is on a base made from a thermoplastic material. Examples of thermoplastic materials suitable for preparing the textured surface include, but are not limited to, poly(methyl methacrylate) (PMMA), polydimethylsiloxane, gelatin, agarose, polyethylene glycol, cellulose nitrate, polyacrylamide, or chitosan. In another aspect, the textured surface is on a base made of polydimethylsiloxane (PDMS).

[0040] A hydrophobic surface is known in the art to be beneficial for cell growth and alignment. Materials like PDMS have a hydrophobic surface and are therefore useful for preparing a textured surface of this invention. A hydrophilic surface, on the other hand, can help cell attachment. In one aspect of the invention, a temporary hydrophilic surface is created by applying an electric charge on the textured surface. The electric charge is removed after the cells attach to the surface.

[0041] In some embodiments, the preparation of the textured surface comprises the steps of: a) depositing a metal onto a pre-stressed thermoplastic material; b) reducing the surface area of the receptive material by at least about 60%; and c) preparing the surface via lithography.

[0042] Steps a) and b) prepare a metal wrinkled surface on the pre-stressed thermoplastic material. Methods for preparing the metal wrinkled surface can be found in PCT Patent Application No. PCT/US2008/1083283, which is incorporated by reference in its entirety.

[0043] In some embodiments, the pre-stressed thermoplastic material is a heat sensitive thermoplastic receptive material. In certain embodiments, the depositing of heat sensitive thermoplastic receptive material is by evaporating, which is a physical vapor deposition method to deposit a thin film of metal on the surface of a substrate. By heating a metal in a vacuum chamber to a hot enough temperature, the vapor pressure of the metal becomes significant and the metal evaporated. It recondenses on the target substrate. The height of the metal is dependent on length of processing time. The thermoplastic substrate must be far enough from the source such that the plastic does not heat up during deposition.

[0044] After the metal is deposited on the thermoplastic, it is placed in an oven, or similar device, to be heated, and upon heating, because of the stiffness incompatibility between the metal and the shrinking thermoplastic, wrinkles form. The spacing between the metal wrinkles can be controlled by the amount of heating, and hence shrinkage.

[0045] Wrinkle height can be controlled by adjusting the metal film thickness. Fig. 17 of the PCT application PCT/US2008/1083283 shows a plot of the maximum average wrinkle height as a function of metal layer thickness. Therefore, one can easily predict the spacing between and height of the metal wrinkles by adjusting the thickness of metal deposited onto the thermoplastic material and the time the thermoplastic material is heated. The thickness of metal deposited onto the thermoplastic material can be easily controlled using the metal deposition methods disclosed herein by adjusting parameters such as time, temperature, and the like. Such methods are well known to one of skill in the art.

[0046] Various heights can be achieved from about 2 nanometers to about 100 nanometers. In an particular embodiment, the height of the metal is about 2 nanometers. In an alternative embodiment, the height of the metal is about 5 nanometers, or alternatively, about 10 nanometers, or alternatively, about 20 nanometers, or alternatively, about 30 nanometers, or alternatively, about 40 nanometers, or alternatively, about 50 nanometers, or alternatively, about 60 nanometers, or alternatively, about 70 nanometers, or alternatively, about 80 nanometers, or alternatively, about 90 nanometers, or alternatively, about 100 nanometers.

[0047] In some embodiments, wrinkle heights can be achieved from about 100

nanometers to about 5 micrometers. In a particular embodiment, the height of the metal is about 200 nanometers. In an alternative embodiment, the height of the metal is about 200 nanometers, or alternatively, about 300 nanometers, or alternatively, about 500 nanometers, or alternatively, about 700 nanometers, or alternatively, about 1 micrometer, or

alternatively, about 2 micrometers, or alternatively, about 3 micrometers, or alternatively, about 4 micrometers, or alternatively, less than about 5 micrometers.

[0048] In addition, the directionality of the wrinkles is controlled by grooving the substrate prior to metal deposition. Alternatively, the directionality of the wrinkles can be controlled by monodirectional shrinking using a uni-axially biasing thermoplastic receptive material. In one embodiment, the method to prepare a textured metal surface further comprises first heating a heat sensitive thermoplastic receptive material under conditions that reduce the size of the thermoplastic receptive material bi-axially by at least about 60%, followed by uni-axially biasing the thermoplastic receptive material to shrink along one axis or dimension prior to depositing a metal onto a heat sensitive thermoplastic receptive material, and reducing the material by at least about 60%, thereby preparing a textured metal surface.

[0049] In one aspect, the size of the textured metal surface is substantially the same as the thermoplastic receptive material before the receptive material was uni-axially biased. In one embodiment, the thermoplastic receptive material is uni-axially biased using heat.

[0050] It is contemplated that any metal can be deposited onto the thermoplastic receptive material to fabricate the metal wrinkles disclosed herein. In some embodiments, the metal is at least one of silver, gold or copper. Depending on the intended use of the metal surface, it may be desired that the metal be deposited in a given pattern or design. The metal can be deposited to only a desired area of the thermoplastic receptive material to form isolated metal sections or 'islands' on the thermoplastic receptive material. Methods for the controlled deposition of metals are well known in the art. [0051] The periodicity of the wrinkle as the wavelength of the wrinkles scale according to the thickness to the 314th power. Therefore, tighter wrinkles are achieved by changing the thickness, or height of the metal layer.

[0052] It is contemplated that any thermoplastic material can be used in the methods disclosed herein. In one aspect of the disclosed invention, the thermoplastic materials are those which shrink uniformly without substantial distortion. Suitable thermoplastic materials for inclusion in the methods of this invention include, for example, high molecular weight polymers such as acrylonitrile butadiene styrene (ABS), acrylic, Poly(methyl methacrylate) (PMMA), celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), ionomers kydex, a trademarked acrylic/PVC alloy, liquid crystal polymer (LCP), polyacetal (POM or Acetal), polyacrylates (Acrylic), polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), Polycyclohexylene Dimethylene Terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polysulfone polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), polyolefm, and spectralon. In one embodiment, the thermoplastic material is polystyrene.

[0053] Alternative embodiments of the methods include, but are not limited to the application of heat to reduce the size of the thermoplastic receptive material by at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively, at least 85%, or alternatively, at least 90%.

[0054] Thus in some embodiments, the pre-stressed thermoplastic material is biaxially biased. In some embodiments, the pre-stressed thermoplastic material is uniaxially biased.

[0055] In some embodiments, the metal is deposited by sputter coating, evaporation or chemical vapor deposition. [0056] In some embodiments, the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity in the range of from about 10 nanometers to about 5 micrometers.

[0057] In some embodiments, the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity in the range of from about 10 nanometers to about 600 nanometers. In one aspect, the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity in the range of from about 15 nanometers to about 100 nanometers. In yet another aspect, the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity selected from the group consisting of about 15 nanometers, about 30 nanometers, about 60 nanometers, and about 600 nanometers.

[0058] In some embodiments, the metal is deposited in a desired pattern.

[0059] In some embodiments, the heat sensitive thermoplastic material is reduced by heating. In some embodiments, the temperature used to heat and reduce the size of the thermoplastic material is from about 100°C to about 250°C, or alternatively from about 120°C to about 220°C, or alternatively from about 150°C to about 200°, or alternatively from about 180°C to about 190°C, or alternatively about 185°C.

[0060] In one aspect, the lithography of step c) comprises soft lithography or imprint lithography. [0061] In one aspect, the lithography of step c) uses a thermoplastic material. In another aspect, the material is selected from the group consisting of polydimethylsiloxane, gelatin, agrose, polyethylene glycol, cellulose nitrate, polyacrylamide, and chitosan.

[0062] In one aspect, the material used in lithography, such as PDMS, is poured onto the textured metal surface, which serves as the mold, as in typical soft lithography, and cured at 110° Celsius for 10 minutes. The cured PDMS device is then peeled off the mold and bonded using a hand-held corona discharger (Haubert et al. (2006) Lab Chip Technical Note 6: 1548-1 549). The whole process from device design conception to working device can be completed within minutes.

[0063] In some embodiments, to induce the formation of cell growth or alignment, the textured surface of the thermoplastic material, such as PDMS, can be soaked in polar and non-polar solvents such as pentane for 12 hours, followed by a solvent change where new pentane is added and is further soaked for 12 hours, next the pentane solvent is replaced with xylene for 7 hours and is replaced with new xylene for another 12 hours, last the microwells are soaked in ethanol for 12 hours prior to use. To simplify the protocol and save time, the first solvent is generally used to swell PDMS as much as possible, then followed by de-swelling gradually. Solvents that swelled PDMS the least: water, nitromethane, dimethyl sulfoxide, ethylene glycol, perfluorotributylamine, perfluorodecalin, acetonitrile, and propylene carbonate. Solvents that swelled PDMS the most:

diisopropylamine, triethylamine, pentane, and xylenes. For an example of swelling and de- swelling procedure, soak in pentane for 24 hours; pentane 7 hours; then xylene isomers plus ethylbenzene 98.5% 1-2 hours; then xylenes for 16 hours; xylenes for 7 hours; then EtOH 1- 2 hours, then EtOH again for 16 hours, and finally EtOH for 7 hours. Then soak in about 1L of sterile DI water overnight and dry at 70°C overnight.

[0064] Various types of cells may be grown or aligned on the textured surface of the present invention. In one aspect, the cell is an isolated prokaryotic or eukaryotic cell. In another aspect, the cell is an isolated eukaryotic cell.

[0065] In one aspect, the isolated eukaryotic cell may be an isolated stem cell. In some embodiments, the isolated stem cell is selected from the group consisting of an embryonic stem cell, a pluriopotent stem cell, a somatic stem cell and an induced pluripotent stem cell (iPS stem cell). Methods to grow and culture such cells are known in the art. See for example, US Patent Publ. Nos. 200910081 784; 200910075374; 200910068742; and 200910047263, each of which is incorporated herein by reference.

[0066] In one aspect, the isolated stem cell is of animal origin. In some embodiments, the animal origin is mammalian, simian, bovine or murine. In one aspect, the animal origin is human.

[0067] In another aspect, the isolated eukaryotic cell is a fetal or neonatal cell.

[0068] In one aspect, the eukaryotic cell is selected from the group consisting of a smooth muscle cell, a bladder smooth muscle cell, a keratocyte, a corneal epithelial cell, an endothelial cell, a vascular endothelial cell, an osteoblast cell, a fibroblast cell, a myoblast cell, a nerve cell, a skin cell, and a cardiac cell. In another aspect, the eukaryotic cell is a fetal or neonatal cardiac cell. [0069] The present invention can be used to form a cardiac patch by aligning cardiac cells on the textured surface. Cardiac patches can be generated with methods known in the art, for example, Stevens et al. discloses a method to grow a human cardiac patch from embryonic stem cells (Stevens et al. (2009) Tissue Engineering

15:DOI: 10.1089/ten.tea.2008.0151).

[0070] Thus in one aspect, fetal or neonatal heart cells of step 2) are allowed to form a cardiac patch.

[0071] In certain aspects, the cardiac patch may be removed from the textured surface and used alone or in conjunction with additional cardiac patches for in vivo cardiac repair.

[0072] Also provided is a method for assaying a potential agent for the ability to affect cell migration, growth and/or differentiation of an isolated stem cell, comprising the steps of placing a cell with an agent on a surface of a thermoplastic material and allowing the cell to migrate or divide on the metal surface thereby assaying for the agent's effect on the cell's migration, growth and/or differentiation, wherein the surface has a texture having an average height of from about 100 nanometers to about 5 micrometers.

[0073] Also provided is a kit for use in preparing an aligned cell population comprising a thermoplastic material having a surface, and instructions to prepare an aligned cell population, which surface has a texture that has an average height of from about 100 nanometers to about 5 micrometers. EXAMPLES

[0074] The present technology is further understood by reference to the following examples. The present technology is not limited in scope by the examples, which are intended as illustrations of aspects of the present technology. Any methods that are functionally equivalent are within the scope of the present technology. Various

modifications of the present technology in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

Example 1. Rapid generation of textured surface for cell alignment

[0075] A piece of unshrunken polystyrene sheet (PS) is cut (approximately 2.5 X 3 cms). It is cleaned with isopropanol and distilled water and allowed to dry. A 60 nm layer of silver is deposited on the PS using a metal sputter. Alternatively, metals can be deposited onto the shrinkable thermoplastic by either thermal evaporation or sputtering. The metal coated PS is fixed by the opposite side to a glass slide using binder clips and baked for 10 minutes at 165°C. This will shrink the PS sheet in one direction creating aligned metal wrinkles. The thickness, or height of the deposited metal is dependent on length of processing time. The plastic substrate should be far enough from the source such that the plastic does not heat up during deposition. A wide range of thicknesses, or heights, of deposited metal are accomplished, from about 5 nanometers to about 90 nanometers.

[0076] Upon heating, because of the stiffness incompatibility between the metal and the shrinking thermoplastic, wrinkles (textures) formed. The spacing between the wrinkles can be controlled by the amount of heating, and hence shrinkage. In addition, the directionality of the wrinkles can be controlled by grooving the substrate prior to metal deposition.

Finally, the periodicity of the wrinkle as the wavelength of the wrinkles scale according to the thickness to the 314th power. Therefore, tighter wrinkles were achieved by changing the thickness, or height of the metal layer. After heating, the PS is cooled to 75°C to avoid cracking. The wrinkled mold is then attached to a petri dish with double-sided tape.

[0077] Then PDMS is poured onto the mold as in typical soft lithography, and cured at 110° Celsius for 10 minutes. The cured PDMS device is then peeled off the mold and bonded using a hand-held corona discharger (Haubert et al. (2006) Lab Chip Technical Note 6: 1548-1 549). The whole process from device design conception to working device can be completed within minutes.

[0078] Alternatively, PDMS (1 : 10) is added to the mold and allowed to polymerize at 75°C for 30 to 45 minutes. A 0.5 by 0.5 cm piece of micropatterned PDMS is cut and cleaned using a piece of tape to remove any dirt or undesired materials. The patterned PDMS is bonded to a piece of glass with uncured PDMS (1 : 10) and left to polymerize at 75°C for 30 to 40 minutes. The PDMS is then treated with a corona discharger for 15 seconds and subsequently sterilized with 70 % ethanol solution for 15 minutes under an ultraviolet light source. The chip is then washed twice with sterile phosphate buffered saline (PBS) and placed on a 48 well-plate. Example 2. Directed Differentiation of hESCs

[0079] Applicants developed a high throughput method for the generation of large numbers of embryoid bodies (EBs) from human embryonic stem cells (hESC). The method incorporates use of a Honeywell microchip that generates large numbers (-20,000 EBs/24 well plate) of embryoid bodies (EBs) with well-controlled sizes from single cells (Figure 3). Human ESC are broken into single cells with rock inhibitor and seeded at high density into the honey well microchips. After 8 days in the wells, the EBs are replated onto gelatin- coated dishes and given to induce CM induction. Spontaneously beating sections of EBs are then manually dissected from the remaining cells in the EB outgrowth. Example 3. Aligning cardiac cells on textured surface

[0080] Applicants have developed a rapid method to fabricate tunable bio-mimetic anisotropic waveguiding wrinkled nano-topographies to align and culture cardiomyocytes. Whereas other approaches to align CM include using electric fields, recent studies have demonstrated the ability to align CM passively. In contrast to their approaches, Applicants' method of creating effective waveguides is very simple, easily tunable for optimal size, and the cells can be detached as intact sheets from the alignment structures.

[0081] Applicants have previously demonstrated such cell alignment with murine neonatal cardiac myocytes (NNCM). This substrate topography is fabricated using polystyrene (PS) biaxially pre-stressed sheets coated with a nanometric layer of metal. The wrinkle length scales arise from a competition between the elastic bending energy of the film and the elastic energy of deformation of the shape memory polymeric substrate.

For wrinkles of wavelength λ and amplitude ζ, the bending energy of the film Fm ~ Ymh 3 ζ 2 / λ4, where Ym is the Young's modulus of the metal and h is the thickness of the film. The elastic deformation energy of the substrate Fps ~ Υρ8ζ /χ , where Yps is the Young's modulus of the polystyrene substrate and χ is the penetration depth of the strain field into the

2 2

substrate. Since the total strain Δ~ ζ / λ is fixed by the overall shrinkage, minimizing the total elastic energy Fm+Fps yields the wrinkle wavelength

Figure imgf000017_0001
[0082] When the thickness of the substrate is large compared to the wavelength χ ~ λ, this yields:
Figure imgf000018_0001

[0083] The scaling relations above have allowed Applicants to achieve desired features on optimal length scales in a systematic fashion. Applicants can thus culture myocytes on these wrinkled patterns as they align within days and follow the waveguiding pattern. Applicants have been developing waveguide patterns from 300-800nm.

[0084] Notably, this wrinkled substrate resemble more the fibrous environment provided by the cardiac tissue the generated metal wrinkle waveguide pattern can serve as either a soft lithography mold to fabricate any conforming polymer substrate or directly, by functionalization of the gold and subsequent deposition of the cells on the gold waveguides.

[0085] Prior to placing cells onto the textured surface of PDMS, the surface was treated with solvent as described previously. Prior to loading the cells, the PDMS device was electrically charged to create a temporary hydrophilic environment facilitating cell attachments. Alternatively, the substrate can be coated with laminin, fibronectin or collagen IV, depending on the cell type. For example, for cardiomyoctyes, laminin and fibronectin were used at a concentration of 1 μg/μl. For stromal cells, fibronectin was used at a concentration of 0.1 μg/cm . The coated substrates were allowed to air-dry in a biohood overnight.

[0086] The dried, coated substrate were rinsed with sterile PBS. 500 μΙ_, of media was added to each well. Cells should be seeded at a high density (for example, for

5 2

cardiomyocytes, use 0.3 X 10 cells/cm ). The cells are incubated in 95 % air/ 5% C02 at 37°C for one (1) hour. Media is changed every 72 hours or at any other time depending on cell type.

[0087] The PDMS device was placed in a well, and fetal cardiac cells isolated from mice were placed on the PDMS device in the well. The wells were examined under microscope after 4 hours, 24 hours, 48 hours, and 72 hours to examine alignment of the cardiac cell.

[0088] As shown in Fig. 1 , cells did not migrate or migrated at a random direction on a flat surface. On the textured PDMS surface, the cells migrated and aligned in the direction of the texture. As shown in Fig. 2, cardiac patches started to form at about 24 hours. The alignment also depended on periodicity of the texture and time of culturing.

Tunable Cardiac Waveguides

[0089] As described herein, Applicants have developed a new approach to create tunable nano to micro sized anisotropic wrinkles to align and culture CM. The substrate topography is fabricated using polystyrene (PS) biaxially pre-stressed sheets coated with a nanometric layer of metal (Figure 4).

[0090] The wrinkle length scales arise from a competition between the elastic bending energy of the film and the elastic energy of deformation of the shape memory polymeric substrate. This then can be used for a re-useable mold for polydimethylsiloxane (PDMS). The PDMS substrate is then coated with fibronectin and then the cells are seeded onto the substrate.

Example 4. Assaying for CM Alignment - Yes tense of text is correct. Keep.

[0091] Applicants will assay for CM alignment by fluorescence staining as well as functional assays. The fluorescence stains will help Applicants visualize actin alignment, cell-to-cell communication by gap junctions, nuclear alignment, and the expression of cardiac troponin.

[0092] After ensuring the cells are properly aligned, Applicants will perform functional assays. In particular, Applicants will perform Ca+ signaling assays and action potential propagation assays. Applicants will measure conduction velocity both in the aligned and in the perpendicular direction. These assays will be performed both prior to and after preconditioning with mechanical and then electrical stimulation. In addition, Applicants will follow of cardiomyocyte cells by assaying the expression of Nkx2.5 to examine

cardiovascular progenitor phenotype (CVP), Mef2c and GATA-4 as evidence of cardiac progenitor phenotype (CP) as well as a-MHC and β-MHC as evidence of myocardiac cell development. The correct alignment and formation of tight junctions will be assayed by immunohistochemistry of N-cadherin and Connexin-43.

[0093] Assay for functional CM phenotype can be accomplished by calcium signaling, action potential propagation, RT-PCR and Immunohistochemistry. Because Applicants hypothesize that providing these structural cues to stem cell derived-CM will better recapitulate the natural micro niche to help maturate them into functional CM, Applicants will monitor the cells for CD- 144 and B-MHC.

Example 5. Mechanical Stretching for Improved Contractile Phenotype

[0094] Myocardial function can be improved by the ability of the transplanted hESC- derived CM to contract properly, restoring the ventricle's ability to pump blood effectively.

[0095] In the native heart, natural growth causes mechanical load on the cardiomyocytes, causing them to be gradually elongated and hypertrophied by mechanical load. It has therefore been hypothesized by many that heart tissue constructs could be similarly strengthened by applied chronic mechanical forces.

[0096] Sustained forces on cardiac myocytes have been shown to improve growth, morphology, orientation, mitogen-activated protein kinase activation, and gene expression. Phasic stretch of planar tissue-engineered heart tissue has been shown to induce

hypertrophic growth and considerable functional improvement, including 2-4x

improvements in force of contraction.

[0097] Cyclic stretching (as experienced in vivo) has been shown to promote the expression of contractile phenotype and increase significantly the production of elastin as well as cell proliferation.

[0098] To chronically stretch the aligned CM sheets, Applicants will employ the Flexcell FX-5000 Tension system, which is compatible with the format our of cell-substrate sheets. Applicants will apply 10% strain load at a frequency of 2Hz for sustained variable durations of 1,3 ,5 , and 7 days. Applicants will monitor cell proliferation, sarcomere shortening, and functional force contraction improvements.

Example 6. Electrical stimulation of CM for electrophysiological matching

[0099] While many existing efforts focus on cardiac differentiation, few studies lead to the generation of functionally mature CM. In contrast to the normally quiescent-yet- excitable adult phenotype, ESC-derived CM exhibit high degrees of spontaneous firing. This spontaneous firing, along with impaired cell-to-cell coupling, has been shown to cause arrhythmias (delays after depolarization). Studies lend promise by demonstrating that hESC-derived CMs can maturate over time during in vitro differentiation (over > 3 months). 2+ 2+

[0100] In adult CMs, Ca entry into the cytosol through sarcolemmal L-type Ca

2_|_

channels triggers the release of Ca from the sarcoplasmic reticulum [SR] during an action

2_|_

potential. This process escalates the cytosolic Ca , activating contraction. For relaxation,

2+ 2+

elevated Ca is pumped back into the SR. Proper Ca handling properties of hESC-CMs are therefore crucial for functional integration and cardiac excitation-contraction coupling with the heart after transplantation.

[0101] Studies suggest that at this immature proarrhythmic developmental stage,

2_|_

contraction may not depend on sarcoplasmic reticulum Ca release; rather, it is thought that

2_|_

voltage-dependent Ca current present in hESC-CM may contribute to mechanical function. Liu et al. discovered that functional SRs are indeed expressed in hESC-CMs, lending hope that driven of Ca handling properties of hESC-CMs may be possible for enhanced contractile functions.

[0102] Radisic et al. paced (2ms square pulses) neonatal rat heart cells at 1 Hz, 5V/cm and showed an increase in cell alignment and coupling.

Example 7. Cardiomyocyte-Sheet Removal

[0103] The approach of Applicants' cell alignment is compatible to the cell-sheet approach pioneered by Nakajima et al. using the temperature-responsive polymer, poly(N- isopropylacrylamide)(PIPAAm). With this approach, the grafted polymer configuration changes such that it is no longer cell adhesive when the temperature is lowered (below 32°C). Upon temperature actuation, the cells are released together with intact membrane proteins. Because this type of release from the surface does not disrupt membrane and adhesive proteins as with protease digestion, studies have demonstrated that cells using PIPA Ammaintain differentiated functions better. After the CM sheets are lifted off, they can be stacked on top of each other to create thicker patches.

[0104] Applicants' cell types consist of a myogenic strategy of repair, using CM for generating contracting cardiac tissue, combined with an angiogenic strategy of repair, using vascular endothelial cells (EC) for building neovessels. Creating thick cardiac patches of several cell layers without vascularization results in necrosis of the inner cells. However, when vasculature is introduced, considerably thicker cardiac patches (up to 8mm) are achievable. [0105] The approach therefore is to create the aligned CM sheets, remove them while retaining their anisotropic orientation and intercellular connectivity, and then interweave them with EC sheets. Soejima et al. demonstrated that a single layer of EC enhances capillary formation in vivo. As such, it is reasonable to hypothesize that heterogenous layering of EC between CM layers may promote neovascularization. To this end,

Applicants will use their microfluidic expertise to create a network of channels to seed the EC for neovascularization.

[0106] Applicants results indicate that neonatal CM align, interact to form cardiac tissue that communicate with each other (apparent through calcium signaling), and continue to beat while maturating and aligning. Applicants can achieve a co-culture of various cell types (CMs, ECs, and fibroblasts), all of which align to the guiding patterns.

[0107] While the present invention is exemplified and illustrated by the use of polystyrene sheets to fabricate channel structures and molds, it would be obvious to those of skill in the art that any thermoplastic receptive material that can be patterned to control the dimensions of the channel defining walls and thereby their size, can be used to fabricate the devices disclosed and claimed herein. In addition, although several other embodiments of the invention are described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.

Claims

What is claimed is:
1. A method for preparing a tissue patch comprising the steps of:
a) placing one or more cells on a surface having a texture, which texture has an average height of from about 100 nanometers to about 5 micrometers;
b) allowing the cells to migrate or divide on the surface, and
c) removing the cells from the surface, thereby forming cardiac patch.
2. The method of claim 1, wherein the texture has an average height selected from the group consisting of about 200 nanometers, about 300 nanometers, about 500 nanometers, about 700 nanometers, about 1 micrometer, about 2 micrometers, about 3 micrometers, about 4 micrometers, and about 5 micrometers.
3. The method of claim 1 or claim 2, wherein the texture has a periodicity in the range of from about 10 nanometers to about 600 nanometers.
4. The method of claim 2, wherein the texture has a periodicity selected from the group consisting of about 15 nanometers, about 30 nanometers, about 60 nanometers, and about 600 nanometers.
5. The method of claim 1, wherein the textured surface is on a material selected from the group consisting of polydimethylsiloxane, gelatin, agrose, polyethylene glycol, cellulose nitrate, polyacrylamide, and chitosan.
6. The method of claim 1, wherein the textured surface is on polydimethylsiloxane.
7. The method of claim 1, wherein preparation of the surface comprises the steps of: a) depositing a metal onto a pre-stressed thermoplastic material;
b) reducing the surface area of the receptive material by at least about 60%; and c) preparing the surface via lithography.
8. The method of claim 7, wherein the pre-stressed thermoplastic material is uniaxially biased.
9. The method of claim 7, wherein the pre-stressed thermoplastic material is biaxially biased.
10. The method of claim 7, wherein the metal is deposited by sputter coating, evaporation or chemical vapor deposition.
11. The method of claim 7, wherein the metal is deposited in a thickness of from about 2 nanometers to about 100 nanometers.
12. The method of claim 7, wherein the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity in the range of from about 10 nanometers to about 5 micrometers.
13. The method of claim 7, wherein the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity in the range of from about 10 nanometers to about 600 nanometers.
14. The method of claim 7, wherein the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity in the range of from about 15 nanometers to about 100 nanometers.
15. The method of claim 7, wherein the pre-stressed thermoplastic material is reduced to achieve a surface texture having a periodicity selected from the group consisting of about 15 nanometers, about 30 nanometers, about 60 nanometers, and about 600 nanometers.
16. The method of claim 7, wherein the metal is deposited in a desired pattern.
17. The method of claim 7, wherein the heat sensitive thermoplastic material is reduced by heating.
18. The method of claim 7, wherein the lithography of step c) comprises soft
lithography or imprint lithography.
19. The method of claim 7, wherein the lithography of step c) uses a material selected from the group consisting of polydimethylsiloxane, gelatin, agrose, polyethylene glycol, cellulose nitrate, polyacrylamide, and chitosan.
20. The method of claim 1, wherein the cell is an isolated prokaryotic or eukaryotic cell.
21. The method of claim 20, wherein the cell is an isolated eukaryotic cell.
22. The method of claim 21, wherein the isolated eukaryotic cell is an isolated stem cell.
23. The method of claim 22, wherein the isolated stem cell is selected from the group consisting of an embryonic stem cell, a pluriopotent stem cell, a somatic stem cell and an iPS stem cell.
24. The method of claim 22, wherein the isolated stem cell is of animal origin.
25. The method of claim 24, wherein the animal origin is mammalian, simian, bovine or murine.
26. The method of claim 25, wherein the animal origin is human.
27. The method of claim 21, wherein the isolated eukaryotic cell is a fetal or neonatal cell.
28. The method of claim 21, wherein the eukaryotic cell is selected from the group consisting of a smooth muscle cell, a bladder smooth muscle cell, a keratocyte, a corneal epithelial cell, an endothelial cell, a vascular endothelial cell, an osteoblast cell, a fibroblast cell, a myoblast cell, a nerve cell, a skin cell, and a cardiac cell.
29. The method of claim 28, wherein the eukaryotic cell is a fetal or neonatal cardiac cell.
30. The method of claim 28, wherein the fetal or neonatal heart cell of step 2) is allowed to form a cardiac patch.
31. The method of claim 30, further comprising layering the cardiac patch on one or more additional cardiac patches.
32. The method of claim 1, wherein removing the cells from the surface comprises exposing the cells to poly(N-isopropylacrylamide)(PIPAAm).
33. A kit for use in preparing a tissue patch comprising a thermoplastic material having a surface, and instructions to prepare a tissue patch, which surface has a texture that has an average height of from about 100 nanometers to about 5 micrometers.
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