WO2007103135A2 - Substrats de croissance de cellules actives et utilisations de ceux-ci - Google Patents

Substrats de croissance de cellules actives et utilisations de ceux-ci Download PDF

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WO2007103135A2
WO2007103135A2 PCT/US2007/005291 US2007005291W WO2007103135A2 WO 2007103135 A2 WO2007103135 A2 WO 2007103135A2 US 2007005291 W US2007005291 W US 2007005291W WO 2007103135 A2 WO2007103135 A2 WO 2007103135A2
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Prior art keywords
substrate
cell
kpa
scaffold
diameter
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WO2007103135A3 (fr
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Michael P. Sheetz
James C. Hone
Monica Tanase
Samuel J. Wind
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Columbia University in the City of New York
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Columbia University in the City of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials 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
    • A61L27/38—Materials 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

  • the shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms. [0005] Throughout the biological kingdom there is a wide diversity of shapes, and this phenomenon has interested physical biologists for a long time (see On Growth and Form. D'Arcy W. Thompson (Dover Publications, 1992)).
  • the invention is based, in part, on the finding that a cell growth substrate having a variable rigidity can be constructed and utilized to control cellular functions and responses.
  • the invention provides for a cell growth substrate comprising: a) a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa; and b) a growth medium, wherein the substrate is permeable to liquids, gases, and cellular by-products.
  • the polymer is not collagen, not hyaluronic acid, not polydimethylsiloxane (PDMS), and not a carbohydrate based gel.
  • the polymer comprises an anionic polymer, a cationic polymer, an amphipathic polymer, a neutral polymer, a synthetic polymer, or any combination thereof.
  • the cationic polymer comprises chitosan or polylysine.
  • the amphipathic polymer comprises gelatin, fibrin, or carboxymethyl chitin.
  • the neutral polymer comprises dextran, agarose, or pullulan.
  • the synthetic polymer comprises a polyester or derivative thereof.
  • the polyester comprises polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, or polyacrylamide.
  • the polymer is functionalized with chemical groups.
  • the substrate further comprise a magnetic material, a Piezo actuator, or a combination thereof. The substrate contains such a material in order to provide a way to cause the substrate to be moved, strained or stretched.
  • the magnetic material comprises nickel or iron.
  • the magnetic material is in the form of a bead.
  • the bead is about 0.1 mm to about 5 mm in diameter, about 0.2 mm to about 4 mm in diameter, about 0.3 mm to about 3 mm in diameter, about 0.4 mm to about 2 mm in diameter, or about 0.5 mm to about 1 mm in diameter. In one embodiment, the bead is about 2.7 mm in diameter.
  • the magnetic material is in the form of a nanowire. In one embodiment, the nanowire is about 2-50 mm in length, about 3-40 mm in length, about 4-30 mm in length, about 5-20 mm in length, or about 6-10 mm in length.
  • the nanowire is about 30 mm in length, hi one embodiment, the nanowire is about 100-500 nm in diameter, about 150-450 nm in diameter, about 200-400 nm in diameter, or about 250-350 nm in diameter. In one embodiment, the nanowire is about 300 nm in diameter.
  • the elastic modulus is about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
  • the invention also provides for an apparatus for applying a perturbation to a cell in vitro, the apparatus comprising: a) the substrate described herein; and b) an external force generator associated with the substrate.
  • the external force generator comprises an electrical force, a magnetic force, a mechanical force, or a combination thereof.
  • the external force generator comprises a pipet-assisted manipulation device, a laser tweezer, an optical trap, or a magnetic field generator.
  • the external force generator stretches the substrate.
  • the substrate is stretched intermittently.
  • the external force generator comprises a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate.
  • the apparatus further comprises a means for quantifying a cellular response to the perturbation.
  • the cellular response quantified is cell growth, cell differentiation, apoptosis, cell movement, cell proliferation, cell morphology changes, or a combination thereof.
  • the invention provides a scaffold for supporting cells, the scaffold comprising: a) portions of the substrate described herein; and b) fibers stretched over the substrate portions.
  • the portions are strips in the form of a fence.
  • the fence is about 0.5 ⁇ m to about 3 ⁇ m thick, about 0.6 ⁇ m to about 2.5 ⁇ m thick, about 0.7 ⁇ m to about 2 ⁇ m thick, about 0.75 ⁇ m to about 1.5 ⁇ m thick, or about 0.8 ⁇ m to about 1 ⁇ m thick. In one embodiment, the fence is about 2 ⁇ m thick.
  • the fence is about 3 ⁇ m to about 12 ⁇ m in height, about 4 ⁇ m to about 11 ⁇ m in height, about 5 ⁇ m to about 10 ⁇ m in height, about 5.5 ⁇ m to about 9 ⁇ m in height, or about 6 ⁇ m to about 8 um in height. In one embodiment, the fence is spaced about 10 ⁇ m to about 200 ⁇ m apart, about 15 ⁇ m to about 150 ⁇ m apart, about 20 ⁇ m to about 100 ⁇ m apart, or about 25 ⁇ m to about 50 ⁇ m apart.
  • the fence is spaced about 25 ⁇ m apart, hi one embodiment, the fence is less than about 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 ⁇ m in length. In one embodiment, the fiber is absorbable.
  • the fiber comprises polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide-lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, or any combination thereof.
  • PGA polyglycolic acid
  • PLA polylactic acid
  • PDA polyglycolide-lactide
  • polycaprolactone polydioxanone
  • polyoxalate polyoxalate
  • a polyanhydride a poly(phosphoester)
  • catgut suture collagen
  • silk chitin
  • chitosan hydroxyapatite
  • bioabsorbable calcium phosphate hyaluronic acid
  • elastin or any combination thereof.
  • the fiber is about 50 nm to about 1500 run in diameter, about 100 nm to about 1250 nm in diameter, about 200 nm to about 1000 nm in diameter, about 250 nm to about 900 nm in diameter, about 300 nm to about 800 nm in diameter, about 350 nm to about 750 nm in diameter, about 400 nm to about 700 nm in diameter, or about 500 nm to about 600 nm in diameter.
  • the fiber is coated with a growth factor, an extracellular matrix molecule, or a combination thereof.
  • the extracellular matrix molecule comprises hyaluronic acid, collagen, chondroitin, or a combination thereof.
  • the invention also provides a method for stimulating a cellular response in vitro, the method comprising: a) plating a cell on, or in, the substrate described herein or the scaffold described herein; b) perturbing the substrate or the scaffold containing the cell; and c) detecting the cellular response, the perturbation of the substrate or the scaffold stimulating the cellular response.
  • the perturbation comprises a mechanical perturbation, an electrical perturbation, a magnetic perturbation, or any combination thereof.
  • perturbing comprises subjecting the substrate or the scaffold to the external force generator.
  • the external force generator stretches the substrate or the scaffold.
  • the substrate or scaffold is perturbed intermittently.
  • the substrate or scaffold is perturbed up to 10 times per hour, up to 5 times per hour, up to 2 times per hour, or 1 time per hour. In one embodiment, the substrate or scaffold is perturbed up to 10 times per day, up to 5 times per day, up to 2 times per day, or 1 time per day. In one embodiment, the substrate or scaffold is perturbed up to 10 times per week, up to 5 times per week, up to 2 times per week, or 1 time per week. In one embodiment, the cellular response is cell growth, cell differentiation, apoptosis, cytokinetics, mo ⁇ hological changes, or a combination thereof.
  • the cellular response is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, mRNA expression levels, a change in cell morphology or a combination thereof.
  • the substrate or the scaffold has an elastic modulus of about 3 kPa. In one embodiment, the substrate or the scaffold promotes a fibroblast cellular response. In one embodiment, the substrate or the scaffold has an elastic modulus of about 2 kPa. In one embodiment, the substrate or the scaffold promotes a myoblast cellular response. In one embodiment, the substrate or the scaffold has an elastic modulus of about 1 kPa. In one embodiment, the substrate or the scaffold promotes a neuronal cellular response. In one embodiment, the cell is obtained from a tissue. In one embodiment, the cell (a) is a primary cell, (b) is a cell from a cell culture that has been passaged, or (b) is a cell from a cell line.
  • the invention also provides a method for promoting growth of a cell in vivo, the method comprising: a) inserting the scaffold into a subject; and b) applying a magnetic field to the subject, wherein the application of the magnetic field promotes growth of the cell.
  • the scaffold has an elastic modulus of about 0.3 kPa to about 8 kPa, about 0.5 kPa to about 7 kPa, about 0.6 kPa to about 6 kPa, about 0.7 kPa to about 6 kPa, about 0.8 kPa to about 5 kPa, about 0.9 kPa to about 4 kPa, or about 1.0 kPa to about 3 kPa.
  • the scaffold has an elastic modulus of about 3 kPa. In one embodiment, the scaffold promotes a fibroblast cellular response. In one embodiment, the scaffold has an elastic modulus of about 2 kPa. In one embodiment, the scaffold promotes a myoblast cellular response. In one embodiment, the scaffold has an elastic modulus of about 1 kPa. In one embodiment, the scaffold promotes a neuronal cellular response. In one embodiment, cell growth comprises wound healing in a subject. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human, a dog, a cat, a mouse, a rat, a horse, a pig, a cow, or a bird.
  • One aspect of the present invention provides for a cell maintenance substrate permeable to liquids, gases, and cellular by-products, that is made up of a growth medium and a polymer with a constant elasticity through stretch and a rigidity where the elastic modulus (E) is in the range of about 0.1 kPa to about 10.0 kPa.
  • the elastic modulus of the substrate is about 0.3 kPa to about 8 kPa.
  • the polymer is cross-linked.
  • the polymer comprises natural polymers and their derivatives, synthetic polymers and their derivatives, or some combination.
  • the natural polymers may include anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers.
  • the anionic polymers comprise hyaluronic acid, alginic acid, carageenan, chondroitin sulfate, dextran sulfate, or pectin.
  • the cationic polymers may include chitosan or polylysine.
  • the amphipathic polymers may include of collagen, gelatin, fibrin, or carboxymethyl chitin.
  • the neutral polymers may inlcude dextran, agarose, or pullulan.
  • the synthetic polymers can be polyesters or derivatives thereof.
  • the polyesters comprise polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyacrylamide, or polydimethylsiloxane.
  • the cell maintenance substrate of the present invention further includes a magnetic material.
  • the magnetic material may comprise nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt.
  • the nanowire is made of nickel.
  • the magnetic material is in the form of a bead.
  • the diameter of the magnetic bead is greater than or equal to about 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, 0.8, 0.9, 1.0, 2.0, 2.5, 3, 4, or 5 ⁇ m in diameter. In particular embodiments, the diameter of the
  • the magnetic material is in the form of a nanowire.
  • the diameter of the magnetic wire is greater than or equal to about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 run in diameter.
  • the diameter of the wire is about 300 nm in diameter.
  • the length of the magnetic wire is greater than or equal to about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ⁇ m in length. In specific embodiments, the length of the magnetic wire is about 30 ⁇ m in length.
  • the invention provides an apparatus for applying a mechanical perturbation to a cell in vitro, wherein the apparatus is made up of the cell maintenance substrate described above and an external force generator associated with the substrate.
  • the external force generator acts as a means for physically manipulating the substrate.
  • the external force generator may entail a pipet-assisted manipulation device.
  • the external force generator may be a laser tweezer.
  • the external force generator comprises an optical trap.
  • the external force generator stretches the substrate.
  • the external force generator may include a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate.
  • the external force generator can be a magnetic field generator.
  • the apparatus of the present invention may further encompass a means for quantitating a cellular response to the mechanical perturbation.
  • the cellular response quantitated is cell growth, cell differentiation, apoptosis, cell movement, cell morphology changes, or some combination of these responses.
  • Elastic modulus (E) refers to tensile elasticity and reflects the measure of the stiffness of a given material.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa.
  • the substrate promotes a fibroblast cellular response.
  • the substrate has an elastic modulus of about 2 kPa.
  • the substrate promotes a myoblast cellular response.
  • the substrate has an elastic modulus of about 1 IcPa.
  • the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention.
  • the cell is excised from a tissue.
  • the cell is excised from the tissue of an animal.
  • the cell is excised from the tissue of a mammal, hi some embodiments, the cell is excised from the tissue of a human.
  • the cell is obtained from a cultured cell line.
  • the invention provides a method for stimulating growth of a cell in vitro, wherein cells are plated on or in the cell maintenance substrate described above, the substrate containing the cells is perturbed (for example, mechanically, electrically, or magnetically), and growth of the cell is subsequently detected.
  • mechanically perturbing the substrate entails subjecting the substrate to an external force generator described above which results in stimulating growth of cells in the substrate.
  • growth of the cell is detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or some combination of these measurements.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
  • the invention provides a method for stimulating cell differentiation in vitro, wherein cells are plated on or in the cell maintenance substrate previously described above, the substrate containing the cells is mechanically perturbed, and differentiation of cells is detected. Mechanical perturbation of the substrate stimulates the differentiation of the cell and entails subjecting the substrate to an external force generator described above.
  • differentiation of the cell is detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or some combination of these measures.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above. [0016] In another aspect, the invention provides for a method of stimulating a cellular response in vitro.
  • the method includes plating a cell on or in the cell maintenance substrate described above, mechanically perturbing the substrate containing the cell, and detecting a cellular response, wherein the mechanical perturbation of the substrate stimulates a cellular response.
  • mechanically perturbing the substrate entails subjecting the substrate to an external force generator described above.
  • the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination.
  • the cellular response can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or some combination.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In another embodiment, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • the current invention provides for a method of promoting growth of a cell in vivo, which includes creating a matrix ex vivo from the cell maintenance substrate described above, inserting the matrix into the body of a subject, and applying a magnetic field to the subject. Application of the magnetic field promotes the growth of the cell within the subject.
  • the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa. In particular embodiments, the substrate has an elastic modulus of about 3 kPa. In a further embodiment, the substrate promotes a fibroblast cellular response. In some embodiments, the substrate has an elastic modulus of about 2 kPa. In other embodiments, the substrate promotes a myoblast cellular response. In particular embodiments, the substrate has an elastic modulus of about 1 kPa. In specific embodiments, the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
  • the invention also provides for a method of treating a wound, which entails creating a matrix ex vivo from the cell maintenance substrate described above, inserting the matrix into the body of a subject, and applying a magnetic field.
  • the exposure of cells to a magnetic field results in the proliferation of the cells.
  • the cellular response is growth, differentiation, apoptosis, cytokinetics, morphological changes, or some combination of the responses.
  • the cell maintenance substrate has an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the substrate has an elastic modulus of about 3 kPa.
  • the substrate promotes a fibroblast cellular response.
  • the substrate has an elastic modulus of about 2 IcPa.
  • the substrate promotes a myoblast cellular response.
  • the substrate has an elastic modulus of about 1 kPa.
  • the substrate promotes a neuronal cellular response.
  • Various cell types can be sustained on the cell maintenance substrate of the invention and can be obtained as described above.
  • FIG. 1. is a schematic that emphasizes the steps involved in the responses of cells to their environment.
  • FIG. 2 illustrates the three basic mechanisms of force sensing.
  • FIG. 2A depicts the conversion of force into biochemical signals by partial protein unfolding (as shown for the fibronectin module III). This can result in the gain or loss of binding sites, increased separation between protein domains, or the gain or loss of enzyme function.
  • FIG. 2B depicts the opening of some mechanosensitive ion channels can be regulated by membrane tension (Ba), whereas the opening of others requires that their intra- and/or extracellular domains are physically connected to force-bearing filaments (Bb).
  • FIG. 2C shows the Stabilizing receptor-ligand bonds by switching them to a long-lived state by force (catch bonds). Yellow arrows indicate forces.
  • FIG. 2A depicts the conversion of force into biochemical signals by partial protein unfolding (as shown for the fibronectin module III). This can result in the gain or loss of binding sites, increased separation between protein domains, or the gain or loss of enzyme function.
  • FIG. 2B depicts the opening of
  • FIG. 3 are schematics that illustrate (a) how inward curvature of the plasma membrane could cause BAR-domain (Bin, amphiphysin, Rvs domain) proteins to release Rac and (b) how outward curvature could activate the opening of an ion channel.
  • FIG. 4 illustrates the position-dependent mechanism of rigidity sensing.
  • FIG. 4A depicts the crucial feature in such a model is that the enzyme, Fyn, and the substrate to be activated by stretch (kinase and substrate in this example) move relative to one another by actin rearward transport.
  • FIG. 4B if the surface is hard, the components would be close enough for modification to occur (causing small displacement).
  • FIG. 4A depicts the crucial feature in such a model is that the enzyme, Fyn, and the substrate to be activated by stretch (kinase and substrate in this example) move relative to one another by actin rearward transport.
  • FIG. 4B if the surface is hard, the components would be close enough for modification to occur (causing small displacement
  • ECM extracellular matrix
  • F applied force
  • RPTPot receptor-like protein tyrosine phosphatase- ⁇ .
  • FIG. 5 shows the steps in mechanosensing over time that involve periodic testing of the substrate, substrate modification and changes in cellular protein content.
  • FIGS. 6A-B represent a local force assay.
  • FIG. 6A depicts magnetic tweezers (tip at top left corner) exerting a force on 2.7 ⁇ m magnetic beads bound to a spreading MEF cell.
  • FIG. 6B is an image showing the rearward flow of actin that displaces the bead from the cell edge, towards the nucleus (purple trace) prior to application of force.
  • the bead moves under the competing action of the two forces, cellular and magnetic. Shown here is the case of a large magnetic force ( 1 - 1.2 nN) and the bead is temporarily pulled back to the cell edge (red trace).
  • FIG. 6C is a graph that demonstrates the cell responding to the local stress by pulling in a contractile manner (positive and negative rearward velocities) and reinforcing the cytoskeletal adhesion to the stress site during the adaptive phase.
  • the end of adaptive phase is marked by the recovery of the bead's constant rate of displacement.
  • FIG. 7 represents the spatio-temporal dynamics of ⁇ Actinin in response to local force.
  • FIG. 7A is an epifluorescent image of an MEF cell expressing ⁇ Actinin-GFP that shows an accumulation of the fluorophore around two 2.7 mm beads.
  • FIG. 7B represents the intensity of fluorescence vs. time and distance from center of the bead, r. Data at constant (r, t) was obtained by averaging over the arcsector shown in FIG. 7A. The frame of reference is centered on the bead and moves with it. Red and blue indicate the highest and respectively lowest levels of intensity. Maximum protein accumulation is observed ⁇ 50 sec from the application of large force. The duration of the adaptive phase is -105 sec.
  • FIG. 8 depicts active substrates.
  • FIG. 8A (left) is a diagram of magnetic nanowires (for example, nickel) that can be fabricated by electrochemical deposition into porous templates. Scanning electron micrographs (SEM) of nickel nanowires are shown in the right panel.
  • FIG 8B is a schematic of soft gels containing self-assembled arrays of magnetic nanowires that can serve as substrates for cells requiring rigid substrates for normal function.
  • FIG. 8C represents external magnets that can actuate the embedded wires and impart quasi-local stresses at the surface of the gels.
  • FIG. 8D demonstrates the restoration of spreading that can occur after 4 - 24 hrs, especially in the regions closer to the actuating magnet where wires deflect more and the local stresses are larger.
  • FIG. 9 represents a whole-cell dynamic environment.
  • FIG. 9A is a schematic of a stress gradient that can be established across the width of free floating elastic substrates by having a varying initial length (diagonal clamp) and constant elongation.
  • FIGS. 9B-E are representative experiments that were performed to test if the cells detect increased rigidity in the high-strain regions of polyacrylimide gels. Images are shown at same magnification.
  • FIGS. 10A-B show laser tweezers (represented by red circle) that were used to place silica beads coated with fibronectin (FN) or vitronectin (VN) at the edge of the active growth cones. The rearward movement of the beads was recorded and further analyzed for reinforcement.
  • FN fibronectin
  • VN vitronectin
  • FIGS. lOC-D are graphs that demonstrate that RPTP ⁇ is required for the reinforcement of FN-specific integrin-cytoskeleton bonds in the neuronal growth cones.
  • FIGS. 10E-F are schematics of the trajectories of individual beads that were generated in cases of non-reinforced (FIG. 10E) and reinforced of rearward movement (FIG. 10F). The laser trap is represented by a red circle. These trajectories are representative of an average bead behavior in both categories.
  • FIGS. 10G-H depict the mean square displacement (MSD) that was calculated for the initial 10s of rearward movement for each given condition. The results (represented as mean+standard error) were statistically significant as confirmed by t-test (p ⁇ 0.01).
  • FIGS. 1 IA-B are graphs showing that Ov ⁇ integrins are required for the reinforcement of the FN-cytoskeleton bonds at the leading edge of the growth cones. Function blocking antibodies to the ots ⁇ i and ⁇ v ⁇ 3 integrins had no effect on binding and the reinforcement of FN-coated beads, but function blocking of the ⁇ v and ⁇ v ⁇ 6 integrins reduced binding and reinforcement to the background levels.
  • FIGS. 1 IC-F are microscopy images representing that localization of RPTP ⁇ and cCv ⁇ integrin in growth cones were ECM-specific. Both RPTP ⁇ and ⁇ v ⁇ integrins were localized to the leading edge of active growth cones in neurons plated on FN-coated glass (FIG. 11C, FIG. 1 IE). On laminin (LN), RPTP ⁇ was localized to the growth cones, while ⁇ v ⁇ integrin was expressed at a very low level along the axons and at even lower levels in the growth cones (FIG. 1 ID, FIG. 1 IF). The scale bar is 5 ⁇ m. Insets provide image of the entire neuron. [0035] FIGS.
  • FIGS. 12A-B represent Hippocampal neurons isolated from Pl brains of wild type and RPTP ⁇ knockout mice that were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 36h in serum-free medium, fixed and visualized by anti-Tau immunofluorescence. Neuronal stages of differentiation were observed on ECM-coated polyacrylamide gels, wherein stage 2 was characterized by many neurites of approximately equal length (FIG. 12A). At stage 3, -one significantly longer axon was differentiated (FIG. 12B). The Scale bar represents approximately lO ⁇ m. [0036] FIGS.
  • FIG. 12C-D are graphs that represent quantification of differentiation stages and neurite lengths, which revealed an effect of rigidity and a role for RPTP ⁇ .
  • Control neurons RPTPa+/+
  • RPTP ⁇ -/- neurons showed a high level of differentiation irrespective of matrix rigidity (FIG. 12C).
  • control neurons extended longer axons on soft than on rigid surfaces; whereas, RPTP ⁇ -/- neurons extended longer axons on both soft and rigid surfaces (FIG. 12D).
  • the results, in both FIG. 12C and 12D, (represented as mean + standard error) were statistically significant as confirmed by t-test (p ⁇ .01).
  • FIGS. 12E-F represent Hippocampal neurons isolated from Pl brains of wild type and RPTP ⁇ knockout mice that were plated on FN-coated polyacrylamide gels of decreasing rigidities, incubated for 36h in serum-free medium, fixed and visualized by anti-Tau immunofluorescence.
  • the axons in RPTP ⁇ -/- neurons appeared wavier than the axons in wt neurons. No other morphological differences between RPTP ⁇ -/- and wild type neurons were observed.
  • the scale bar represents approximately 15 ⁇ m.
  • FIG. 13 are graphs depicting that the laminin (LN) rigidity response is RPTP ⁇ - independant in neurons.
  • FIG. 13A is a graph that shows that axon differentiation is inhibited by increasing rigidities of LN -coated substrates in both contol and knockout neurons.
  • FIG. 13B is a graph that shows neurite extension is stimulated by soft LN-coated substrates, and loss of RPTP ⁇ had no effect on this behavior
  • FIGS. 14A-B are graphs that show rigidity response in the growth cones is SFK- dependent.
  • Primary wild type neurons were plated on FN-coated substrates of varying rigidities, and a SFK inhibitor (lO ⁇ M SU6656) was added after cell adhered to the substrate. After 36h incubation, there was no difference in wild type neurons on rigid versus soft surfaces. However, axon elongation and differentiation were inhibited compared to controls.
  • FIGS. 14C-D are fluorescent micrographs that depict immunofluorescent staining of
  • FIGS. 14E-F are fluorescent micrographs that demonstrate phosphorylation of pl30Cas, a known substrate for SFKs, requires RPTP ⁇ activity and rigid matrix.
  • Immunofluorescent staining of phosphorylated pl30Cas showed high levels of phospho-pl30Cas in the presence of RPTP ⁇ and rigid matrices. In RPTPa+/+ neurons plated on soft matrix, and in RPTP ⁇ -/- neurons regardless of the matrix rigidity, the observed levels of phopho-pl30Cas were significantly lower.
  • FIG. 15 represents a proposed model for the molecular mechanism of the FN-specific reinforcement and rigidity response in hippocampal neurons.
  • FIG. 16 is a photographic representation of 3D substrates depicting coated silk fibers
  • Cell-cell contacts are dynamic, and cells seem to evaluate the level of force and make adjustments, as the cytoskeleton filaments and their linkages to transmembrane proteins assemble, break down and reassemble.
  • Sensing of geometry at the sub-cellular level is a crucial component of the cellular sensing of two-dimensional (2D) versus 3D matrices (FIG. 1).
  • 2D two-dimensional
  • 3D matrices FIG. 1
  • Recent studies have determined that the same matrix protein will elicit a different response when it is organized in filaments from when it is displayed on a flat surface (Cukierman, E. , Pankov, R. , Stevens, D. R. & Yamada, K. M. Science 294, 1708-1712 (2001); Cukierman, E. , Pankov, R. & Yamada, K. M. Curr. Opin. Cell Biol. 14, 633-639 (2002); Katz, B. Z. et al. MoI Biol.
  • Complicated transduction processes such as rigidity responses involve several steps that can combine the transduction of force and/or geometry sensing with time (reviewed in Refs 24,25) (Discher, D. E. , Janmey, P. & Wang, Y. L. Science 310, 1139-1 143 (2005); Giannone, G. & Sheetz, M. P. Trends Cell Biol, (in the press). Guanine nucleotide-exchange factors, Ca 2+ ion channels, receptor-like protein tyrosine phosphatases, Src-family kinases and membrane receptors have all been invoked as early steps in force or geometry transduction (von Wichert, G. et al. J.
  • FIG. 2 These include the force-induced exposure of otherwise cryptic peptide sequences (FIG. 2A), the opening of mechanosensitive ion channels (FIG.
  • the isometric ATPase activity of motors keeps the tension in the cytoskeleton constant, and the recruitment of other motors will then increase the overall tension in the cytoskeleton. Because the cytoskeleton filaments are dynamic and undergo assembly— disassembly cycles on the timescale of seconds to minutes, filament tension will be rapidly lost and must constantly be maintained by motor activity.
  • Conformational strain may be capable of increasing the activity of some enzymes.
  • the question of whether the activity of some enzymes can be upregulated by strain if they or their substrates are physically integrated into a force-bearing structure is relatively unexplored in the context of mechanosensing. Force might open up enzymatic cleavage sites through partial unravelling.
  • Fibronectin for example, has a partially cryptic disulphide-isomerase (Langenbach, K. J. & Sottile, J. J. Biol. Chem. 11$, 7032-7038 (1999) and a cryptic metalloprotease activity (Schnepel, J. & Tschesche, H J. Protein Chem.
  • Titin contains a module that can show kinase activity and computational studies indicated a mechanical opening of its active site (Grater, F., Shen, J., Jiang, H., Gautel, M. & Grubmuller, H. Biophys. J. 88, 790-804 (2005)). Furthermore, it is well known that enzymes exert strain on their substrates on binding — thereby catalysing the reaction (reviewed in Ref. 7) (Bustamante, C, Chemla, Y.R., Forde, N.R. & Izhaky, D. Annu. Rev. Biochem.
  • Mechanosensitive ion channels The structural diversity of mechanosensitive channels seems to have been driven by the physiological necessity to detect mechanical stimuli — from thermal energy to high pressures — as changes in conductive state (FIG. 2B) (reviewed in Sukharev, S. & Anishkin, A. Trends Neurosci. 27, 345—351 (2004)). Some channels respond to stress in the lipid bilayer, whereas others must be physically connected to the cytoskeleton and/or the extracellular matrix to transmit forces to the channel.
  • the bacterial mechanosensitive K + channel, MscL represents the first case and has been structurally analysed.
  • Membrane-tension forces are mainly concentrated in the interfacial polar headgroup regions of MscL, thereby inducing helix tilting that opens and wets the pore interior (Kung, C. Nature 436, 647-654 (2005); Sotomayor, M. & Schulten, K. Biophys. J. 87, 3050-3065 (2004))
  • Bacterial channels are relatively force-insensitive and only open at high tensions that are approaching the lytic tensions for the lipid bilayer.
  • Plant channels seem to operate at tensions that are of an order of magnitude lower than bacterial channels, whereas typical membrane tensions in animal cells are a thousandfold lower than the activating bacterial tensions (Sheetz, M. P. Nature Rev. MoI. Cell Biol. 2, 392-396 (2001)).
  • the bond-survival time of the strongest non-covalent bond is diminished from more than a day under static conditions to about 1 minute or 0.001 second at bond stresses of 5 pN or 170 pN, respectively (Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Nature 397, 50-53 (1999)); whereas 5 pN can be exerted onto a protein complex by a single motor protein. Therefore, non-covalent bonds will fail under any level of tensile stress if held for sufficient time periods (Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E.
  • Adhesion sites form in response to tensile forces acting on the membrane, and one consequence of the shortened bond lifetimes under force must be high turnover rates of the constituents in newly formed adhesion sites. Turnover rates on the timescale of seconds have been observed for acti ⁇ filaments and the two focal-contact proteins, paxillin and vinculin, which might serve as signalling molecules after their release from the cell contacts (Vallotton, P., Gupton, S.L., Waterman- Storer, C. M. & Danuser, G. Proc. Natl Acad. ScL USA 101, 9660-9665 (2004); Von Wichert, G. , Haimovich, B. , Feng, G. S.
  • Cells may be able to form force-sustaining adhesion sites. Because force is transmitted across the membrane, not by single integrins, but by integrin clusters that increase in size with time in a force-dependent manner (Riveline, D. et al. J. Cell Biol. 153, 1175—1186 (2001); Bershadsky, A. D. et al. Eur. J. Cell Biol. 14 Dec 2005 (10.1016/j.ejcb.2005.11.001); Wehrle-Haller, B.
  • ECM of cells is a complex 3D fibrous meshwork with a wide distribution of fibres and gaps that provide complex biochemical and physical cues, which are very different from uniformly coated 2D surfaces. Cell reactions to 3D matrices are altered from their reaction to 2D matrices of the same material (Cukierman, E. , Pankov, R. , Stevens, D. R. & Yamada, K. M.
  • Geometry sensing can refer to the formation of signalling complexes by changes in the spacing of molecular-recognition sites the geometrical shape of the substrates. For example, surface steps as small as 11 run can lead to contact guidance (Curtis, A. & Wilkinson, C. Biochem. Soc. Symp. 65, 15—26 (1999)). Restricting cells to spreading on adhesive micropatterns of various shapes can regulate cell proliferation and cell death (Chen, C. S. , Mrksich, M. , Huang, S., Whitesides, G. M. & Ingber, D.E.
  • micropatterns also regulates whether mesenchymal stem cells differentiate into adipocytes or osteoblasts (McBeath, R. , Pirone, D. M. , Nelson, C. M. , Bhadriraju, K. & Chen, C. S. Dev. Cell 6, 483-495 (2004)). Because micropatterns confine cell shape, thereby causing an integrated cellular response, the ability of cells to sense nanoscale surface features, including the size of nanoscale fibres and topographies, as well as the spatial presentation of molecular- recognition sites will be considered.
  • Fibroblasts move on collagen fibres by a specific mode of motility that involves one myosin isoform (Meshel, A. S., Wei, Q., Adelstein, R. S. & Sheetz, M. P. Nature Cell Biol. 1, 157-164 (2005).
  • the same myosin isoform was shown to be involved in in vivo-like organization and morphogenesis of fibroblasts grown on electrospun nanofibrous matrices (Schindler, M. et al. Biomaterials 26, 5624—5631 (2005). It has also been shown that 3D networks of nanofibres presenting the neurite-promoting laminin epitope promoted the selective differentiation of neural progenitor cells (Silva, G. A.
  • the BAR domains of arfaptins were shown to bind to the small GTPases Rac, adenosine-ribosylation factor- 1 (ARFl), ARF3 and ARF6, as well as to the ARF-like protein- 1 (ARLl ; Refs 93-96) (Lu, L., Horstmann, H., Ng, C. & Hong, W. J. Cell ScL 114, 4543-4555 (2001); Ta ⁇ cone, C. et al. Nature 41 1, 215-219 (2001); Van Aelst, L. , Joneson, T. & Bar- Sagi, D. EMBO J, 15, 3778-3786 (1996); Williger, B. T. , Ostermann, J.
  • BAP2 ⁇ /IRSp53 insulin-receptor substrate protein of 53 IcDa
  • WAVE Wiskott-Aldrich syndrome protein
  • K + channels are opened by a convex curvature of the membrane (Patel, A. J. , Lazdunski, M. & Honore, E. Curr. Opin. Cell Biol. 13, 422-428 (2001)).
  • Concave and convex membrane curvatures might be sensed by two different mechanisms.
  • BAR domains might sense the concave membrane curvature that is formed in contact with external posts and fibres, whereas membrane channels might selectively be opened if membranes come into contact with surface indentations.
  • BAR-domain proteins do indeed bind preferentially to concave surfaces, such as posts or fibres, the local release of Rac and its subsequent activation, for example by integrin-linked kinase (Filipenko, N. R. , Attwell, S. , Roskelley, C. & Dedhar, S. Oncogene 24, 5837-5849 (2005)), might lead to a local enhancement of traction forces.
  • Rhen activation is known to enhance focal- complex assembly (Filipenko, N.R., Attwell, S., Roskelley, C. & Dedhar, S. Oncogene 24, 5837-5849 (2005); Burridge, K. & Wennerberg, K Cell 116, 167-179 (2004); Civelekoglu- Scholey, G. et al. J. Theor. Biol. 232, 569-585 (2005); Machacek, M. & Danuser, G. Biophys. J. 90, 1439—1452 (2006)) and cells adhere more weakly on surfaces with nanopits than on those with nanoposts (Dalby, M. J., Riehle, M. O., Sutherland, D. S.
  • the rigidity of the external ligand linkage will determine how far the cytoskeleton-linked complexes will be displaced in a given time period before reaching isometric conditions and whether the force will be sufficient to activate any of the force sensors through the exposure of otherwise cryptic peptide sequences.
  • the separation between the fixed and moving components is greater, and might be sufficient to prevent the sensor from interacting with the stationary component (FIG. 4).
  • Extracellular signal-regulated kinases (ERKs) and Rho constitute part of an integrated mechanoregulatory circuit that links matrix stiffness, through integrin clustering, to cytoskeletal tension and, ultimately, regulation of tissue phenotype (Silver, F. H. & Siperko, L. M. Crit. Rev. Biomed. Eng. 31, 255-331 (2003); Ingber, D. E. Proc. Natl Acad. Sd. USA 102, 11571-11572 (2005); McBeath, R., Pirone, D.M., Nelson, C.
  • Transcription factors that are recruited to the adhesion sites could have an important role in translating the physical stimulus that is sensed at the periphery into biochemical signals that alter gene expression. Transcription factors might be modified in a force-dependent manner and transported to the nucleus; for example, paxillin is modified at focal-contact sites and is then transported to the nucleus (Woods, A. J. et al. J. Biol. Chem. 277, 6428-6437 (2002)). This mechanism provides an obvious way to transform force on specific intracellular-adhesion sites to a change in protein expression.
  • Tyrosine kinases that interact with G proteins of the Ras family to stimulate cell proliferation and differentiation are also downregulated, as is ⁇ -actinin (Dalby, M.J., Riehle, M.O., Sutherland, D. S., Agheli, H. & Curtis, A. S. Eur. Cell Mater. 9, 1-8 (2005)).
  • Many of the tyrosine kinases and phosphatases that have been linked to changes in cell and tissue shape are also linked to the early events of force and rigidity sensing (reviewed in Ref. 25) (Giannone, G. & Sheetz, M. P. Trends Cell Biol. (in the press)).
  • Cell behavior may be determined by cell dynamics and the state of the cellular environment. The latter parameters can allow a better understanding of the feedback mechanism between cell contractility and mechanosensing, as well as the role of pulsatory activity in cell function and replication. Such studies can also be motivated by the need for better tissue scaffolds, and implantable materials (Vogel, V. & Baneyx, G. Annu Rev Biomed Eng 5, 441-63 (2003)). Elucidating the dynamic nature of the interactions of the cell with its environment can clarify the requirements for better biomedical devices and drive the subsequent phases of research in engineered materials and technologies.
  • the mechanical perturbations can be generated using one of the following non-limiting examples of devices: pipet-assisted manipulation device, laser tweezer, optical trap, magnetic field generator, or a substrate stretching device, wherein a stationary clamp is attached to a first end of the substrate and a mobile clamp is attached to a second end of the substrate.
  • these devices can physically manipulate an engineered environment, such as a cell growth substrate or scaffold of the invention.
  • cell growth can be assessed via measuring a gross change in cell size, an increase in cell proliferation, an increase in focal adhesion assembly, or a combination thereof.
  • cell differentiation can be determined via examining the expression of target genes utilizing standard molecular biology methods practiced in the art.
  • apoptosis can be investigated via examining cell viability or the expression and/or downregulation of target genes.
  • cell movement can be assessed via motility assays utilized in the art.
  • cell morphology changes can be examined via light, fluorescent, or electron microscopy methods utilized by one skilled in the art.
  • a cell growth substrate of the invention can be permeable to liquids, gases, and cellular by-products. It can comprise a growth medium in addition to a cross-linked polymer, wherein increased strain on the polymer does not substantially increase rigidity of the polymer, and wherein the polymer has an elastic modulus (E) of about 0.1 kPa to about 10.0 kPa (E is described in Engler at al., (2004) Biophys J S6: 617-28, which is hereby incorporated by reference). Polymers are long chain organic molecules that are assembled from smaller molecules called monomers.
  • Polymers comprise many repeating monomer units in long chains, and can be classified as synthetic or natural polymers (for example, those of biological in nature that can comprise proteins, carbohydrates, and nucleic acids).
  • the polymer is cross-linked and can comprise natural polymers and their derivatives, synthetic polymers and their derivatives, or a combination thereof.
  • These natural polymers can be anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers.
  • Non-limiting examples of anionic polymers can include hyaluronic acid, alginic acid, carageenan, chondroitin sulfate, dextran sulfate, and pectin.
  • Some examples of cationic polymers include but are not limited to, chitosan or polylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).
  • amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin.
  • Non-limiting examples of neutral polymers can include dextran, agarose, or pullulan.
  • polyesters can also be used to generate the cell growth substrate of the invention.
  • polyesters can include polyethylene glycol, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyacrylamide, and polydimethylsiloxane.
  • a magnetic tweezers apparatus uses expertise in condensed matter physics with a focus in magnetism and magnetic materials, as well as interdisciplinary training in materials science, microfabrication, and cell biology, systems can be engineered for measuring the limits of cellular responses to mechanical perturbations.
  • a magnetic tweezers apparatus generates forces as large as 20 nN with frequencies from 0 to 3 kHz, values that match and exceed those found in tissues.
  • This system allows application of local forces at the position of interest: lamellipodium, lamella, and perinuclear region, via beads (for example, magnetic beads) attached to specific receptors on the cellular dorsal surface.
  • the bead can be a magnetic bead.
  • the diameter of the magnetic bead can be greater than or equal to about 0.1, 0.2, 0.3, 0.5, 0.6, 0.75, 0.8, 0.9, 1.0, 2.0, 2.5, 3, 4, or 5 ⁇ m in diameter.
  • the diameter of the bead is about 2.7 ⁇ m in diameter.
  • the cell growth substrate described above also comprises a magnetic material, such as a magnetic bead, wire and the like.
  • Non- limiting examples of magnetic material include nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt.
  • the trajectory of the beads in the example above is the result of the interplay between the magnetic force and the force exerted by the cell on the bead.
  • f ⁇ bronectin-coated beads were placed on laminin coated glass substrates and mouse embryonic fibroblasts (MEFs) were subsequently allowed to spread.
  • MEFs mouse embryonic fibroblasts
  • the adaptive phase is defined by the presence of fluctuations in the bead's velocity and direction of movement, and can last between 20 and 140 seconds. Since the magnetic force is constant, the variations in bead velocity indicate variations in the cell force. This can be interpreted as a cyclic testing of the site of mechanical stimulation. As the cell adapts to the local stress, it generates pulsatory traction forces at the site of the mechanical signal, i.e. the bead. This is reminiscent of the periodic lamellipodial contractions in spreading and migrating cells (Dobereiner, H. G., et al.,. J Appl Physiol 98, 1542-6 (2005); Dubin-Thaler, B.
  • Whole cell assays can allow the cellular adaptive response to be studied when the environment (adhesive substrate) has different rigidities or is pulsating.
  • two assays can be utilized: active and stretchable substrates.
  • Active substrates such as cell growth substrates
  • embedded magnetic materials such as magnetic
  • the nanowires comprise a magnetic material.
  • Some non- limiting examples of magnetic material include nickel, iron, cobalt, or alloys of gold, platinum, copper, zinc, or silver that are combined with nickel, iron, or cobalt.
  • the magnetic nanowires were nickel nanowires.
  • the diameter of the magnetic wire can be greater than or equal to about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm in diameter.
  • the diameter of the wire is about 300 nm in diameter.
  • the length of the magnetic wire (for example, the nanowire) can be greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ⁇ m in length, hi another embodiment, the length of the magnetic wire is about 30 ⁇ m in length.
  • nanowire is about 300 nm in diameter, 30 ⁇ m long, and is comprised of nickel.
  • Synthetic biodegradable, matrixes such as the cell grwoth substrate of the invention described in Example 1 or the microfence scaffold described in Example 2, can be manufactured using synthetic polymers (such as those described above), in addition to naturally occurring, absorbable materials, such as polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide- lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester), catgut suture, collagen, silk, chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid, elastin, and the like.
  • synthetic polymers such as those described above
  • absorbable materials such as polyglycolic acid (PGA), polylactic acid (PLA), polyglycolide- lactide, polycaprolactone, polydioxanone, polyoxalate, a polyanhydride, a poly(phosphoester
  • An active substrate can be fabricated via suspending the wires in unpolymerized cell growth substrates (for example those made of polyacrylimide gels) and aligning them by a uniform magnetic field. Ferromagnetic nanowires are highly responsive to magnetic fields (Tanase, M. et al., (2001) Nano Letters 1, 155-158) and can be directed to self-assemble in arrays perpendicular to the gels' surface. Polymerization is then induced, and the wires remain in this configuration even after the aligning field is removed.
  • the active substrate can be fabricated via suspending magnetic beads in unpolymerized cell growth substrates (for example those made of polyacrylimide gels) and aligning them by a uniform magnetic field.
  • soft gels can be employed that do not support normal cell growth (FIG. 8B).
  • An external oscillating magnetic field will tilt the embedded magnetic nanowires, and transmit local oscillatory displacements at the surface of the gels and at the same time to the basal side of the cells (FIG. 8C).
  • the displacements achieved at the surface of the gels were on the order of hundreds of nanometers.
  • the cells may be able to sense the local displacements as sites of increased rigidity (FIG. 8D). This assay may allow the spatial correlation of mechano-response, and its controlled initiation.
  • the stress at the surface of the gels can be reliably modulated, and the density of nanowires in the array determines the distance between the wires and ultimately the number of oscillating sites per cell.
  • the mechano-induced cellular functions can be controlled.
  • substrates can be developed (such as those described above) that support many cells while being stretched. Cells can be plated on soft gels that will be subject to oscillatory stresses. A system that provides a stress gradient within the same substrate can therefore generate parallel data for cells in different mechanical environments.
  • the invention provides an apparatus for applying a mechanical perturbation to a cell (for example, a cell in vitro), wherein the apparatus comprises the cell growth substrate described above in addition to an external force generator associated with the substrate.
  • the apparatus can further comprise a means for quantitating a cellular response to the mechanical perturbation.
  • cell responses can include cell growth; cell differentiation; apoptosis; cell movement/cytokinetics; and cell morphology changes.
  • cell growth can be assessed via measuring a gross change in cell size, an increase in cell proliferation, an increase in focal adhesion assembly, or a combination thereof.
  • gross changes in cell size can be determined with microscopy methods (light, fluorescence, electron, and the like) by obtaining cell size measurements.
  • Cell proliferation can be examined via FACS analysis, cell density readings (i.e., OD ⁇ oo absorption readings), or other methods commonly used in the art.
  • cell differentiation can be determined via examining the expression of target genes (for example, via examining mRNA or protein levels) utilizing standard molecular biology methods practiced in the art.
  • apoptosis can be investigated via examining cell viability or the expression and/or downregulation of target genes (for example, via examining mRNA or protein levels).
  • cell movement can be assessed via motility assays utilized in " the art.
  • cell morphology changes can be examined via light, fluorescent, or electron microscopy methods utilized by one skilled in the art.
  • the external force generator is a means for physically manipulating the cell growth substrate.
  • external force generating devices include a pipet-assisted manipulation device, laser tweezers, a magnetic twisting cytometry device, an optical trap, a magnetic field generator, and a substrate-stretching device (for example, a stationary clamp attached to a first end of the substrate and a mobile clamp attached to a second end of the substrate) (Sniadecki et al., (2006) Annals Biomed Eng 34(1): 59-74). . . .
  • a purpose of the whole-cell assay consisting of stretchable substrates is to investigate the long-term effects of pulsatory signal in soft environments on cell proliferation and migration.
  • the invention provides a method for stimulating growth of a cell in vitro.
  • cells can be plated on or in the cell growth substrate described above, the substrate containing the cell is perturbed (for example, mechanically, electrically, or magnetically), and the growth of the cell of the cell is detected, wherein the perturbation of the substrate stimulates the growth of the cell.
  • the invention also provides a method for stimulating cell differentiation in vitro where cells can be plated on or in the cell growth substrate described above, the substrate containing the cell is perturbed (for example, mechanically, electrically, or magnetically), and the differentiation of the cell of the cell is detected, wherein the perturbation of the substrate stimulates the differentiation of the cell.
  • an elastic modulus is the mathematical description of an object or substance's tendency to be deformed along an axis when an opposing force is applied along that axis.
  • (E) describes tensile elasticity and thus reflects the measure of the stiffness of a given material. It is defined as the ratio of tensile stress to tensile strain and can be experimentally determined from the slope of a stress-strain curve created during tensile tests conducted on a sample of the material (described in Engler et al., (2004) Biophys J 86: 617-28, which is hereby incorporated by reference).
  • the cell growth substrate can have an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the cell growth substrate can promote a fibroblast cellular response (such as growth, differentiation, apoptosis, cytokinetics, morphological changes, and the like).
  • the substrate can promote a myoblast cellular response, such as those previously described.
  • the cell growth substrate can promote a neuronal cellular response, such as those described above.
  • the growth of the cell can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or a combination thereof. Examples of methods used to detect cell growth have been described above.
  • differentiation of the cell can be detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or a combination thereof.
  • the -expression of target genes can be ascertained by examining mRNA or protein-levels utilizing standard molecular biology and biochemical methods practiced in the art. Cell morphology changes in addition to changes in size can be examined via light, fluorescence, or electron microscopy methods utilized by one skilled in the art.
  • perturbing the cell growth substrate entails subjecting the substrate harboring cells to an external force generator previously described.
  • the cell can be excised from a tissue (for example muscular tissue, such as skeletal, cardiac, or smooth; neuronal; connective; epithelial; or haemopoietic).
  • the tissue can come from a cultured cell line or an animal (for example a mammal, such as a dog, cat, human, bird, and the like).
  • the invention also provides for a method of promoting the growth of a cell in vivo.
  • the method can comprise the ex vivo creation of a matrix from the cell growth substrate described above followed by insertion of the matrix into the body of a subject and subsequently applying a magnetic field to the subject, wherein the application of the magnetic field can promote the growth of the cell.
  • the invention provides for a method of treating a wound, where the method can comprise the ex vivo creation of a matrix from the cell growth substrate described.above followed by insertion of the matrix into the body of a subject and subsequently applying a magnetic field to the subject, wherein exposure to the magnetic field can result in the proliferation of cells.
  • the subject can be an animal (for example a mammal, such as a dog, cat, human, horse, cow, sheep, rabbit, bird, and the like).
  • the cell growth substrate can have an elastic modulus greater than or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, or 10 kPa.
  • the substrate can have an elastic modulus of about 3 kPa, wherein the cell substrate can promote a fibroblast cellular response.
  • the substrate can have an elastic modulus of about 2 kPa, wherein the substrate can promote a myoblast cellular response.
  • the substrate can have an elastic modulus of about 1 kPa, wherein the cell substrate can promote a neuronal cellular response.
  • Mechanical perturbations can stimulate a cellular response.
  • perturbing the cell substrate entails subjecting the substrate harboring cells to an external force generator previously described.
  • the growth of the cell can be detected by measuring cell proliferation, an increase in cell size, an increase in focal adhesion assembly, or a combination thereof. Examples of methods used to detect cell growth have been described above.
  • Cell differentiation can be detected by measuring mRNA expression levels, an increase in cell size, a change in cell morphology, or a combination thereof.
  • the expression of target genes can be ascertained by examining mRNA or protein levels utilizing standard molecular biology and biochemical methods practiced in the art.
  • Cell morphology changes in addition to changes in size can be examined via light, fluorescence, or electron microscopy methods utilized by one skilled in the art.
  • EXAMPLE 1 - RPTP ⁇ is required for the fibroncctin-specific rigidity response in hippocampal neurons
  • RPTP ⁇ receptor-like protein tyrosine phosphatase alpha
  • RPTP ⁇ was shown to mediate fibronectin-specific rigidity responses in hippocampal neurons in an SFK-dependent process that leads to the recruitment of fibronectin and pl30Cas phosphorylation at the leading edge of the growth cone.
  • the results show that the rigidity of a cell growth substrate can be manipulated to affect a cellular response, such as cell growth and differentiation (for example, as shown with neurons).
  • RPTP ⁇ is reported to be required for reinforcement of fibronectin-cytoskeleton bonds and the rigidity response in hippocampal neurons.
  • Neurons were plated on coverglass or gels that were pre-incubated with 20 ⁇ g/mL polylysine and subsequently coated with 50 ⁇ g/mL laminin (BD Biosciences) or 50 ⁇ g/mL fibronectin (Roche).
  • Optical gradient laser trap set at 10OmW (40pN/ ⁇ m) (Axiovert TV 100; Carl Zeiss Microimaging, Inc) was equipped with a 10Ox objective, and calibrated as described previously (Choquet, D., et al., (1997). Cell 88, 39-48).
  • the beads were held at the smooth lamelipodium-like edges of the growth cones for approximately 3 -5 s, and then, the laser trap was turned off to check for binding. If the bead was bound, the trap was turned back on and the rearward movement of the bead was recorded using a cooled CCD camera.
  • the fraction of the beads bound and moving rearward was calculated as mean + standard error for at least three independent experiments and statistical significance of the results confirmed by t-test (p ⁇ .01).
  • the total number of beads included in analysis was at least 35 beads for each condition.
  • the rearward movements of the beads were further analyzed using Nanotracker plug-in, with a tracking accuracy of 3-5nm for 0.64 ⁇ m beads.
  • the MSD values were calculated using an algorithm modified from (Qian, H., et al., (1991). Biophys J 60, 910-921).
  • the uniformity of coating on the substrate surface was examined by coating the gels with proteins conjugated to Cy5 fluorophore (Amersham Biosciences) according to manufacturer's instructions and visualized by confocal microscopy. Experiments were performed 36h after the neurons were plated on the polyacrylamide gels. Neurite extension was quantified for at least 50 neurites for each condition and statistical significance of the results confirmed by t-test (p ⁇ .01). Data is presented as mean+standard error of at least two independent experiments. In experiments with SFK inhibitor, lO ⁇ M SU6656 (Calbiochem) was added after neurons were adhered to the substrate, and further incubated for total time of 36h.
  • BD Transduction Laboratories BD Transduction Laboratories
  • X v ⁇ antibody USBiological
  • a mouse monoclonal anti-Tau antibody Biosource
  • an affinity purified polyclonal rabbit anti-phoshoY165Cas antibody Cell Signaling Technology
  • anti-cts ⁇ i Chemicon
  • anti- ⁇ v BD Pharmingen
  • anti- ⁇ v ⁇ USBiological antibodies
  • the fraction of breaking events was reciprocally proportional to the rigidity of the trap and hence to the rigidity response (Jiang, G., et al., (2003). Nature 424, 334-337). As predicted, the number of breaking events was significantly higher in RPTP ⁇ -/- neurons than in controls (Fig. 10C). Therefore, the reinforcement of FN-clustered integrin-cytoskeleton bonds was impaired in RPTP ⁇ -/- growth cones, indicating that the rigidity response was impaired as well. [00141] The effect of RPTP ⁇ deletion on reinforcement was further determined by quantifying the diffusivity of the beads through calculation of the mean square displacement (MSD).
  • MSD is used as a measure of the stiffness of the bead-cytoskeleton contact and is inversely proportional to the bead reinforcement (Choquet, D., et al., (1997). Cell 88, 39-48; Qian, H., et al., (1991). Biophys J 60, 910-921). Individual trajectories of the beads were generated (FIG. 1OE, 10F), and MSD was determined as described previously (Qian, H., et al., (1991). Biophys J 60, 910-921). The average MSD of the beads moving rearwards was determined during the initial period of time after the beads moved outside of the trap.
  • the average MSD of the beads was two to threefold higher during early rearward movement on RPTP ⁇ -/- growth cones compared to RPTPa+/+ growth cones (FIG. 10G). MSD was on average higher for FN-coated beads bound to RPTP ⁇ -/- growth cones compared to RPTPa+/+ growth cones (FIG. 10G); however, no significant difference was observed for VN-coated beads (FIG. 10H).Thus, the greater bead diffusion along perpendicular axis in RPTP ⁇ -/- growth cones supports the hypothesis that RPTP ⁇ is required for reinforcement of integrin-cytoskeleton bonds.
  • VN vitronectin
  • RPTP ⁇ may be involved in a signaling pathway upregulated by the activation of (a) FN-specific integrin(s).
  • FN-stimulated RPTPa signaling is activated through a v ⁇ 6 integrin
  • the particular integrin involved in the RPTP ⁇ -mediated reinforcement in neurons was investigated next. The expression of a variety of FN-specific integrin subunits was reported in the hippocampus (Pinkstaff, J. K., et al., (1999). J Neurosci 19, 1541-1556), including ⁇ v ⁇ e integrins (Chan, C. S., et al., (2003). J Neurosci 23, 7107-7116), whose expression was previously believed to be limited to epithelial cells.
  • RPTP ⁇ was abundantly present in the growth cones of neurons plated both on FN and LN.
  • FIG. 1 1C, 1 ID an increased fraction of OC v ⁇ integrins accumulated at the edge of the growth cone upon interaction with FN may cause RPTP ⁇ activation, leading to the upregulation of the downstream rigidity response pathway.
  • RPTPcc ⁇ neurons are deficient in FN-specific rigidity response
  • Fibronectin and laminin show differential distribution in the mammalian brain during development (Chun, J. J., and Shatz, C. J. (1988). J Cell Biol 106, 857-872; Hagg, T., et al., (1989). Neuron 3, 721-732).
  • stage 1 is characterized by the absence of neurites; at stage 2, neurites of approximately equal lengths are extended (FIG. 12A); and at stage 3, the significantly longer axons are differentiated (FIG. 12B).
  • Neurons were isolated from the brains of neonate mice (Pl) and plated on FN -coated polyacrylamide gels of varying rigidities. After 48h of incubation in serum-free medium, the lengths of extended neurites were measured and the differentiation stages of the neurons were determined (FIG. 12C).
  • RPTPa+/+ neurons differentiated faster on soft than stiffer FN-coated substrates (38.7%+4.8% neurons at stage 3 on rigid, 39.5%+3.7% on intermediate, and 62.7%+2.3% on soft)
  • RPTP ⁇ -/- showed no preference for soft substrates (62.0%+6.5% neurons at stage 3 on rigid, 64.1%+6.6% on intermediate, and 66.3%+5.6% on soft) and differentiated at a rate similar to wild type neurons plated on soft FN-coated gels.
  • the average lengths of the neurites both axons and dendrites were also reflective of the absence of a FN rigidity response in the absence of RPTP ⁇ .
  • the soft substrate stimulated neurite extension in RPTPa+/+ neurons
  • SFKs and in particular Fyn, have been previously implicated as RPTP ⁇ substrates in a variety of processes including the FN rigidity response in fibroblasts, SFKs may be involved in this process in neurons as well. Therefore, the effect of a broad SFK inhibitor (10 ⁇ M SU6656) on the neurite extension and rigidity response to FN-coated substrates was examined. Similar to RPTP ⁇ -/- neurons, neurons cultured in the presence of the SFK inhibitor, showed no preference for the soft FN matrices, and there was no difference in neurite extension between substrates of different rigidities in the presence of inhibitor (FIG. 14A).
  • pl30Cas was known as an indispensable component in the regulation of actin cytoskeleton organization, focal contact formation, and migration of fibroblasts (Cary et al., (1998). J Cell Biol. 140(l):211-21; Cho and Klemke (2000). J Cell Biol. 149(l):223-36; Honda et al., (1999.) Biochem Biophys Res Commun. 262(l):25-30), its role in neuronal motility has been poorly understood.
  • dissociated hippocampal neuronal cultures displayed different growth properties on polyacrylamide gels of different rigidities.
  • RPTP ⁇ -/- mice display a severe hippocampal phenotype (Petrone, A., et al., (2003). Embo J 22, 4121-4131), and that RPTP ⁇ was implicated in force transduction and the rigidity response in fibroblasts (Jiang, G., et al., (2006). Biophys J 90, 1804-1809; Von Wichert, G., et al., (2003). J Cell Biol 161, 143- 153), RPTP ⁇ ablation may affect the rigidity response in neurons as well.
  • RPTP ⁇ -/- neurons lacked the ability to distinguish between FN-coated substrates of varying rigidities, unlike RPTPa+/+ neurons which differentiated faster and grew longer neurites on softer than on stiffer substrates, hi contrast, the LN rigidity response was not affected by the absence of RPTP ⁇ , indicating FN specificity of the integrin(s) that activate RPTP ⁇ .
  • the correlation between the rigidity response and the reinforcement of integrin-cytoskeleton bonds was confirmed (Choquet, D., et al., (1997). Cell 88, 39-48). Motile growth cones were logical candidates to test for the impairment of reinforcement.
  • the RPTP ⁇ -mediated rigidity response in hippocampal neurons was shown to be SFK-dependent and that Fyn, as well as its direct substrate pl30Cas localize to the leading edge of the growth cones in a rigidity-dependant manner.
  • the role of Fyn in the neuronal rigidity response might seem contradictory, since the broad inhibitor of SFKs had not only inhibited the rigidity response, but also overall neurite extension. This can be explained by a specific and non-redundant role that Fyn plays in the rigidity response. Therefore, the molecular mechanism of the FN rigidity response in neurons may be mediated through a pathway similar to the one proposed in fibroblasts (Jiang, G., et al., (2006).
  • FIG. 15 shows that rigidity could be sensed by the relative displacement of RPTP ⁇ -immobilized Fyn and a liganded integrin complex with pl30Cas that depends upon the rigidity of the surrounding matrix.
  • the rigidity of the matrix triggers force-dependent activation of the RPTP ⁇ , followed by activation of Fyn, that consequently phosphorylates stretch-sensitive pl30Cas. This results in the further recruitment of the focal contact proteins causing the reinforcement of the interaction between the growth cone and the substrate. This reinforcement has a negative effect on the neurite extension.
  • the force exerted by the actin-myosin network in response to the substrate rigidity does not reach critical threshold necessary for the reinforcement of the FN-cytoskeleton bonds and subsequent focal contact formation. Therefore, the neurite extension is stimulated on soft matrices.
  • This model proposes that Fyn plays a critical role through its immobilization by palmitate groups to lipid domains near the leading edge. This is speculative but the phenotype of the Fyn knockout mice is similar to that of the RPTP ⁇ -/- in several respects (Grant et al., (1992) Science 258(5090): 1903-10). Similarly, pl30Cas is an important component in many motility pathways including rigidity response (Kostic and Sheetz, (2006) MoI Biol Cell. 17(6):2684-95; Tamada, M., et al., (2004). Dev Cell 7, 709-718; Vuori and Ruoslahti (1995) J Biol Chem.
  • pl30Cas-/- mice die in utero before the brain has functionally developed making it difficult to determine the effect of pl30Cas ablation on brain development and function (Honda et al., (1998) Nat Genet. 19(4):309-l 1). Nevertheless, studies in dissociated cerebellar neurons showed that pl30Cas is required for neurite extension (Huang, J., et al., (2006). MoI Biol Cell 17, 3187-3196). Further, phosphorylation of p 130Cas has been recently shown to be directly related to force transduction (Sawada et al., (2006). Cell. 127(5):1015-26).
  • pl30Cas could signal to a variety of different pathways that normally promote growth and not differentiation.
  • the model is plausible but much more is needed to prove the exact roles of the components.
  • the same molecular components would be implicated in the rigidity responses of such different cells as fibroblasts and neurons, particularly since in the two cases the cells have different rigidity responses.
  • the response to the rigid matrix is needed for growth and motility, whereas, in neurons, the response to rigid matrix inhibits differentiation and mobility of the growth cones.
  • the response to rigid matrix appears to promote proliferation, and not differentiation.
  • the response to soft matrices is apoptosis in the case of the fibroblasts and increased differentiation in the case of neurons.
  • Neuronal growth cones are smaller than lamellae of the fibroblasts and they are known to pull the neurites forward. Reinforcement of the integrin-cytoskeleton bonds and focal contact formation stabilizes the lamellipodia and supports cell spreading in fibroblasts (Choquet, D., et al., (1997). Cell 88, 39-48; Giannone, G. et al. (2004). Cell 116, 431-43). Growth cones distinguish FN from LN by forming contacts on FN similar to focal contacts characterized in fibroblasts (Gomez, T. M., et al., (1996). J Neurobiol 29, 18-34).
  • the stiff substrates may support formation of these focal contacts, which in turn stabilize interactions between growth cones and the substrate. This might result in reduced velocity of the growth cone progression and eventually in shorter neurites (FIG. 15). Similar rigidity responses could lead to different cellular responses in different cell backgrounds.
  • the FN-rigidity response in neurons may require RPTP ⁇ activation through CC v ⁇ ⁇ integrins, and subsequent recruitment of Fyn and its substrate pi 30Cas to the leading edge.
  • This pathway appears to be critical for matrix rigidity-dependent regulation of neurite extension and axon differentiation, which can explain at least some of the abnormal aspects of hippocampal structure and function in RPTPot-/- mice. Since FN plays an important regulatory role in both normal development and variety of pathological processes in the brain, one could use these findings as a basis for further understanding of the basis for those diseases.
  • fences are 1--2 micrometers in thickness, 5-10 micrometers in height, and up to lmm in length with spacing of 20 to 100 micrometers (see FIG. 16).
  • Piezo actuators P ⁇ ezo Systems, Cambridge, MA
  • magnetic wires in the fences that will move them maximally 1-2 micrometers.
  • Fibers will be electrospun of silk or other polymers and stretched over the fences in one-dimensional arrays (FIG. 16). Fiber diameters of 100 to 1000 nm will be used and fibers will be coated with growth factors, adhesion proteins, extracellular matrix molecules or fragments, or any combination thereof. In a typical experiment, cells will be applied to the fibers and followed over time to determine growth and differentiation patterns along with forces and morphology. [00165]

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Abstract

La présente invention concerne un substrat de croissance de cellules qui comprend un milieu de croissance et un polymère réticulé. Une sollicitation plus importante exercée sur ledit polymère n'implique pas d'augmentation sensible de la rigidité de ce polymère. Ledit polymère présente un module d'élasticité (E) situé entre environ 0,1 kPa et environ 10,0 kPa. La présente invention concerne également un appareil conçu pour soumettre une cellule in vitro à une perturbation. Cet appareil comprend ledit substrat de croissance de cellules et un générateur de force externe qui est associé au substrat. Cette invention concerne aussi des procédés pour stimuler la croissance d'une cellule in vitro, une différenciation cellulaire in vitro et une réponse cellulaire in vitro, pour favoriser la croissance d'une cellule in vitro et pour traiter une plaie.
PCT/US2007/005291 2006-03-02 2007-03-02 Substrats de croissance de cellules actives et utilisations de ceux-ci Ceased WO2007103135A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102650077A (zh) * 2011-02-24 2012-08-29 中国科学院合肥物质科学研究院 修饰有壳聚糖的铁纳米线及其制备方法和用途
US9157070B2 (en) 2010-04-12 2015-10-13 Spiber Technologies Ab Methods and combination comprising eukaryotic cells and recombinant spider silk protein
US10156561B2 (en) 2012-10-05 2018-12-18 The Regents Of The University Of California Mechanical stress response analysis of cells and tissues
WO2019025070A1 (fr) 2017-07-31 2019-02-07 Università Degli Studi Di Genova Échafaudage d'hydrogel tridimensionnel pour culture cellulaire et sa méthode de production
WO2022250614A3 (fr) * 2021-05-28 2023-01-12 National University Of Singapore Plateforme de culture cellulaire et matériau magnétique pour une telle plateforme

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DE10041988B4 (de) * 2000-08-26 2006-02-09 Artmann, Gerhard, Prof. Dr. Vorrichtung und Verfahren zur Messung von Kräften von belebtem Material
US20040078090A1 (en) * 2002-10-18 2004-04-22 Francois Binette Biocompatible scaffolds with tissue fragments
US7713923B2 (en) * 2003-06-25 2010-05-11 Massachusetts Institute Of Technology Self-assembling peptides incorporating modifications and methods of use thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9157070B2 (en) 2010-04-12 2015-10-13 Spiber Technologies Ab Methods and combination comprising eukaryotic cells and recombinant spider silk protein
CN102650077A (zh) * 2011-02-24 2012-08-29 中国科学院合肥物质科学研究院 修饰有壳聚糖的铁纳米线及其制备方法和用途
US10156561B2 (en) 2012-10-05 2018-12-18 The Regents Of The University Of California Mechanical stress response analysis of cells and tissues
WO2019025070A1 (fr) 2017-07-31 2019-02-07 Università Degli Studi Di Genova Échafaudage d'hydrogel tridimensionnel pour culture cellulaire et sa méthode de production
WO2022250614A3 (fr) * 2021-05-28 2023-01-12 National University Of Singapore Plateforme de culture cellulaire et matériau magnétique pour une telle plateforme

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