Surfaces having desirable cell adhesive effects
First Claim
1. A cell culture substrate containing less than about 45% water comprising a surface with covalently linked peptide at a concentration density of between about 0.001 picomoles and about 50 picomoles/cm2, wherein said peptide is attached through an N-terminal amine to a hydroxyl group at the substrate surface, wherein said peptide is at least one peptide selected from the group consisting of:
- GRGD;
GYIGSRY;
GRGDF, GRGDY;
GREDV;
GYIGSR;
GREDVY; and
GRGDS;
wherein said substrate is capable of supporting cell focal adhesion formation and cell spreading.
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Accused Products
Abstract
An absorbed protein-independent cell-adhesive surface is disclosed. The treated surface comprises a chemically derivatized material to which small peptides, having less than 12 amino acid residues and including YIGSR, RGD or REDV amino acid sequence are covalently linked to. The peptides of the present invention include a terminal glycine amino acid. Tresyl chloride activation of surface hydroxyl moieties provides the active surface sites by which a terminal glycine arm of a selected peptide attaches to form covalent bonds between the substrate and peptide. Peptides high with cell adhesive properties are bound in high efficiency.
By way of example, surfaces which may be used in conjunction with the present invention include polymer, metal, and ceramic surfaces. The most preferred polymer surfaces include PHEMA and PET polymer surfaces, with the most preferred glass surfaces being glycophase glass.
The present methods also include a pretreatment method which provides hydroxyl moieties to surfaces devoid of readily available hydroxyl moieties. The pretreatment, by way of example, comprises immersion of the surface in a mixture of formaldehyde and acetic acid.
Methods of preparing the treated surfaces are also included in the present invention.
Also included are surface-treated biomedical implant devices and cell culturing devices. The treated surface promotes an enhanced rate and an enhanced amount of cell adhesion to the surface, independent of media serum concentrations or other absorbed proteins. The treated surfaces of the present invention are thermally stable, reusable, peptide efficient (attached to surface only) and resistant to cell proteolysis.
The invention further concerns polymeric substrates with a surface having physically interpenetrating water-soluble polymer chains, and methods for production thereof.
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Citations
57 Claims
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1. A cell culture substrate containing less than about 45% water comprising a surface with covalently linked peptide at a concentration density of between about 0.001 picomoles and about 50 picomoles/cm2, wherein said peptide is attached through an N-terminal amine to a hydroxyl group at the substrate surface, wherein said peptide is at least one peptide selected from the group consisting of:
GRGD;
GYIGSRY;
GRGDF, GRGDY;
GREDV;
GYIGSR;
GREDVY; and
GRGDS;wherein said substrate is capable of supporting cell focal adhesion formation and cell spreading. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method of derivatizing a substrate surface comprising:
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chemically derivatizing a surface with GREDVY, GRGD or GYIGSR peptide, wherein said peptide is covalently attached through an N-terminal amine to a hydroxyl group on the substrate surface; and plating cells on the peptide derivatized surface, wherein the peptide is at a concentration of about 0.001 picomoles/cm2 to about 50 picomoles/cm2, wherein said substrate supports cell focal adhesion formation and cell spreading. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38)
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32. A cell culture modification method comprising:
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activating hydroxyl moieties of a substrate surface; and covalently coupling an N-terminal amine of a peptide to a hydroxyl group on the substrate surface, wherein said peptide is at least one peptide selected from the group consisting of; GRGD;
GYIGSRY;
GRGDF;
GRGDY;
GREDV;
GYIGSR;
GREDVY; andwherein said peptide is included at a concentration density of between about 0.001 picomoles/cm2 to about 50 picomoles/cm2, and wherein said surface is capable of supporting cell focal adhesion formation and cell spreading. - View Dependent Claims (33, 39)
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40. A method for derivatizing a substrate surface of poly (hydroxyethyl methacrylate) comprising:
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derivatizing the poly (hydroxyethyl methacrylate) surface with GRGD peptides, wherein said peptides are chemically attached by a covalent chemical bond between the N-terminal amine of the peptide and a hydroxyl moiety of the surface; and plating cells on the peptide derivatized surface, wherein the GRGD peptide is at a concentration of between about 0.001 picomoles/cm2 to about 50 picomoles/cm2 on the surface, and wherein said substrate supports cell focal adhesion formation and cell spreading.
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- 41. A method for enhancing selective endothelial cell adhesion to a surface comprising covalently attaching GREDV, or GREDVY peptide through the N-terminal amine of the peptide to surface hydroxyl groups on a substrate surface, and plating cells on the surface, wherein said surface supports selective endothelial cell focal adhesion formation and cell spreading.
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45. A method for enhancing endothelial cell adhesion and spreading to a surface comprising chemically derivatizing the surface with a peptide consisting essentially of GREDV whereby the peptide is covalently bound to the surface through the N-terminal glycine of the peptide;
- and contacting the derivatized surface with a population of cells containing endothelial cells.
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46. A method for enhancing fibroblast adhesion to a surface comprising chemically derivatizing the surface with a peptide consisting essentially of GYIGSR whereby the peptide is covalently bound to the surface through the N-terminal glycine of the peptide;
- and contacting the derivatized surface with a population of cells containing fibroblasts.
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47. A cell culture substrate comprising a surface with a covalently linked peptide at a concentration density of less than about 50 picomoles/cm2, said peptide being attached through an N-terminal glycine to hydroxyl groups at the substrate surface, wherein said peptide is at least one peptide selected from the of peptides group consisting of:
GRGD;
GYIGSRY;
GRGDF;
GRGDY;
GREDV;
GYIGSR;
GREDVY; andwherein said substrate is capable of supporting cell focal adhesion formation and cell spreading. - View Dependent Claims (48)
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49. A method of preparing a cell substrate surface comprising:
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covalently attaching a GRGD, GRGDY, GYIGSR, or GYIGSRY peptide through the N-terminal glycine to surface hydroxyl moieties of a substrate surface; and plating cells on the substrate surface, wherein the peptide is at a concentration of between about 0.001 picomoles/cm2 to about 50 picomoles/cm2 on the substrate surface supports cell focal adhesion formation and cell spreading.
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50. A method for promoting selective endothelial cell and fibroblast cell adhesion without platelet adhesion to a surface comprising:
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covalently attaching GYIGSR or GYIGSRY peptide to surface hydroxyl groups; and exposing the surface to a population of cells that includes endothelial cells, wherein said surface supports the selective adhesion of endothelial cells and fibroblast cells and excludes platelet adhesion.
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51. A method for promoting selective endothelial cell and fibroblast cell adhesion without platelet adhesion to a surface comprising;
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covalently attaching the peptide GPDSGRY through the N-terminal amine to a surface hydroxyl group; and exposing the surface to a population of cells that includes endothelial cells, wherein said surface supports the selective adhesion of endothelial cells and fibroblast cells and excludes platelet adhesion.
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52. A surface for the selective adhesion of endothelial cells and fibroblast cells without platelet adhesion comprising a surface with a covalently attached GYIGSR peptide, wherein said peptide is attached through the N-terminal amine to a hydroxyl group on the surface and wherein said surface supports the selective adhesion of endothelial cells and fibroblast cells and excludes platelet adhesion.
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53. A surface for the selective adhesion of endothelial cells and fibroblasts without platelet adhesion comprising a surface with a covalently attached peptide, GPDSGRY, wherein said peptide is attached through the N-terminal amine to hydroxyl groups on the surface, and wherein said surface supports the selective adhesion of endothelial cells and fibroblast cells and excludes platelet adhesion.
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54. A method for promoting selective endothelial cell adhesion without fibroblast or platelet adhesion to a surface comprising:
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covalently attaching the peptide GREDVY through the N-terminal amine to a surface hydroxyl group; and exposing the substrate to a population of cells that includes endothelial cells, wherein said surface supports the selective adhesion of endothelial cells excludes fibroblast and platelet adhesion.
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55. A surface for the selective adhesion of endothelial cells without fibroblast or platelet adhesion comprising a surface with a covalently attached peptide GREDVY, wherein said peptide is attached through the N-terminal amine to a hydroxyl group on the surface, and wherein said surface supports the selective adhesion of endothelial cells and excludes fibroblast and platelet adhesion.
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56. A cell culture modification method comprising:
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activating hydroxyl moieties of a poly (hydroxyethyl methyl acrylate) surface of a substrate; and covalently coupling GRGD peptides through their N-terminal amine to hydroxyl groups of the surface of the substrate, wherein the GRGD peptides are included at a concentration density of between about 0.001 picomoles/cm2 to about 50 picomoles/cm2, and wherein the surface is capable of supporting cell focal adhesion formation and cell spreading.
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57. A method of derivatizing a substrate surface to support cell focal adhesion formation and cell spreading comprising:
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chemically derivatizing a surface with GREDVY peptides, wherein said peptides are covalently attached through their N-terminal amines to hydroxyl groups on the substrate surface; and plating cells on the peptide derivatized surface, wherein the GREDVY peptide is included at a concentration density of between about 0.001 picomoles/cm2 and about 50 picomoles/cm2, and wherein said surface is capable of supporting cell focal adhesion formation and cell spreading.
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Specification