US5330911A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Surfaces having desirable cell adhesive effects
Assignee: The University of Texas System
•
Filing: 1990-05-21
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Granted: 1994-07-19
•
Expires: 2010-05-21
Abstract
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.
Inventors
- 👤 Jeffrey A. Hubbell
- 👤 Stephen P. Massia
- 👤 Neil P. Desai
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 04:31
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/0068; A; A61; A61L; A61L17/00; A61L17/14; A61L17/145; A61L27/00; A61L27/14; A61L27/22; A61L27/227; A61L27/28; A61L27/34; A61L29/00; A61L29/04; A61L29/044; A61L29/048; A61L29/08; A61L29/085; A61L31/00; A61L31/04; A61L31/043; A61L31/047; A61L31/08; A61L31/10; C07; C07K; C07K17/00; C07K17/02; C07K17/08; C12N2533/00; C12N2533/50
Claims Analysis
Total Claims: 74
Claim 1
Independent Claim
86 words
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/cm 2 , 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.
Claim 2
Dependent Claim
86 words
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/cm 2 , 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.
Claim 3
Dependent Claim
16 words
The cell culture substrate of claim 1, wherein said peptide is GRGDY, GYIGSRY, GRGD or GYIGSRY.
Claim 4
Dependent Claim
15 words
The cell culture substrate of claim 1, or 2, wherein the substrate comprises a ceramic.
Claim 5
Dependent Claim
12 words
The cell culture substrate of claim 3, wherein the ceramic is glass.
Claim 6
Dependent Claim
14 words
The cell culture substrate of claim 4, wherein the glass comprises glycerol propylsilane-bonded glass.
Claim 7
Dependent Claim
14 words
The cell culture substrate of claim 5, wherein the peptide is GRGDY or GYIGSRY.
Claim 8
Dependent Claim
17 words
The cell culture substrate of claim 1, wherein said peptide has 4 or 6 amino acid residues.
Claim 9
Dependent Claim
21 words
The cell culture substrate of claim 7, wherein the peptide has 4 amino acid residues and is further defined as GRGD.
Claim 10
Dependent Claim
21 words
The cell culture substrate of claim 7, wherein the peptide has 6 amino acid residues and is further defined as GYIGSR.
Claim 11
Dependent Claim
12 words
The cell culture substrate of claim 7 wherein the peptide is GREDVY.
Claim 12
Dependent Claim
26 words
The cell culture substrate of claim 1, wherein the substrate comprises a material selected from the group consisting of: a ceramic; a polymer; and a metal.
Claim 13
Dependent Claim
12 words
The cell culture substrate of claim 11 wherein the metal is titanium.
Claim 14
Dependent Claim
24 words
The cell culture substrate of claim 1, wherein the substrate comprises a material selected from the group consisting of a polymer; or a ceramic.
Claim 15
Dependent Claim
39 words
The cell culture substrate of claim 1, wherein the substrate comprises a polymer selected from the group consisting of: a plastic; a poly (hydroxyethyl methylacrylate); a poly (ethylene terephthalate); a poly (tetrafluoroethylene); a fluorinated ethylene; and a poly(dimethyl siloxane).
Claim 16
Dependent Claim
13 words
The cell culture substrate of claim 14, wherein the polymer is poly(hydroxyethyl methacrylate).
Claim 17
Dependent Claim
12 words
The cell culture substrate of claim 15, wherein the peptide is GRGD.
Claim 18
Dependent Claim
13 words
The cell culture substrate of claim 14, wherein the polymer is poly(ethylene terephthalate).
Claim 19
Dependent Claim
14 words
The cell culture substrate of claim 17, wherein the peptide is GRGD or GYIGSR.
Claim 20
Dependent Claim
31 words
The cell culture substrate of claim 1, wherein the peptide is attached to the substrate at a surface concentration of between about 0.001 picomoles/cm 2 and about 20 picomoles/cm 2 .
Claim 21
Dependent Claim
28 words
The cell culture substrate of claim 1, wherein the peptide is attached to the substrate at a concentration of between 1 picomoles/cm 2 to 12 picomoles/cm 2 .
Claim 22
Dependent Claim
24 words
The cell culture substrate of claim 1 wherein the peptide is GREDV and provides for the selective adhesion of endothelial cells to the substrate.
Claim 23
Independent Claim
73 words
A method of derivatizing a substrate surface comprising: 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/cm 2 to about 50 picomoles/cm 2 , wherein said substrate supports cell focal adhesion formation and cell spreading.
Claim 24
Independent Claim
73 words
A method of derivatizing a substrate surface comprising: 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/cm 2 to about 50 picomoles/cm 2 , wherein said substrate supports cell focal adhesion formation and cell spreading.
Claim 25
Dependent Claim
15 words
The method of claim 22, wherein the cells are human, murine, insect or porcine cells.
Claim 26
Dependent Claim
36 words
The method of claim 23, wherein a hydroxyl group on the surface is activated by exposing the surface to a chemical selected from the group consisting of: glutaraldehyde; cyanuric chloride; sulfonylchloride; cyanogen bromide; and trifluoroethanesulfonyl chloride.
Claim 27
Dependent Claim
11 words
The method of claim 22 wherein the cells are human cells.
Claim 28
Dependent Claim
26 words
The method of claim 22 or 25 wherein the surface comprises a peptide concentration of between about 0.001 picomoles/cm 2 and about 20 picomoles/cm 2 .
Claim 29
Dependent Claim
26 words
The method of claim 22 or 25, wherein the surface comprises a peptide concentration of between about 0.5 picomoles/cm 2 and about 20 picomoles/cm 2 .
Claim 30
Dependent Claim
20 words
The method of claim 22 or 25, wherein the surface comprises a peptide concentration of about 12 picomoles/cm 2 .
Claim 31
Dependent Claim
16 words
The method of claim 22, wherein the cells are 3T3 cells, aortic cells, or foreskin cells.
Claim 32
Dependent Claim
11 words
The method of claim 22, wherein the cells comprise NIH/3T3 fibroblasts.
Claim 33
Dependent Claim
13 words
The method of claim 22, wherein the cells comprise human foreskin fibroblast cells.
Claim 34
Dependent Claim
88 words
A cell culture modification method comprising: 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; and wherein said peptide is included at a concentration density of between about 0.001 picomoles/cm 2 to about 50 picomoles/cm 2 , and wherein said surface is capable of supporting cell focal adhesion formation and cell spreading.
Claim 35
Dependent Claim
88 words
A cell culture modification method comprising: 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; and wherein said peptide is included at a concentration density of between about 0.001 picomoles/cm 2 to about 50 picomoles/cm 2 , and wherein said surface is capable of supporting cell focal adhesion formation and cell spreading.
Claim 36
Dependent Claim
36 words
The method of claim 32 wherein the surface comprises the cell culture surface of a device selected from the group consisting of: a tissue-culture flask; a Petri dish; microcarriers; macrocarriers; fibers; monolith supports; and roller bottles.
Claim 37
Dependent Claim
26 words
The method of claim 22 or 32, wherein the surface comprises a material selected from the group consisting of: a ceramic; a polymer; and a metal.
Claim 38
Dependent Claim
10 words
The method of claim 34, wherein the metal is titanium.
Claim 39
Dependent Claim
11 words
The method of claim 34, wherein the surface comprises a polymer.
Claim 40
Dependent Claim
32 words
The method of claim 36, wherein a terminal primary amine of a glycine linker arm on the peptide is covalently coupled to the hydroxyl groups of polymer surface by tresyl chloride activation.
Claim 41
Dependent Claim
11 words
The method of claim 36, wherein the polymer is poly(hydroxyethyl methylacrylate).
Claim 42
Dependent Claim
21 words
The method of claim 32, wherein the peptide of covalently coupled to the substrate surface hydroxyl groups by tresyl chloride activation.
Claim 43
Independent Claim
88 words
A method for derivatizing a substrate surface of poly (hydroxyethyl methacrylate) comprising: 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/cm 2 to about 50 picomoles/cm 2 on the surface, and wherein said substrate supports cell focal adhesion formation and cell spreading.
Claim 44
Independent Claim
88 words
A method for derivatizing a substrate surface of poly (hydroxyethyl methacrylate) comprising: 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/cm 2 to about 50 picomoles/cm 2 on the surface, and wherein said substrate supports cell focal adhesion formation and cell spreading.
Claim 45
Independent Claim
52 words
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.
Claim 46
Independent Claim
52 words
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.
Claim 47
Dependent Claim
17 words
The method of claim 41 wherein the peptide is GREDV and the surface is a glass surface.
Claim 48
Dependent Claim
27 words
The method of claim 41 wherein the peptide is attached at the N-terminus of the peptide to the surface, wherein said surface excludes the attachment of platelets.
Claim 49
Dependent Claim
24 words
The method of claim 41 wherein the surface comprises a material selected from the group consisting of: a ceramic; a polymer; and a metal.
Claim 50
Independent Claim
53 words
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.
Claim 51
Independent Claim
53 words
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.
Claim 52
Independent Claim
49 words
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.
Claim 53
Independent Claim
49 words
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.
Claim 54
Dependent Claim
77 words
A cell culture substrate comprising a surface with a covalently linked peptide at a concentration density of less than about 50 picomoles/cm 2 , 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; and wherein said substrate is capable of supporting cell focal adhesion formation and cell spreading.
Claim 55
Dependent Claim
77 words
A cell culture substrate comprising a surface with a covalently linked peptide at a concentration density of less than about 50 picomoles/cm 2 , 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; and wherein said substrate is capable of supporting cell focal adhesion formation and cell spreading.
Claim 56
Dependent Claim
12 words
The cell culture substrate of claim 47 wherein the peptide is GRGDY.
Claim 57
Independent Claim
67 words
A method of preparing a cell substrate surface comprising: 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/cm 2 to about 50 picomoles/cm 2 on the substrate surface supports cell focal adhesion formation and cell spreading.
Claim 58
Independent Claim
67 words
A method of preparing a cell substrate surface comprising: 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/cm 2 to about 50 picomoles/cm 2 on the substrate surface supports cell focal adhesion formation and cell spreading.
Claim 59
Independent Claim
58 words
A method for promoting selective endothelial cell and fibroblast cell adhesion without platelet adhesion to a surface comprising: 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.
Claim 60
Independent Claim
58 words
A method for promoting selective endothelial cell and fibroblast cell adhesion without platelet adhesion to a surface comprising: 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.
Claim 61
Independent Claim
62 words
A method for promoting selective endothelial cell and fibroblast cell adhesion without platelet adhesion to a surface comprising; 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.
Claim 62
Independent Claim
62 words
A method for promoting selective endothelial cell and fibroblast cell adhesion without platelet adhesion to a surface comprising; 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.
Claim 63
Dependent Claim
58 words
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.
Claim 64
Dependent Claim
58 words
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.
Claim 65
Dependent Claim
56 words
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.
Claim 66
Dependent Claim
56 words
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.
Claim 67
Independent Claim
59 words
A method for promoting selective endothelial cell adhesion without fibroblast or platelet adhesion to a surface comprising: 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.
Claim 68
Independent Claim
59 words
A method for promoting selective endothelial cell adhesion without fibroblast or platelet adhesion to a surface comprising: 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.
Claim 69
Dependent Claim
56 words
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.
Claim 70
Dependent Claim
56 words
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.
Claim 71
Dependent Claim
74 words
A cell culture modification method comprising: 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/cm 2 to about 50 picomoles/cm 2 , and wherein the surface is capable of supporting cell focal adhesion formation and cell spreading.
Claim 72
Dependent Claim
74 words
A cell culture modification method comprising: 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/cm 2 to about 50 picomoles/cm 2 , and wherein the surface is capable of supporting cell focal adhesion formation and cell spreading.
Claim 73
Independent Claim
86 words
A method of derivatizing a substrate surface to support cell focal adhesion formation and cell spreading comprising: 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/cm 2 and about 50 picomoles/cm 2 , and wherein said surface is capable of supporting cell focal adhesion formation and cell spreading.
Claim 74
Independent Claim
86 words
A method of derivatizing a substrate surface to support cell focal adhesion formation and cell spreading comprising: 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/cm 2 and about 50 picomoles/cm 2 , and wherein said surface is capable of supporting cell focal adhesion formation and cell spreading.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-07-18 | 1998-01-17 | 1998-07-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-07-18 | 2002-01-17 | 2002-07-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-07-18 | 2006-01-17 | 2006-07-19 | $7700.00 | Window Closed / Lapsed |