Cell culture medium and solidified preparation of cell adhesion protein or peptide

• US 20060263878A1
• Filed: 03/24/2004
• Published: 11/23/2006
• Est. Priority Date: 03/24/2003
• Status: Active Grant

• Current Assignee: Katsumi Mochitate
• Original Assignee: Katsumi Mochitate, National Institute For Environmental Studies
• Inventors: Mochitate, Katsumi
• Granted Patent: US 8,304,238 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

The present invention provides a cell culture substrate which effectively adsorbs to a cell culture substrate such as a well, which is excellent in cell adhesion reproducibility, and whose surface is coated with a hydrophobic binding-adsorptive polymer and an immobilized preparation of cell adhesion proteins or peptides which effectively binds to the cell culture substrate and which is excellent in cell adhesion reproducibility, and further, an artificial tissue prepared by seeding and culturing cells on the immobilized preparation of cell adhesion peptides.

Claims

1. A cell culture substrate coated with a hydrophobic binding-absorptive polymer having a hydrophobic linear skeleton and a functional group that can react to a protein or a peptide in a molecule.

2. The cell culture substrate according to claim 1, wherein a base material of the cell culture substrate comprises a biobased polymer, plastic, natural or synthetic rubber, an inorganic material or metal.

3. The cell culture substrate according to claim 2, wherein the biobased polymer is collagen, gelatin, cellulose, agarose, alginic acid, chitin, chitosan, or a biodegradable polymer, which is, polylactic acid, polybutylente succinate, or polycaprolactone.

4. The cell culture substrate according to claim 2, wherein the plastic is a thermoplastic resin or a thermosetting resin.

5. The cell culture substrate according to claim 4, wherein the thermoplastic resin is an acryl resin, a polyvinyl chloride resin, a polyethylene resin, a polystyrene resin, a polypropylene resin, a polymethylpentene resin or a fluorocarbon resin.

6. The cell culture substrate according to claim 4, wherein the thermosetting resin is a phenolic resin, a urea formaldehyde resin, an epoxy resin, a melamine resin or a silicone resin.

7. The cell culture substrate according to claim 2, wherein the synthetic rubber is butadiene-styrene rubber, butadiene-acrylonitrile rubber, butyl rubber, polysulfide-based synthetic rubber, fluorocarbon rubber or silicone rubber.

8. The cell culture substrate according to claim 2, wherein the inorganic material is glass, hydroxyapatite, IC substrate or carbon nanotube.

9. The cell culture substrate according to claim 2, wherein the metal is inert gold, platinum, titanium, indium, or an oxide thereof which is titanium oxide, indium oxide, or ITO (indium tin oxide).

10. The cell culture substrate according to claim 1, wherein the cell culture substrate is a well, a printed-wiring board, or an artificial organ.

11. The cell culture substrate according to claim 10, wherein the artificial organ is an artificial blood vessel, an artificial heart lung, or an artificial kidney.

12. The cell culture substrate according to claim 1, wherein the cell culture substrate is a well comprising silicone as a base material.

13. The cell culture substrate according to claim 1, wherein the hydrophobic binding-adsorptive polymer is shown by the following formula [I]:

14. The cell culture substrate according to claim 13, wherein the hydrophobic binding-adsorptive polymer shown by the formula [I] is a copolymer made of a vinyl-based compound and maleic anhydride.

15. The cell culture substrate according to claim 14, wherein the vinyl-based compound is methyl vinyl ether, ethyl vinyl ether, butyl ether, hexyl vinyl ether or styrene.

16. An immobilized preparation of a cell adhesion protein or peptide wherein the cell adhesion protein or peptide is bound to the cell culture substrate according to claim 1.

17. The immobilized preparation according to claim 16, wherein the binding is covalent bonding formed by a reaction between a functional group, which is capable of reacting to a protein or a peptide, of a hydrophobic binding-adsorptive polymer and a reactive group of a cell adhesion protein or peptide.

18. The immobilized preparation according to claim 17, wherein the covalent bonding is amide bonding.

19. The immobilized preparation according to claim 16, wherein the cell adhesion protein is fibronectin (FN), collagen (Col), laminin (LN) or vitronectin (VN).

20. The immobilized preparation according to claim 16, wherein the cell adhesion peptide is a peptide in a region relating to cell adhesion in an amino acid sequence of the cell adhesion protein.

21. The immobilized preparation according to claim 20, wherein the peptide in a region relating to cell adhesion of fibronectin (FN) protein is a peptide having a specific Arg-Gly-Asp (RGD) amino acid sequence which binds to an integrin receptor on a cell side.

22. The immobilized preparation according to claim 21, wherein the peptide having an RGD amino acid sequence is Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser (FIB-1).

23. The immobilized preparation according to claim 20, wherein the peptide in a region relating to cell adhesion of laminin (LN) protein is an a-chain G-domain peptide.

24. The immobilized preparation according to claim 23, wherein the G-domain peptide is

25. The immobilized preparation according to claim 20, wherein the cell adhesion peptide is a peptide comprising 3 to 20 amino acid residues.

29. An artificial tissue prepared by seeding a desired cell on the immobilized preparation of a cell adhesion protein or peptide according to claim 16, and culturing the cell.

30. The artificial tissue according to claim 29, wherein the desired cell is an epithelial cell, an endothelial cell or a mesenchymal cell.

31. The artificial tissue according to claim 30, wherein the epithelial cell is an epidermal cell, a corneal epithelial cell, an alveolar epithelial cell, a mucosal epithelial cell of digestive system, a renal glomerular epithelial cell or a hepatic parenchymal cell.

32. The artificial tissue according to claim 30, wherein the endothelial cell is a renal glomerular ciliated cell, a vascular endothelial cell, a pulmonary arterial vascular endothelial cell, a placental venous vascular endothelial cell or an aortic endothelial cell.

33. The artificial tissue according to claim 30, wherein the mesenchymal cell is a muscle cell, an adipocyte, a glial cell, a Schwann cell or a neural cell (neuron).

34. The artificial tissue according to claim 29, wherein the artificial tissue is an artificial epidermal tissue, an artificial corneal epithelial tissue, an artificial alveolar epithelial tissue, an artificial respiratory epithelial tissue, an artificial renal glomerular tissue, an artificial hepatic parenchymal tissue or an artificial vascular endothelial tissue, or an artificial blood vessel, an artificial lung, an artificial liver, an artificial kidney, an artificial skin or an artificial cornea.

26. A method for producing an immobilized preparation wherein a functional group, which is capable of reacting to a protein or a peptide, of a hydrophobic binding-adsorptive polymer coated on a cell culture substrate reacts to a cell adhesion protein or peptide.

27. A method for producing an immobilized preparation wherein a functional group, which is capable of reacting to a protein or a peptide, of a hydrophobic binding-adsorptive polymer reacts to a cell adhesion protein or peptide, and a cell culture substrate is coated with the reactant.

28. A reactant obtained by reacting a functional group, which is capable of reacting to a protein or a peptide, of a hydrophobic binding-adsorptive polymer, to cell adhesion proteins or peptides.