Surface modification of polysaccharide, the modified polysaccharide, and method of culturing and recovery cells using the same

• US 20070148768A1
• Filed: 03/21/2006
• Published: 06/28/2007
• Est. Priority Date: 12/27/2005
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Hsu, Yuan-Hua, Wu, Yung-chih, Huang, Hsiang-Ming, Chiang, Shu-Fang, Tsai, Chen-Chi, Liao, Chun-Jen
• Granted Patent: US 7,906,333 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/10 Metals; Metal chelators cobalamine C12N2500/38
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/70 Polysaccharides
• CPC Class Codes: C12N 2533/72 Chitin, chitosan
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

A surface modification method of polysaccharide, the modified polysaccharide, and a method of culturing and recovery cells using the same are provided. The surface modification method of polysaccharide comprises (a) immersing a polysaccharide material in an acid, (b) immersing the polysaccharide material in an acidic solution containing a protein, and (c) immersing the polysaccharide material in an alkaline solution containing bivalent metal ions.

Claims

1. A surface modification method of polysaccharide, comprising:

(a) immersing a polysaccharide material in an acid;

(b) immersing the polysaccharide material in an acidic solution containing a protein; and

(c) immersing the polysaccharide material in an alkaline solution containing bivalent metal ions.

2. The method as claimed in claim 1, wherein the polysaccharide comprises alginate, N,O-carboxymethyl chitosan, or carboxymethyl cellulose.

3. The method as claimed in claim 2, wherein the polysaccharide is alginate.

4. The method as claimed in claim 1, wherein the polysaccharide material is a film, a microbead, or a porous matrix.

5. The method as claimed in claim 4, wherein the polysaccharide material is a film with a thickness of 20 μ

m to 5,000 μ

m.

6. The method as claimed in claim 4, wherein the polysaccharide material is a microbead with a diameter of 20 μ

m to 2,000 μ

m.

7. The method as claimed in claim 4, wherein the polysaccharide material is a porous matrix with a pore size of 20 cm to 2,000 μ

m.

8. The method as claimed in claim 4, wherein the polysaccharide material is a porous matrix with a porosity of 30% to 95%.

9. The method as claimed in claim 1, wherein the protein comprises collagen, gelatin, or chitosan.

10. The method as claimed in claim 9, wherein the protein is collagen.

11. The method as claimed in claim 1, wherein the alkaline solution containing bivalent metal ions comprises calcium hydroxide, magnesium hydroxide, or strontium hydroxide solution.

12. The method as claimed in claim 11, wherein the alkaline solution containing bivalent metal ions is calcium hydroxide solution.

13. The method as claimed in claim 1, further comprising a step (d) of washing the polysaccharide material with water after step (c).

42. A film for cell culture prepared by the method as claimed in claim 1.

43. The film as claimed in claim 42, which is with a thickness of 20 μ

m to 5,000 μ

m.

44. A microbead for cell culture prepared by the method as claimed in claim 1.

45. The microbead as claimed in claim 44, which is with a diameter of 20 μ

m to 2,000 μ

m.

46. A porous matrix for cell culture prepared by the method as claimed in claim 1.

47. The porous matrix as claimed in claim 46, which is with a pore size of 20 μ

m to 2,000 μ

m.

48. The porous matrix as claimed in claim 46, which is with a porosity of 30% to 95%.

14. A modified polysaccharide comprising a protein embedded on the surface thereof.

15. The modified polysaccharide as claimed in claim 14, wherein the polysaccharide comprises alginate, N,O-carboxymethyl chitosan, or carboxymethyl cellulose.

16. The modified polysaccharide as claimed in claim 15, wherein the polysaccharide is alginate.

17. The modified polysaccharide as claimed in claim 14, wherein the protein comprises collagen, gelatin, or chitosan.

18. The modified polysaccharide as claimed in claim 17, wherein the protein is collagen.

19. The modified polysaccharide as claimed in claim 14, which is a film, a microbead, or a porous matrix.

20. The modified polysaccharide as claimed in claim 19, which is a film with a thickness of 20 μ

m to 5,000 μ

m.

21. The modified polysaccharide as claimed in claim 19, which is a microbead with a diameter of 20 μ

m to 2,000 μ

cm.

22. The modified polysaccharide as claimed in claim 19, which is a porous matrix with a pore size of 20 μ

m to 2,000 μ

m.

23. The modified polysaccharide as claimed in claim 19, which is a porous matrix with a porosity of 30% to 95%.

24. The modified polysaccharide as claimed in claim 14, which is prepared by:

(a) immersing a polysaccharide material in an acid;

(b) immersing the polysaccharide material in an acidic solution containing a protein; and

(c) immersing the polysaccharide material in an alkaline solution containing bivalent metal ions.

25. The modified polysaccharide as claimed in claim 24, wherein the polysaccharide comprises alginate, N,O-carboxymethyl chitosan, or carboxymethyl cellulose.

26. The modified polysaccharide as claimed in claim 25, wherein the polysaccharide is alginate.

27. The modified polysaccharide as claimed in claim 24, wherein the protein comprises collagen, gelatin, or chitosan.

28. The modified polysaccharide as claimed in claim 27, wherein the protein is collagen.

29. The modified polysaccharide as claimed in claim 24, wherein the alkaline solution containing bivalent metal ions comprises calcium hydroxide, magnesium hydroxide, or strontium hydroxide solution.

30. The modified polysaccharide as claimed in claim 29, wherein the alkaline solution containing bivalent metal ions is calcium hydroxide solution.

31. The modified polysaccharide as claimed in claim 24, wherein the preparation further comprises a step (d) of washing the polysaccharide material with water after step (c).

32. A method of culturing and recovering cells, comprising:

seeding cells into the modified polysaccharide as claimed in claim 14;

cultivating the cells;

dissolving the modified polysaccharide in an ion chelating agent; and

collecting the cells.

33. The method as claimed in claim 32, wherein the cells are anchorage-dependent.

34. The method as claimed in claim 32, wherein the cells are liver cells, fibroblast cells, cartilage cells, Vero cells, CHO cells, osteoblasts, bone marrow cells, or umbilical blood cells.

35. The method as claimed in claim 32, wherein cultivation step comprises cultivating the cells in a static flat culturing system.

36. The method as claimed in claim 32, wherein the cultivation step comprises cultivating the cells in a dynamic bioreactor culturing system.

37. The method as claimed in claim 32, wherein the ion chelating agent comprises EDTA (ethylenediminetetraacetic acid), sodium citrate, or EGTA (ethyleneglycol-bis(2-aminoethylether)-N′

,N′

,N′

,N′

-tetraacetic acid).

38. The method as claimed in claim 37, wherein the ion chelating agent is EDTA.

39. The method as claimed in claim 38, wherein EDTA is in a concentration of 50 to 300 mM.

40. The method as claimed in claim 37, wherein the ion chelating agent is sodium citrate.

41. The method as claimed in claim 40, wherein the sodium citrate is in a concentration of 50 to 500 mM.