Chondrocyte media formulations and culture procedures

• US 6,150,163 A
• Filed: 01/13/1999
• Issued: 11/21/2000
• Est. Priority Date: 07/25/1996
• Status: Expired due to Term

• Current Assignee: Vericel Corporation
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Binette, Francois, McPherson, John M., Brown, Marie E., Yaeger, Peter C., Hanlon, James G.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US09/229,430
• Time in Patent Office: 678 Days
• Field of Search: 435/384, 435/383, 435/405, 435/406, 435/404
• US Class Current: 435/384
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

One object of the present invention is based upon the development and use of a serum-free defined cell culture medium comprising a supplement mixture, a component mixture, a vitamin mixture, an inorganic salt mixture and amino acid mixture that avoids the problems inherent in the use of serum. In particular, the defined medium is useful in culturing fibroblasts, especially chondrocytes. Another object of the present invention is to claim a method of enhancing the differentiation of chondrocytes and enhancing the synthesis of a cartilage specific matrix using tumor growth factor beta (TGF-β). Another object of the present invention is to claim a method of enhancing the differentiation of chondrocytes using the combination of TGF-βand IGF.

Claims

1. A method for enhancing the rate of re-differentiation of passaged, de-differentiated, human articular chondrocytes, comprising the step of culturing said passaged, de-differentiated chondrocytes in a medium supplemented with TGF-β

and a growth factor selected from the group consisting of;

IGF and insulin.

2. The method of claim 1, wherein the TGF-β

is TGF-β

1.

3. The method of claim 1, wherein the TGF-β

is TGF-β

2.

4. The method of claim 1, wherein the TGF-β

is present at 0.2 to 5.0 ng/ml.

5. The method of claim 1, wherein the growth factor is insulin.

6. The method of claim 1, wherein the growth factor is IGF.

7. The method of any one of claims 1-6, wherein the medium is defined.

8. The method of claim 5, wherein the insulin is supplied by ITS.

9. The method of claim 7, wherein the defined medium further comprises human serum albumin.

10. The method of claim 5, wherein the medium is defined and comprises DME, human serum albumin, insulin and TGF-β

.

11. A composition comprising passaged, de-differentiated human articular chondrocytes in a medium supplemented with TGF-β

and a growth factor selected from the group consisting of;

IGF and insulin.

12. The composition of claim 11, wherein the TGF-β

is TGF-β

1.

13. The composition of claim 12, wherein the TGF-β

is TGF-β

2.

14. The composition of claim 12, wherein the TGF-β

is present at 0.2 to 5.0 ng/ml.

15. The composition of claim 11, wherein the growth factor is insulin.

16. The composition of claim 11, wherein the growth factor is IGF.

17. The composition of any one of claims 11-16, wherein the medium is defined.

18. The composition of claim 14, wherein the insulin is supplied by ITS.

19. The composition of claim 16, wherein the defined medium further comprises human serum albumin.

20. The composition of claim 15, wherein the medium is defined and comprises DME, human serum albumin, insulin and TGF-β

.