Serum-free cell growth medium

• US 6,617,161 B2
• Filed: 05/09/2001
• Issued: 09/09/2003
• Est. Priority Date: 06/25/1997
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Erlacher, Ludwig, Luyten, Frank P.
• Primary Examiner(s): Tate, Christopher R.
• Assistant Examiner(s): LEITH, PATRICIA A
• Application Number:
  US09/851,921
• Publication Number:
  US 20010039050A1
• Time in Patent Office: 853 Days
• Field of Search: 424/93.7, 435/325, 435/366, 435/374, 435/375, 435/383, 435/384, 435/388, 435/389, 435/404, 435/405
• US Class Current: 435/375
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/33 other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
• 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 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/20 Cytokines; Chemokines
• CPC Class Codes: C12N 2501/37 Parathyroid hormone [PTH]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

A chemically defined-serum free growth medium for the in vitro and ex vivo of cells and cell lines. The medium consists of about a one to one ratio (v/v) of two basal growth media containing α-ketoglutarate, insulin, transferrin, selenium, bovine serum albumin, linoleic acid, ceruloplasmin, cholesterol, phosphatidyl-ethanolamine, α-tocopherol acid succinate, reduced glutathione, taurine, triiodothyronine, hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate, β-glycerophosphate, PDGF, EGF and FGF. Chondrocytes, when cultured in this medium in the presence of a cartilage derived morphogenetic protein or bone morphogenetic protein, retain their cartilaginous phenotype.

Claims

1. A method of maintaining a cartilaginous phenotype in chondrocytes in vitro comprising contacting a culture comprising chondrocytes, which have not lost and subsequently re-expressed a cartilaginous phenotype, with a serum-free cell growth medium comprising about a 1:

1 ratio (v/v) of two basal cell culture media, said medium containing effective cell growth-promoting concentrations of α

-ketoglutarate, insulin, transferrin, selenium, bovine serum albumin, linoleic acid, ceruloplasmin, cholesterol, phosphatidylethanolamine, α

-tocopherol acid succinate, reduced glutathione, taurine, triiodothyronine, hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate, β

-glycerophosphate, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bEGF) and at least one morphogenetic protein selected from the group consisting of cartilage-derived morphogenetic proteins and bone morphogenetic proteins, whereby a cartilaginous phenotype is maintained in said chondrocytes in vitro.

3. The method of claim 1 or 2, wherein two basal cell culture media are selected and mixed in equal proportion and wherein said basal cell culture media are selected from the group consisting of Ham'"'"'s F-12, Dulbecco'"'"'s modified Eagle'"'"'s medium (DMEM), Essential modified Eagle'"'"'s medium (EMEM) and RPMI-1640.

4. The method of claim 1 or 2, wherein said morphogenetic protein is cartilage-derived morphogenetic protein and is selected from the group consisting of CDMP-1 and CDMP-2.

5. The method of claim 1 or 2, wherein said morphogenetic protein is bone morphogenetic protein and is selected from the group consisting of OP-1, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6.

6. The method of claim 1 or 2, wherein the concentration of α

-ketoglutarate is about 1×

10<sup>−

4</sup> M.

7. The method of claim 1 or 2, wherein the concentration of ceruloplasmin is about 0.25 U/ml.

8. The method of claim 1 or 2, wherein the concentration of cholesterol is about 5 μ

g/ml.

9. The method of claim 1 or 2, wherein the concentration of phosphatidyl-ethanolamine is about 2 μ

g/ml.

10. The method of claim 1 or 2, wherein the concentration of α

-tocopherol acid succinate is about 9×

10<sup>−

7</sup> M.

11. The method of claim 1 or 2, wherein the concentration of reduced glutathione is about 10 μ

g/ml.

12. The method of claim 1 or 2, wherein the concentration of taurine is about 1.25 μ

g/ml.

13. The method of claim 1 or 2, wherein the concentration of triiodothyronine is about 1.6×

10<sup>−

9</sup> M.

14. The method of claim 1 or 2, wherein the concentration of hydrocortisone is about 1×

10<sup>−

9</sup> M.

15. The method of claim 1 or 2, wherein the concentration of parathyroid hormone is about 5×

10<sup>−

10</sup> M.

16. The method of claim 1 or 2, wherein the concentration of L-ascorbic acid 2-sulfate is about 50 μ

g/ml.

17. The method of claim 1 or 2, wherein the concentration of PDGF is about 4 ng/ml.

18. The method of claim 1 or 2, wherein the concentration of EGF is about 10 ng/ml.

19. The method of claim 1 or 2, wherein the concentration of bFGF is about 10 ng/ml.

2. A method of repairing a joint surface defect in a mammal in need thereof comprising the steps of:

removing normal cartilage in the vicinity of said surface defect;

isolating chondrocytes from said cartilage;

contacting a culture comprising said chondrocytes, which have not lost and subsequently re-expressed a cartilaginous phenotype, with a serum-free cell growth medium comprising about a 1;

1 ratio (v/v) of two basal cell culture media, said medium containing effective cell growth-promoting concentrations of α

-ketoglutarate, insulin, transferrin, selenium, bovine serum albumin, linoleic acid, ceruloplasmin, cholesterol, phosphatidylethanolamine, α

-tocopherol acid succinate, reduced glutathione, taurine, triiodothyronine, hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate, β

-glycerophosphate, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bEGF) and at least one morphogenetic protein selected from the group consisting of cartilage-derived morphogenetic proteins and bone morphogenetic proteins, whereby a cartilaginous phenotype is maintained in said chondrocytes; and

implanting said chondrocytes into said surface defect.

20. The method of claim 2, wherein said isolating chondrocytes step comprises digestion of said cartilage with collagenase.