Serum-free chemically defined cell culture medium

• US 10,287,550 B2
• Filed: 05/27/2016
• Issued: 05/14/2019
• Est. Priority Date: 06/17/2010
• Status: Active Grant

• Current Assignee: Stemrd, Inc.
• Original Assignee: Stemrd, Inc.
• Inventors: An, Songzhu, Zhu, Yanan
• Primary Examiner(s): Yamasaki, Robert J
• Application Number:
  US15/166,965
• Publication Number:
  US 20160281060A1
• Time in Patent Office: 1,082 Days
• Field of Search: None
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• 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 2501/105 Insulin-like growth factors [IGF]
• 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 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/825 Serotonine (5-HT); Melatonine
• CPC Class Codes: C12N 2506/1346 from mesenchymal stem cells
• CPC Class Codes: C12N 5/0037 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage
• CPC Class Codes: C12N 5/0662 : Stem cells
• CPC Class Codes: C12N 5/0663 : Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 5/0665 : Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
• CPC Class Codes: C12N 5/0667 : Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Abstract

Embodiments of chemically defined cell culture media containing nutrients and growth factors free of any serum for culturing cells such as mesenchymal stem cells and methods of using embodiments of the cell culture medium for expanding cell populations such as mesenchymal stem cells while maintaining a pluripotent phenotype and methods of inducing chondrogenesis and osteogenesis of mesenchymal stem cells by admixing differentiation factors into embodiments of the cell culture medium.

Claims

1. A serum-free chemically defined cell culture medium composition, the composition comprising:

a serum free base medium comprising Dulbecco'"'"'s Modified Eagle Medium and MCDB 201 medium;

a regulated iron source;

an electron transport activator;

an antioxidant;

a steroid;

5-hydroxytryptamine;

a human serum albumin;

a phospholipid growth factor selected from lysophosphatidic acid and sphingosine 1-phosphate;

a cell growth factor;

human wingless/integrated 3a; and

R-spondin-1, said composition adjusted to a pH in a range of about 7.3 to about 7.5.

2. The composition of claim 1, wherein said Dulbecco'"'"'s Modified Eagle Medium and said MCDB 201 medium have a ratio in the range of about 0.75:

1.25 v/v to about 1.25;

0.75 v/v.

3. The composition of claim 2, wherein said ratio of Dulbecco'"'"'s Modified Eagle Medium to MCDB201 medium is in a range selected from the group consisting of:

about 0.75;

1.25 to about 0.85;

1.15, about 0.80;

1.20 to about 0.90;

1.10, about 0.85;

1.15 to about 0.95;

1.05, about 90;

1.10 to about 1.05;

0.95, about 1.00;

1.00 to about 1.10;

0.90, about 1.05;

0.95 to about 1.15;

0.85, about 1.10;

0.90 to about 1.20;

0.80, and about 1.15;

0.85 to about 1.25;

0.75.

4. The composition of claim 3, wherein said steroid comprises dexamethasone and hydrocortisone wherein said dexamethasone has a concentration in a range of about 2.0 μ

g/mL to about 5.0 μ

g/mL, and wherein said hydrocortisone has a concentration in a range of about 0.5 mg/mL to about 1.5 mg/mL.

5. The composition of claim 4, wherein said regulated iron source comprises transferrin having a concentration in the range of about 2 mg/L to about 10 mg/L.

6. The composition of claim 5, wherein said concentration of transferrin is in a range selected from the group consisting of:

about 2 mg/L to about 4 mg/L, about 3 mg/L to about 5 mg/L, about 4 mg/L to about 6 mg/L, about 5 mg/L to about 7 mg/L, about 6 mg/L to about 8 mg/L, about 7 mg/L to about 9 mg/L, and about 8 mg/L to about 10 mg/L.

7. The composition of claim 6, wherein said human serum albumin has concentration in a range of about 100 μ

g/mL to about 350 μ

g/mL.

8. The composition of claim 7, wherein said 5-hydroxytryptamine has a concentration in the range of about 0.001 g/L to about 0.003 g/L.

9. The composition of claim 8, wherein said electron transport activator comprises selenous acid having a concentration in the range of about 0.0000025 g/L to about 0.0000050 g/L.

10. The composition of claim 9, wherein said antioxidant comprises α

-tocopherol acetate having a concentration in the range of about 0.0001 g/L to about 0.0003 g/L.

11. The composition of claim 10, wherein said antioxidant comprises ascorbic acid-2 phosphate having a concentration in the range of about 0.02 g/L to about 0.04 g/L.

12. The composition of claim 11, wherein said cell growth factor comprises human basic fibroblast growth factor having a concentration in the range of about 5 ng/mL to about 20 ng/mL.

13. The composition of claim 12, wherein said cell growth factor comprises platelet-derived growth factor bb having a concentration in the range of about 5 ng/mL to about 20 ng/mL.

14. The composition of claim 13, wherein said cell growth factor comprises epidermal growth factor having a concentration in the range of about 15 ng/mL to about 25 ng/mL.

15. The composition of claim 14, wherein said cell growth factor comprises insulin like growth factor-1 having a concentration in the range of about 2 ng/mL to about 10 ng/mL.

16. The composition of claim 15, wherein said cell growth factor comprises insulin having a concentration in a range about 0.004 g/L and about 0.006 g/L.

17. The composition of claim 16, wherein said phospholipid growth factor comprises lysophosphatidic acid having a concentration in the range of about 80 nM to about 200 nM.

18. The composition of claim 17, wherein said phospholipid growth factor comprises sphingosine 1-phosphate having a concentration in the range of about 80 nM and about 200 nM.

19. The composition of claim 18, wherein said one or more phospholipid growth factors has a concentration in a range selected from the group consisting of:

about 100 nM to about 120 nM, about 110 nM to about 130 nM, about 120 nM to about 140 nM, about 130 nM to about 150 nM, about 140 nM to about 160 nM, about 150 nM to about 170 nM, about 160 nM to about 180 nM, and about 190 nM to about 200 nM.

20. The composition of claim 19, further comprising a transforming growth factor beta 1 having a concentration in a range of about 0.5 ng/mL to about 5 ng/mL.

21. The composition of claim 20, wherein said wingless/integrated 3a has a concentration in the range of about 20 ng/mL to about 200 ng/mL.

22. The composition of claim 21, wherein said R-spondin-1 has a concentration in the range of about 20 ng/mL to about 200 ng/mL.

23. A serum-free chemically defined cell culture medium composition, the composition comprising:

an amount of MCDB 201 medium of about 0.5 L/L;

an amount of Dulbecco'"'"'s modified eagle medium of about 0.5 L/L;

an amount of sodium bicarbonate of about 3.7 g/L;

an amount of human serum albumin of about 0.25 g/L;

an amount of transferrin of about 0.005 g/L;

an amount of insulin of about 0.005 g/L;

an amount of selenous acid of about 0.0000037 g/L;

an amount of ascorbic acid-2phosphate of about 0.032205 g/L;

an amount of 5-hydroxytryptamine of about 0.002127 g/L;

an amount of dexamethasone of about 0.000003925 g/L;

an amount of hydrocortisone of about 0.001 g/L; and

an amount of α

-tocopherol acetate of about 0.0002 g/L;

an amount of platelet-derived growth factor bb of about 10 ng/mL;

an amount of human basic fibroblast growth factor of about 10 ng/mL;

an amount of epiderman growth factor of about 20 ng/mL;

an amount of insulin like growth factor-1 of about 5 ng/mL;

an amount of lysophosphatidic acid of about 100 nM;

an amount of sphingosine 1-phosphate of about 150 nM;

an amount of human wingless/integrated 3a of about 20 ng/mL; and

an amount of R-spondin-1 of about 40 ng/mL.