USE OF ADIPOSE TISSUE-DERIVED STROMAL CELLS FOR CHONDROCYTE DIFFERENTIATION AND CARTILAGE REPAIR

• US 20130017604A1
• Filed: 09/30/2011
• Published: 01/17/2013
• Est. Priority Date: 08/19/1999
• Status: Active Grant

• Current Assignee: Cognate BioServices, Inc.
• Original Assignee: Artecel Sciences Inc.
• Inventors: Halvorsen, Yuan-Di C., Wilkison, William O., Gimble, Jeffrey Martin
• Granted Patent: US 8,911,994 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• 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: A61L 2430/06 for cartilage reconstruction, e.g. meniscus
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3852 Cartilage, e.g. meniscus
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/1305 Adipocytes
• CPC Class Codes: C12N 2506/1384 from adipose-derived stem cells [ADSC], from adipose stromal stem cells
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0653 Adipocytes; Adipose tissue
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage are disclosed. The invention further provides a variety of chondroinductive agents which can be used singly or in combination with other nutrient components to induce chondrogenesis in adipose-derived stromal cells either in cultivating monolayers or in a biocompatible lattice or matrix in a three-dimensional configuration. Use of the differentiated chondrocytes for the therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo is disclosed.

Claims

1. A medium for differentiating adipose tissue derived stromal cells into chondrocyte cells, comprising:

a chemically defined culture medium having or supplemented with (i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype (ii) an antibiotic (iii) a nutrient supplement such as 1-20% fetal bovine serum or 1-20% horse serum or any other biological or synthetic equivalent combination of proteins (iv) ascorbate or related vitamin C analogue and (v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor.

2. The medium of claim 1, wherein the chondroinductive agent or stromal cell mitogen is selected individually or in combination from the group consisting of:

a glucocorticoid;

a member of the transforming growth factor-beta superfamily;

a collagenous extracellular matrix molecule; and

a vitamin A analog.

3. The medium of claim 1, wherein said antibiotic is penicillin.

4. The medium of claim 1, wherein said antibiotic is streptomycin.

5. The medium of claim 3, wherein the concentration of penicillin is about 10 to about 200 units per ml.

6. The medium of claim 4, wherein the concentration of streptomycin is about 10 to about 200 μ

g per ml.

7. The medium of claim 2, wherein the glucocorticoid is hydrocortisone.

8. The medium of claim 2, wherein said glucocorticoid is dexamethasone.

9. The medium of claim 8, wherein the concentration of dexamethasone is about 1 to about 100 nM.

10. The medium of claim 7, wherein the concentration of hydrocortisone is about 1 to about 100 nM.

11. The medium of claim 2, wherein said transforming growth factor-beta is selected from the group consisting of:

bone morphogenic protein-2, bone morphogenic protein-4, TGF-β

1, TGF-β

2, TGF-β

3, IGF, PDGF, EGF, aFBF, bFBF, HGF, KGF, inhibin A, and chondrogenic stimulating factor.

12. The medium of claim 11, wherein the concentration of transforming growth factor-beta is about 1 to about 100 ng per ml.

13. The method according to claim 11, further comprising TGFβ

-1 at concentrations from about 1 ng/ml to about 10 ng/ml.

14. The medium of claim 2, wherein the collagenous extracellular matrix molecule is collagen I.

15. The medium of claim 2, wherein the vitamin A analog is retinoic acid.

16. The medium of claim 15, wherein the concentration of retinoic acid is about 0.1 ng per ml to about 1 μ

g per ml.

17. A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising:

a) pelleting said stromal cells by centrifuging between. 50,000 to 5 million cells at 500×

g for 2 to 20 minutes in sterile tubes containing a medium such as Dulbecco'"'"'s Modified Eagle'"'"'s Medium (DMEM) or alpha modified Minimal Essential Medium (α

MEM) or Roswell Park Memorial Institute media 1640 (RPMI Media

     1640);

b) plating isolated stromal cells at a density of 500 to 20,000 cells/cm² in a differentiating medium;

c) supplementing said medium with;

(i) a chondroinductive agent capable of activating any cellular signal transduction pathway leading to the mature chondrocyte phenotype;

(ii) an antibiotic;

(iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;

(iv) ascorbate or related vitamin C analog;

(v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and

d) incubating said cells at about 31°

C. to 37°

C. for about 3-4 weeks in with 5% CO₂ and between 1% and 20% oxygen.

18. A method for differentiating adipose tissue derived stromal cells into chondrocytic cells, comprising:

a) suspending stromal cells at a concentration of 0.5 to 10 million cells per ml in calcium alginate or any other biocompatible lattice or matrix of supporting chondrogenesis in a three-dimensional configuration;

b) transferring cells to 35 mm culture dishes and plating cells at a density of 500 to 20,000 cells/cm² in a differentiating medium comprising a chemically defined culture medium having or supplemented with;

(i) a chondroinductive agent capable of activating any cellular transduction pathway leading to the mature chondrocyte phenotype;

(ii) an antibiotic;

(iii) a nutrient supplemented with 1 to 20% fetal bovine serum or 1 to 20% horse serum or any other biological or synthetic equivalent combination of proteins;

(iv) ascorbate or related vitamin C analog;

(v) a glucocorticoid or other chemical agent capable of activating the cellular glucocorticoid receptor; and

c) incubating said cells at about 31 to 37°

C. for about 3-4 weeks in an incubator with 5% CO₂ and between 1% and 20% oxygen.