IN VITRO EXPANSION OF POSTPARTUM-DERIVED CELLS USING MICROCARRIERS

• US 20080166328A1
• Filed: 11/13/2007
• Published: 07/10/2008
• Est. Priority Date: 11/13/2006
• Status: Active Grant

• Current Assignee: Depuy Synthes Products Incorporated (Johnson & Johnson)
• Original Assignee: Ethicon Incorporated (Johnson & Johnson)
• Inventors: Harmon, Alexander M., Gosiewska, Anna, Kihm, Anthony J., Hong, L.S. Klaudyne
• Granted Patent: US 9,102,915 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: C12M 25/16 Particles; Beads; Granular material; Encapsulation chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

Compositions and methods for the growth and expansion of mammalian cells in culture are provided. In particular, methods for the growth and expansion of postpartum-derived cells in vitro are provided using surfaces such as microcarrier beads.

Claims

1. A method of culturing anchorage-dependent postpartum cells comprising,

providing at least one anchorage-dependent postpartum cell;

providing a cell growth medium for growing the postpartum cell;

providing at least one carrier particle for attachment of the anchorage-dependent postpartum cell; and

contacting the anchorage-dependent cell with the carrier particle in the presence of the growth medium under conditions permitting attachment and growth of the cell,

thereby culturing the anchorage-dependent postpartum cell.

2. The method of claim 1, wherein the cells are umbilical derived cells.

3. The method of claim 1, wherein the cells are placental derived cells.

4. The method of claim 1 wherein the carrier particle contains a bioactive agent.

5. The method of claim 1 further comprising a second cell type co-cultured with said anchorage-dependent postpartum cells.

6. The method of claim 1 wherein the carrier particle is a microcarrier.

7. The method of claim 6 wherein the microcarrier is comprised of a material selected from the group consisting of collagen, dextran, cellulose, glass, ceramic, metal, polystyrene, poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, and sodium hyaluronate.

8. The method of claim 6 wherein the microcarrier is protein free.

9. The method of claim 6 wherein the microcarrier is coated with poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, sodium hyaluronate, fibronectin, laminin, elastin, lysine, n-isopropyl acrylamide, vitronectin, or collagen.

10. The method of claim 6 wherein the microcarrier comprises a textured or plasma-coated surface.

11. The method of claim 6 wherein the microcarrier possesses a microcurrent or is paramagnetic.

12. The method of claim 1 wherein the carrier particle is a porous microcarrier.

13. The method of claim 12 wherein the porous microcarrier is comprised of a material selected from the group consisting of collagen, dextran, cellulose, glass, ceramic, metal, polystyrene, poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, and sodium hyaluronate.

14. The method of claim 12 wherein the porous microcarrier is protein free.

15. The method of claim 12 wherein the porous microcarrier is coated with poly(monostearoylglyceride co-succinic acid), poly-D,L-lactide-co-glycolide, sodium hyaluronate, fibronectin, laminin, elastin, lysine, n-isopropyl acrylamide, vitronectin, or collagen.

16. The method of claim 12 wherein the porous microcarrier comprises a textured or plasma-coated surface.

17. The method of claim 1 wherein the cells are phenotypically the same as cells grown in static cultures for one or more of the markers CD10, CD13, CD31, CD34, CD44, CD45, CD73, CD90, CD117, CD141, PDGFr-α

, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.

18. The method of claim 17 wherein the cells are phenotypically the same as cells grown in static cultures for each of the markers CD10, CD13, CD31, CD34, CD44, CD45, CD73, CD90, CD117, CD141, PDGFr-α

, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.

19. The method of claim 18 wherein the phenotype of the cells comprises CD10+, CD13+, CD31−

, CD34−

, CD44+, CD45−

, CD73+, CD90+, CD117−

, CD141−

, PDGFr-α

+, HLA-A+, HLA-B+, HLA-C+, HLA-DR−

, HLA-DP−

, and HLA-DQ−

.

20. The method of claim 1 which results in at least about five population doublings over about twenty days.

21. The method of claim 1 wherein the doubling time for the population is less than about 100 hours.

22. The method of claim 21 wherein the doubling time for the population is less than about 70 hours.

23. The method of claim 1 wherein conditions which permit attachment and growth of the anchorage dependent postpartum cell comprise a temperature of about 37°

C.

24. The method of claim 23 wherein conditions further comprise a spinner flask or bioreactor.

25. The method of claim 1 wherein the cell growth medium comprises a serum concentration from about 7% to about 15%.

26. A composition comprising postpartum-derived cells grown in accordance with the method of claim 1.

27. The composition of claim 26 wherein the phenotype of the cells comprises CD10+, CD13+, CD31−

, CD34−

, CD44+, CD45−

, CD73+, CD90+, CD117−

, CD141−

, PDGFr-α

+, HLA-A+, HLA-B+, HLA-C+, HLA-DR−

, HLA-DP−

, and HLA-DQ−

.

28. A bioreactor comprising the cells of claim 26.

29. A composition for cell therapy comprising the cells of claim 26.

30. A composition comprising human umbilical tissue-derived cells grown in accordance with the method of claim 6.