CELL COMPOSITION AND METHODS OF MAKING THE SAME

• US 20100047213A1
• Filed: 08/20/2009
• Published: 02/25/2010
• Est. Priority Date: 08/20/2008
• Status: Active Grant

• Current Assignee: Celularity, Inc.
• Original Assignee: Celularity, Inc.
• Inventors: Murphy, Brian, Chen, Hong J., Zeitlin, Andy, Shorr, Ryan, Brieva, Thomas, He, Shuyang, Russotti, Gregory, Pal, Ajai
• Granted Patent: US 10,104,880 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid

Abstract

Provided herein are improved methods for the formulation of compositions comprising placental stem cells, and improved compositions and cell formulations produced thereby.

Claims

1. A method of making a composition comprising cells, comprising:

(a) contacting said cells with a solution comprising dextran and human serum albumin (HSA) to form a cell-containing solution;

(b) filtering the cell-containing solution;

(c) if said cell-containing solution comprises greater than about 10±

3×

10⁶ cells per milliliter, diluting said cells to no more than about 10±

3×

10⁶ cells per milliliter with a first dilution solution comprising dextran; and

(d) optionally diluting the cell-containing solution with a second dilution solution comprising dextran but not comprising HSA, thereby making a composition comprising cells.

2. The method of claim 1, wherein said cells are cryopreserved after step (c) and before step (d).

3. The method of claim 1, wherein said dextran in said first dilution solution or said second dilution solution is dextran 40.

4. The method of claim 1, wherein said dextran in said first dilution solution and said second dilution solution is dextran 40.

5. The method of claim 1, wherein said dextran 40 in said first dilution solution is 5.5% dextran 40.

6. The method of claim 1, wherein said HSA in said solution comprising HSA is 10% HSA.

7. The method of claim 1, wherein said first dilution solution comprises HSA.

8. The method of claim 7, wherein said HSA in said first dilution solution is 10% HSA.

9. The method of claim 1, wherein said first dilution solution further comprises a cryoprotectant.

10. The method of claim 9, wherein said cryoprotectant is DMSO.

11. The method of claim 1, wherein said dextran 40 in said second dilution solution is 10% dextran 40.

12. The method of claim 1, wherein said solution in step (a) comprises a cryoprotectant.

13. The method of claim 1, wherein said composition comprising cells comprises about 7.5% to about 9% dextran.

14. The method of claim 1, wherein said composition comprising cells comprises about 1.0±

0.3×

10⁶ cells per milliliter to about 5.0±

1.5×

10⁶ cells per milliliter.

15. The method of claim 1, wherein said cells are placental cells.

16. The method of claim 15, wherein said stem cells are isolated human adherent placental cells.

20. The method of claim 1, wherein said filter is a 70 μ

M filter.

21. The method of claim 1, wherein said filter is a 100 μ

M filter.

22. The method of claim 1, wherein said cells are isolated human adherent placental cells.

23. The method of claim 22 wherein said isolated human adherent placental cells are CD10⁺, CD34⁻

and CD105⁺.

24. The method of claim 23, wherein said CD10⁺, CD34⁻

and CD105⁺ cells are CD200⁺.

25. The method of claim 24, wherein said CD10⁺, CD34⁻

, CD105⁺ and CD200⁺ cells are either CD45⁻

or CD90⁺.

26. The method of claim 24, wherein said CD10⁺, CD34⁻

, CD105⁺ and CD200⁺ cells are CD45⁻

and CD90⁺.

27. A composition made by the method of claim 1.

41. The method of claim 1, further comprising concentrating the composition comprising cells to about 5×

10⁶ cells per milliliter to 1×

10⁸ cells per milliliter.

42. The method of claim 41, further comprising subcutaneously administering the composition comprising cells to a subject.

17. A method of making a composition comprising cells, comprising:

(a) suspending a plurality of isolated human adherent placental cells in a 5.5% dextran 40, 10% HSA solution to form a cell-containing solution;

(b) filtering the cell-containing solution through a 70 μ

M filter;

(c) diluting the cell-containing solution in 5.5% dextran 40, 10% HSA, and 5% DMSO to no more than about 10±

3×

10⁶ cells/mL;

(d) cryopreserving the cells;

(e) thawing the cells; and

(f) optionally diluting the cell-containing solution 1;

1 to 1;

11 with 10% dextran 40 to produce said composition comprising cells.

18. A method of making a composition comprising cells, comprising:

(a) centrifuging a plurality of isolated human adherent placental cells to collect the cells;

(b) resuspending the cells in 5.5% dextran 40;

(c) centrifuging the cells to collect the cells;

(d) resuspending the cells in a 5.5% dextran 40 solution that comprises 10% HSA to form a cell-containing solution;

(e) filtering the cell-containing solution through a 70 μ

M to 100 μ

M filter;

(f) diluting the cell-containing solution in 5.5% dextran 40, 10% HSA, and 5% DMSO to no more than about 10±

3×

10⁶ cells/mL;

(g) cryopreserving the cells;

(h) thawing the cells; and

(i) optionally diluting the cell-containing solution 1;

1 to 1;

11 with 10% dextran 40 to produce said composition comprising cells.

19. A method of making a composition comprising isolated human adherent placental cells, comprising:

(a) providing a plurality of isolated human adherent placental cells in a solution comprising 5.5% dextran 40 and 10% human serum albumin (HSA) to form a solution comprising isolated human adherent placental cells;

(b) filtering said solution comprising isolated human adherent placental cells with filter that removes visible cell clumps to produce filtered isolated human adherent placental cells;

(c) diluting said filtered isolated human adherent placental cells with an amount of a solution comprising 5.5% dextran 40, 10% HSA and 5% dimethylsulfoxide (DMSO) sufficient to bring said filtered isolated human adherent placental cells to about 10±

3×

10⁶ cells per milliliter; and

(d) diluting said isolated human adherent placental cells with 10% dextran 40 at a ratio of about 1;

1 to about 1;

11 isolated human adherent placental cells;

dextran 40 to produce said composition.

28. A method of making a composition comprising isolated human adherent placental cells, comprising contacting said isolated human adherent placental cells with a solution comprising 5.5% dextran and 10% HSA;

filtering the isolated human adherent placental cells; and

diluting said isolated human adherent placental cells to about 10±

3.x10⁶ cells per milliliter with a solution comprising 10% dextran 40 (w/v).

29. A composition comprising a plurality of isolated human adherent placental cells in a solution comprising 10% dextran 40, wherein said composition comprises between about 1.0±

0.3×

10⁶ cells per milliliter to about 5.0±

1.5×

10⁶ cells per milliliter, and wherein said composition comprises no macro cell clumps.

30. The composition of claim 29, wherein said placental stem cells are thawed cryopreserved cells.

31. The composition of claim 29, wherein said composition is contained within a bag.

32. The composition of claim 29, wherein said composition comprises no macro cell clumps.

33. The composition of claim 29, wherein said composition comprises fewer than about 200 micro cell clumps per 10⁶ cells.

34. The composition of claim 29, wherein said composition comprises fewer than about 150 micro cell clumps per 10⁶ cells.

35. The composition of claim 29, wherein said composition comprises fewer than about 100 micro cell clumps per 10⁶ cells.

36. The composition of claim 29, wherein said isolated human adherent placental cells are CD10⁺, CD34⁻

and CD105⁺.

37. The composition of claim 36, wherein said CD10⁺, CD34⁻

and CD105⁺ cells are CD200⁺.

38. The composition of claim 37, wherein said CD10⁺, CD34⁻

, CD105⁺ and CD200⁺ cells are either CD45⁻

or CD90⁺.

39. The composition of claim 37, wherein said CD10⁺, CD34⁻

, CD105⁺ and CD200⁺ cells are CD45⁻

and CD90⁺.

40. The composition of claim 29, wherein said composition is a pharmaceutical composition.