Culture of pluripotent and multipotent cells on microcarriers

• US 9,150,829 B2
• Filed: 03/12/2010
• Issued: 10/06/2015
• Est. Priority Date: 03/20/2009
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Oh, Steve, Chen, Allen, Choo, Andre, Reuveny, Shaul
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/257,674
• Publication Number:
  US 20120009645A1
• Time in Patent Office: 2,034 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

A method is disclosed for culturing pluripotent or multipotent cells in vitro, the method comprising attaching pluripotent or multipotent cells to a plurality of microcarriers to form microcarrier-cell complexes, and culturing the microcarrier-cell complexes in suspension culture in the presence of a ROCK inhibitor.

Claims

1. A method of culturing human pluripotent cells in vitro, the method comprising:

(i) attaching human pluripotent cells to a plurality of uncoated microcarriers to form microcarrier-cell complexes, and

(ii) culturing the microcarrier-cell complexes in suspension culture in the presence of a ROCK inhibitor;

(iii) passaging the cultured cells from (ii), wherein cells after passaging are pluripotent, and

(iv) repeating steps (i)-(iii) through at least 7 passages;

wherein human pluripotent cells are expanded between each passage; and

whereby stable, long-term culture and expansion of human pluripotent cells is achieved in suspension in vitro.

2. The method of claim 1 wherein the ROCK inhibitor is chosen from:

Y-27632, HA-1077 (Fasudil), HA-1100 (HydroxyFasudil), H-1152, 3-(4-Pyridyl)-1H-indole, N-(4-Pyridyl)-N′

-(2,4,6-trichlorophenyl) urea, Aurothioglucose, LY294002 or a salt, base, ester or prodrug thereof.

3. The method of claim 1 wherein the cells are stem cells, embryonic stem cells or induced pluripotent stem cells.

4. The method of claim 1 wherein all or a substantial portion of cells in the culture after step (iv) retain a normal karyotype, preferably wherein said substantial portion is 70% or more.

5. The method of claim 1 wherein in step (iv), steps (i)-(iii) are repeated through at least 9 passages.

6. The method of claim 1 wherein the microcarriers comprise or consist of one or more of cellulose, dextran, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, or silicone.

7. The method of claim 1 wherein the microcarriers are macroporous or microporous carboseed microcarriers.

8. The method of claim 1 wherein the microcarriers are coupled with protamine or polylysine.

9. The method of claim 1 wherein the microcarriers are positively charged, preferably having a positive surface charge.

10. The method of claim 1 wherein the microcarriers are hydrophilic.

11. The method of claim 1 wherein the microcarriers are rod-shaped or have a substantially spherical shape.

12. The method of claim 1 wherein in step (ii) the cells are cultured for a period of time sufficient to expand the number of cells in the culture.

13. The method of claim 1 wherein after step (iv) at least 60% of the cells in the culture are pluripotent or at least 60% of the cells in the culture express one, two, three or all of Oct4, SSEA4, TRA-1-60 and Mab84.

14. The method of claim 1 wherein the method comprises culturing the cells in serum free media, or stem cell conditioned media, or feeder cell free conditions, optionally where feeder cells are also attached to the microcarriers.

15. The method of claim 1 wherein the culture further comprises feeder cells attached to microcarriers which are different from the microcarriers to which the pluripotent cells are attached.

17. The method of claim 1 wherein in step (iv), steps (i)-(iii) are repeated through at least 15 passages.

18. The method of claim 1 wherein in step (iv), steps (i)-(iii) are repeated through at least 30 passages.

16. A method of propagating human pluripotent cells comprising the steps of:

(a) providing an uncoated microcarrier;

(b) allowing a human pluripotent cell to attach to the uncoated microcarrier; and

(c) aggregating microcarriers with human pluripotent cells attached thereon to thereby propagate the human pluripotent cells,

wherein in one or more, or all, of steps (a), (b) or (c) the microcarrier and/or cells are contacted with a ROCK Inhibitor.