AUTOMATED METHOD AND APPARATUS FOR EMBRYONIC STEM CELL CULTURE

• US 20090029462A1
• Filed: 06/30/2008
• Published: 01/29/2009
• Est. Priority Date: 06/29/2007
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: Fitzgerald, Megan, Beardsley, Nathaniel, Bergendahl, Veit, Daigh, Christine
• Granted Patent: US 8,815,585 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12M 23/50 Means for positioning or orientating the apparatus C12M41/08 takes precedence
• CPC Class Codes: C12M 33/06 for multiple inoculation or multiple collection of samples
• CPC Class Codes: C12M 33/07 Dosage or metering devices therefore
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• 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

Abstract

The invention concerns methods for automated culture of embryonic stem cells (ESCs) such as human ESCs. In some aspects, methods of the invention employ optimized culture media and limited proteolytic treatment of cells to separate cell clusters for expansion. Automated systems for passage and expansion of ESCs are also provided.

Claims

1. A method for automated expansion of pluripotent stem cells under defined media conditions comprising:

(a) obtaining a first population of pluripotent cells in a defined growth media;

(b) separating the pluripotent cells with an automated separation system; and

(c) suspending the separated cells in fresh defined growth media to provide an expanded population of pluripotent cells.

2. The method of claim 1, wherein the pluripotent cells are ES cells or induced pluripotent cells (iPS).

3. The method of claim 1, wherein the cells are human ES cells.

4. The method of claim 1, wherein no or essentially no differentiation occurs in at least 97% of the cultured expanded pluripotent ES cells.

5. The method of claim 1, wherein the growth media comprises TeSR media.

6. The method of claim 1, wherein the first population of pluripotent cells is comprised on a cell culture plate.

7. The method of claim 6, wherein the cell culture plate comprises a gel matrix.

8. The method of claim 6, wherein the first population of pluripotent cells is between about 50% and about 99% confluent at the time of cell separation.

9. The method of claim 8, wherein the first population of pluripotent cells is about 60%, 70%, 80% or 90% confluent at the time of cell separation.

10. The method of claim 6, wherein suspending the separated cells in fresh growth media comprises seeding the cells in one or more new cell culture plate(s).

11. The method of claim 10, wherein the surface area of the new cell culture plate(s) is between from about 5 to about 35 times greater than the surface area of the plate comprising the first population of ES cells.

12. The method of claim 11, wherein the surface area of the new cell culture plate(s) is between from about 10 to about 35 times greater than the surface area of the plate comprising the first population of ES cells.

13. The method of claim 1, wherein the automated separation protocol comprises contacting the first population of pluripotent cells with a proteolytic enzyme.

14. The method of claim 13, wherein the proteolytic enzyme is trypsin.

15. The method of claim 13, wherein the proteolytic enzyme is recombinant trypsin, a trypsin-like proteinase, or TRYPLE.

16. The method of claim 13, wherein the proteolytic enzyme is a recombinant enzyme.

17. The method of claim 1, wherein the fresh growth media comprises an inhibitor of a proteolytic enzyme.

18. The method of claim 13, wherein the fresh growth media comprises an inhibitor of the proteolytic enzyme used for cell separation.

19. The method of claim 18, wherein the proteolytic enzyme inhibitor is a trypsin inhibitor.

20. The method of claim 19, wherein the proteolytic enzyme inhibitor is a soybean trypsin inhibitor.

21. The method of claim 20, wherein the fresh growth media comprises about 0.5 mg/ml of soybean trypsin inhibitor.

22. The method of claim 1, wherein the separation system is automated by a liquid handler robot.

23. The method of claim 13, wherein the automated separation system comprises:

(i) removing the media from the first pluripotent cell population;

(ii) contacting the pluripotent cells with a proteolytic enzyme; and

(iii) incubating the cells with a proteolytic enzyme to separate cell clusters.

24. The method of claim 23, wherein a defined media comprising a proteolytic enzyme inhibitor and a Rho-associated kinase (ROCK) inhibitor is added to the solution after step (iii).

25. The method of claim 23, wherein the ES cells are incubated with the proteolytic enzyme for from about 2 to about 10 minutes.

26. The method of claim 23, wherein the ES cells are incubated with the proteolytic enzyme at between about 25°

C. and about 40°

C.

27. The method of claim 26, wherein the ES cells are incubated with the proteolytic enzyme at about 37°

C.

28. The method of claim 23, wherein the automated separation system further comprises:

(iv) subjecting the incubated cells to mechanical agitation to further separate cell clusters.

29. The method of claim 28, wherein the mechanical agitation comprises subjecting the ES cells to shear forces or aspiration.

30. The method of claim 17, further comprising replacing the growth media comprising the proteolytic enzyme inhibitor with a growth media that is essentially free of said inhibitor.

31. The method of claim 1, wherein the population of pluripotent cells is free or essentially free from non-pluripotent cells.

32. The method of claim 31, wherein the population of pluripotent cells is human ES cells, and wherein population of human ES cells is free or essentially free from non-human cells.

33. The method of claim 1, further defined as a method for automated serial expansion of embryonic stem (ES) cells comprising:

(a) obtaining a first population of ES cells in a growth media;

(b) separating the ES cells with an automated separation system;

(c) suspending the separated cells in fresh growth media to provide an expanded population of ES cells.

(d) incubating the expanded ES cell population under conditions supporting cell growth; and

(e) repeating steps b-d one or ore times to provide a serially expanded population of ES cells.

34. The method of claim 33, wherein (d) incubating the expanded ES cell population under conditions supporting cell growth comprises incubating the cells in a media perfusion culture.

35. The method of claim 1, wherein the growth media comprises a effective amount of a Rho-associated kinase (ROCK) inhibitor.

36. The method of claim 35, wherein the Rho-associated kinase (ROCK) inhibitor is HA-100.

37. The method of claim 36, wherein the HA-100 is present in a concentration of about 10 μ

M.

38. The method of claim 35, wherein the Rho-associated kinase (ROCK) inhibitor is H-1135.

39. The method of claim 38, wherein the H-1135 is present in a concentration of about 1-3 μ

M.

40. An apparatus for automated maintenance and expansion of pluripotent cells comprising:

a) an incubator;

b) a liquid handler unit;

c) one or more reservoirs, said reservoirs comprising i) a viable population of pluripotent cells, ii) a defined media, the defined media being one in which pluripotent cells can be cultured and maintained in an undifferentiated state, and iii) one or more of a protease, protease inhibitor and a Rho-associated kinase (ROCK) inhibitor; and

d) a controller in communication with the liquid handler unit, wherein the controller is configured to direct the liquid handler unit to effect the automated expansion and maintenance of the pluripotent cells.

41. The apparatus of claim 40, wherein the controller comprises a computer-readable medium including an operating program.

42. The apparatus of claim 40, wherein the controller directs at least one of:

the liquid handler unit, the one or more reservoirs, and the incubator to i) remove media from a pluripotent cell population;

ii) contact pluripotent cells with a proteolytic enzyme; and

iii) incubate pluripotent cells with a proteolytic enzyme to separate cell clusters.

43. The apparatus of claim 40, wherein the apparatus further comprises a mechanical agitator and aspirator, and the controller further directs at least one of:

the liquid handler unit, the one or more reservoirs, and the incubator to subject the pluripotent cells to mechanical agitation or aspiration to further separate cell clusters.

44. The apparatus of claim 40, wherein the controller further directs at least one of:

the liquid handler unit, the one or more reservoirs, and the incubator to contact incubated pluripotent cells with a protease inhibitor.

45. The apparatus of claim 44, wherein the controller further directs at least one of:

the liquid handler unit, the one or more reservoirs, and the incubator to contact incubated pluripotent cells with a Rho-associated kinase (ROCK) inhibitor.

46. The apparatus of claim 40, wherein the liquid handler unit comprises at least a first reservoir and a second reservoir, wherein the first reservoir comprises a TeSR media, and wherein the second reservoir comprises a TeSR media, a Rho-associated kinase (ROCK) inhibitor, and a protease inhibitor.

47. The apparatus of claim 40, wherein the Rho-associated kinase (ROCK) inhibitor is H-1152 or H-100.

48. The apparatus of claim 40, wherein the pluripotent cells are ES cells or induced pluripotent cells (iPS).

49. The apparatus of claim 40, wherein the liquid handler comprises a gripper tool and a liquid handling tool.

50. The apparatus of claim 40, wherein the pluripotent cells are human ES cells.

51. The apparatus of claim 40, further comprising a robotic device configured to facilitate fluid communication between the liquid handler unit and the incubator.

52. The apparatus of claim 40, further comprising a second or more reservoirs in communication with the liquid handler unit.

53. The apparatus of claim 52, wherein the second reservoir comprises cell culture plates, cell growth media, or a proteolytic enzyme solution.

54. The apparatus of claim 40, wherein the liquid handler is in communication with at least a first reservoir and a second reservoir, wherein the first reservoir comprises a TeSR media, and wherein the second reservoir comprises a TeSR media which further comprises a Rho-associated kinase (ROCK) inhibitor and a protease inhibitor.

55. The apparatus of claim 40, wherein the controller is comprised in a computer.

56. The apparatus of claim 40, wherein said incubator comprises a portion of said pluripotent cells, and wherein the apparatus is configured to provide for expansion of the pluripotent cells with no or essentially no further differentiation of the cells in at least 97% of the expanded pluripotent cells.

57. The apparatus of claim 40, wherein the incubator comprises a portion of said pluripotent cells, and wherein the pluripotent cells are human ES cells.