Scalable process for cultivating undifferentiated stem cells in suspension

• US 7,790,456 B2
• Filed: 09/09/2005
• Issued: 09/07/2010
• Est. Priority Date: 09/07/2004
• Status: Active Grant

• Current Assignee: Rheinische Friedrich-Wilhelms Universitã¤T Bonn
• Original Assignee: Rheinische Friedrich-Wilhelms Universitã¤T Bonn
• Inventors: Terstegge, Stefanie, Brüstle, Oliver
• Primary Examiner(s): CHEN, SHIN LIN
• Application Number:
  US11/574,804
• Publication Number:
  US 20070264713A1
• Time in Patent Office: 1,824 Days
• Field of Search: 424/93.21, 435/289.1, 435/325, 435/383, 435/384, 435/395, 435/403
• US Class Current: 435/383
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/70 Polysaccharides
• 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 relates to a process for cultivating undifferentiated stem cells in suspension and in particular to a method for cultivating stem cells on microcarriers in vessels. The method enables stem cells to be cultivated in a scalable process for the first time, without losing their pluripotentiality. Different types of vessels are suitable for cultivation, such as e.g. spinners and bioreactors. The option of precisely setting the cultivation conditions in said vessels aids the process of obtaining a pluripotentiality of the stem cells. the inventive method also permits large yields of stem cells of an unvarying quality to be produced.

Claims

1. A method for proliferating stem cells, comprising:

introducing medium, microcarriers and stem cells into a container, cultivating the stem cells on microcarriers in said container, wherein the cells do not lose their stem cell characteristics after having initiated the culturing, and removing the cells on the microcarriers from the container, wherein after having initiated the culturing the medium in the container is moved and not moved in an alternating manner, wherein the medium is moved between 10 and 100 minutes and is not moved between 10 and 60 minutes in an alternating manner.

2. The method according to claim 1, wherein the stem cells are incubated with the microcarriers before being introduced into the container to allow the stem cells to adhere to the microcarriers.

3. The method according to claim 1, wherein the stem cells are introduced into the container after medium and microcarrier have been introduced into the container.

4. The method according to claim 3, wherein following the introduction of the stem cells into the container a resting phase occurs, wherein during said resting phase the medium in the container is not moved for a maximum period of one day to allow the adhesion of the cells to the microcarriers.

5. The method according to claim 1, wherein the stem cells are embryonic stem cells.

6. The method according to claim 1, wherein the stem cells are adult stem cells.

7. The method according to claim 1, wherein the medium is moved between 15 and 45 minutes and is not moved between 15 and 45 minutes in an alternating manner.

8. The method according to claim 1, wherein said container is a spinner flask.

9. The method according to claim 8, wherein the moving in said container is effected by agitation with an agitator.

10. The method according to claim 9, wherein said agitation is performed at a speed of between 10 and 200 rpm of the agitator.

11. The method according to claim 1, wherein said container is a regulated bioreactor.

12. The method according to claim 1, wherein the medium is conditioned.

13. The method according to claim 12, wherein the medium is conditioned by cultivating the medium with non-stem cells for between 1 and 3 days.

14. The method according to claims 13, wherein the non-stem cells are removed from the medium before the stem cells are introduced.

15. The method according to claims 13, wherein the non-stem cells are fibroblasts.

16. The method according to claim 15, wherein the fibroblasts are genetically modified to express recombinant proteins and secrete said proteins into the culture medium.

17. The method according to claim 12, wherein the medium is conditioned by cultivating the medium with non-stem cells for one day.

18. The method according to claim 1, wherein the microcarriers consist of a carrier material and a coating.

19. The method according to claim 1, wherein the microcarriers consist of a uniform material.

20. The method according to claim 1, wherein the culturing continues for at least two weeks.

21. The method according to claim 1, wherein the culturing is performed at an oxygen partial pressure of between 1% and 25%, preferably at 21%.

22. The method according to claim 1, wherein the culturing is performed without fetal calf serum.

23. The method according to claim 1, wherein the culturing is performed with a serum substitute.

24. The method according to claim 1, wherein the culturing is performed in the presence of basic fibroblast growth factor (bFGF).

25. The method according to claim 1, wherein by the removal of cells from the container only a portion of the medium containing the microcarriers loaded with cells is removed and this portion of the medium is replaced by the introduction of fresh medium and new microcarriers not loaded with cells.

26. The method according to claim 1, wherein at least 90% of the cells maintain stem cell characteristics after the start of culturing.

27. The method according to claim 1, wherein at least 99% of the cells maintain stem cell characteristics after the start of culturing.

28. The method according to claim 1, wherein the number of cells increases by at least 50% per week of cultivation compared to the initial number of cells.

29. The method according to claim 1, wherein the number of cells increases by at least 600% per week of cultivation compared to the initial number of cells.

30. The method according to claim 1, wherein the cultivating of cells is initiated with a concentration of below 10⁶ cells per milliliter.

31. The method according to claim 1, wherein the cultivating of cells is initiated with a concentration of below 10⁵ cells per milliliter.

32. The method according to claim 1, wherein the cultivating of cells is initiated with a concentration of below 10⁴ cells per milliliter.

33. The method according to claim 1, wherein the cells are separated from the microcarriers after removing the cells with the microcarriers from the container.

34. The method according to claim 1, wherein the removing of cells is performed for assessing the maintenance of the stem cell characteristics.