Suspension Culture of Human Embryonic Stem Cells

• US 20100144033A1
• Filed: 06/20/2006
• Published: 06/10/2010
• Est. Priority Date: 06/22/2005
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Li, Yan, Nadeau-Demers, Isabelle, Mandalam, Ramkumar
• Granted Patent: US 9,074,181 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• 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

This disclosure provides an improved system for culturing human embryonic stem cells. The cells are cultured in suspension so as to maximize the production capacity of the culture environment. The new culture system of this invention allows for bulk proliferation of hES cells in a more cost-effective manner, which facilitates commercial production of important products for use in human therapy.

Claims

1. A culture of human embryonic stem (hES) cells in suspension, wherein the hES cells are substantially undifferentiated.

2. The culture of claim 1, wherein the cells are cultured in a medium containing fibroblast growth factor at a concentration of at least about 40 ng/mL.

3. The culture of claim 2, wherein the medium also contains transforming growth factor beta (TGFβ

), stem cell factor (SCF), or Flt3 ligand (Flt3L).

4. The culture of any preceding claim, wherein the cells are cultured in a medium containing one or more soluble or suspended extracellular matrix components.

5. The culture of claim 4, wherein the extracellular matrix component(s) include human laminin and/or human fibronectin.

6. The culture of any preceding claim, wherein the cells are cultured in suspension with solid microparticle.

7. The culture of claim 7, wherein the microparticle are coated with one or more soluble or suspended extracellular matrix components.

8. A method for culturing hES cells, comprising:

a) suspending the cells in a nutrient medium;

b) maintaining the cells in suspension while culturing;

c) changing the medium periodically;

d) optionally splitting the culture from time to time so as to reduce cell density; and

finally

e) harvesting cells from the culture.

9. The method of claim 8, wherein the cells are cultured in a medium containing fibroblast growth factor at a concentration of at least about 40 ng/mL.

10. The method of claim 9, wherein the medium also contains transforming growth factor beta (TGFβ

), stem cell factor (SCF), or Flt3 ligand (Flt3L).

11. The method of any of claims 8-10, wherein the cells are cultured in a medium containing one or more soluble or suspended extracellular matrix components.

12. The method of claim 11, wherein the extracellular matrix component(s) include human laminin and/or human fibronectin.

13. The method of any of claims 8-12, wherein the cells are cultured in suspension with solid microparticle.

14. The method of claim 13, wherein the microparticle are coated with one or more soluble or suspended extracellular matrix components.

15. The method of any of claims 8-14, wherein the cells are cultured in suspension for at least two months.

16. The method of any of claims 8-15, wherein the cells undergo at least a 3-fold expansion while in suspension culture.

17. The method of any of claims 8-16, further comprising plating the harvested cells back onto a solid surface and continuing to culture the cells so as to maintain a substantially undifferentiated cell population.

18. The method of any of claims 8-17, further comprising differentiating the harvested cells.

19. A system or kit for culturing hES cells in suspension, comprising a nutrient medium that contains at least 40 ng/mL fibroblast growth factor.

20. The system or kit of claim 19, also comprising an extracellular matrix component or microparticle for adding to the medium.