Suspension culture of human embryonic stem cells

• US 10,676,714 B2
• Filed: 02/12/2018
• Issued: 06/09/2020
• 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: Mandalam, Ramkumar, Li, Yan, Nadeau-Demers, Isabelle
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/894,842
• Publication Number:
  US 20190017014A1
• Time in Patent Office: 848 Days
• 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 method for culturing undifferentiated human pluripotent stem (hPS) cells in suspension, the method comprising:

a) suspending undifferentiated hPS cells in a cell culture medium comprising basic fibroblast growth factor (bFGF);

b) maintaining the hPS cells substantially undifferentiated while culturing in suspension;

c) changing the cell culture medium periodically; and

d) optionally splitting the culture from time to time, so as to reduce the density of the hPS cells in culture;

wherein the hPS cells are not grown attached to a solid substrate.

2. The method of claim 1, wherein the concentration of bFGF is at least 40 ng/mL.

3. The method of claim 1, wherein the concentration of bFGF is about 40 ng/mL.

4. The method of claim 1, wherein the concentration of bFGF is about 80 ng/mL.

5. The method of claim 1, wherein the cell culture medium further comprises at least one factor selected from transforming growth factor beta (TGF β

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

6. The method of claim 1, wherein the hPS cells are cultured in suspension for a period of at least two months.

7. The method of claim 1, further comprising plating the undifferentiated hPS cells onto a solid substrate and continuing to culture the hPS cells in a substantially undifferentiated state.

8. The method of claim 1, further comprising harvesting the undifferentiated hPS cells from the culture and differentiating them.

9. The method of claim 7, further comprising differentiating the plated hPS cells.

10. The method of claim 1, wherein the undifferentiated hPS cells are cultured in suspension inside a cell culture vessel that is being agitated, rocked, or spun.

11. The method of claim 1, wherein the undifferentiated hPS cells are cultured in suspension inside a cell culture vessel selected from a spinner flask, shaker flask, a fermentor and a bioreactor.