Suspension culture of human embryonic stem cells

• US 9,074,181 B2
• Filed: 06/20/2006
• Issued: 07/07/2015
• 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:
  US11/917,993
• Publication Number:
  US 20100144033A1
• Time in Patent Office: 3,304 Days
• Field of Search: 435/366, 435/383, 435/394
• US Class Current: 1/1
• 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 of culturing undifferentiated hES cells, comprising:

a) suspending human embryonic stem (hES) cells in a nutrient medium;

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

c) changing the nutrient medium periodically; and

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

wherein, the nutrient medium comprises FGF2 and does not comprise added extracellular matrix; and

wherein, the hES cells are not grown on a solid substrate.

2. The method of claim 1, wherein the concentration of FGF2 in the nutrient medium is at least 40 ng/mL.

3. The method of claim 1, wherein the concentration of FGF2 in the nutrient medium is about 40 ng/mL.

4. The method of claim 1, wherein the nutrient medium further comprises at least one of transforming growth factor beta (TGFB), stem cell factor (SCF), and Flt3 ligand (FIt3L).

5. The method of claim 1, wherein the hES cells are cultured in suspension in nutrient medium comprising FGF2 at a concentration of at least 40 ng/mL for at least two months.

6. The method of claim 1, further comprising harvesting the cells and plating the harvested cells back onto a solid surface comprising a matrix and continuing to culture the cells in a substantially undifferentiated state.

7. The method of claim 1, further comprising harvesting the undifferentiated cells from the culture and differentiating the harvested cells.