Method of dynamically culturing embryonic stem cells

• US 7,947,499 B2
• Filed: 11/30/2003
• Issued: 05/24/2011
• Est. Priority Date: 11/29/2002
• Status: Expired due to Fees

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: Gerecht-Nir, Sharon, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Ford; Allison M
• Application Number:
  US10/536,439
• Publication Number:
  US 20060148078A1
• Time in Patent Office: 2,732 Days
• Field of Search: None
• US Class Current: 435/394
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

The present invention is of a method of dynamically generating human embryoid bodies which can be used for generating lineage specific cells and cell lines. Specifically, the present invention can be used to generate ESC-differentiated cells for cell-replacement therapy.

Claims

1. A method of dynamically generating embryoid bodies comprising culturing embryonic stem cells in slow turning lateral vessel bioreactor which subjects cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in said fluid to thereby obtain dynamically generated embryoid bodies.

2. The method of claim 1, wherein said bioreactor exerts a microgravitational force on said embryonic stem cells selected from the range of 0.05-0.9 G on said cells cultured therein.

3. The method of claim 2, wherein said microgravitational force is 0.1 G.

4. The method of claim 1, wherein said bioreactor maintains said embryonic stem cells in a state of free fall.

5. The method of claim 1, wherein said bioreactor holds a fluid volume selected from the range of 1-500 ml.

6. The method of claim 1, wherein a diameter of said bioreactor is selected from range of 2.54-15.24 centimeters.

7. The method of claim 6, wherein said culturing of said embryonic stem cells is effected in said bioreactor under a rotational speed selected from a range of 15-20 rpm.

8. The method of claim 1, wherein said bioreactor enables oxygenation of said fluid via controlled diffusion of dissolved gasses.

9. The method of claim 1, wherein said embryonic stem cells are cultured at an initial concentration of 0.5-0.7×

10⁶ cells per milliliter medium.

10. The method of claim 1, wherein said culturing of said embryonic stem cells is effected in a culture medium selected as being suitable for embryoid bodies formation.

11. The method of claim 10, wherein said culture medium includes 80% DMEM, 20% serum, 0.5% Penicillin-Streptomycin, 1 mM L-glutamine, 0.1 mM β

-mercaptoethanol and 1% non-essential amino acid stock.

12. The method of claim 1, wherein said culturing is effected over a culturing period of 1-35 days.

13. The method of claim 1, wherein said culturing is effected for 30 days.