Serum free cultivation of primate embryonic stem cells

• US 7,005,252 B1
• Filed: 03/09/2000
• Issued: 02/28/2006
• Est. Priority Date: 03/09/2000
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A
• Primary Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/522,030
• Time in Patent Office: 2,182 Days
• Field of Search: 435/325, 435/326, 435/1.1, 435/405, 435/70.1, 435/283.1, 424/93.1, 800/8
• US Class Current: 435/1.1
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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

Disclosed herein are methods for culturing primate embryonic stem cells. These cells are cultured on a prolonged and stable basis in the presence of exogenously supplied fibroblast growth factor and in the absence of animal serum. Preferably there is also a fibroblast feeder layer. Also disclosed is a culture media containing fibroblast feeder layer and the fibroblast growth factor.

Claims

1. A method of culturing primate embryonic stem cells in defined media without serum, the method comprising:

culturing the primate embryonic stern cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, and at least one insulin or insulin substitute, the culture medium being essentially free of mammalian fetal serum and containing exogenously supplied human fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer, so that the stern cells proliferate in culture and remain undifferentiated in the absence of serum in the medium.

2. The method of claim 1, wherein the culture is essentially free of any animal serum.

3. The method of claim 2, wherein the culture also comprises a fibroblast feeder layer.

4. The method of claim 2, wherein the fibroblast growth factor is basic fibroblast growth factor.

5. The method of claim 4, wherein the fibroblast growth factor is human basic fibroblast growth factor which has been produced from a recombinant gene.

6. The method of claim 5, wherein the human basic fibroblast growth factor is present in the culture in a concentration of at least 0.1 ng/ml for at least a portion of the method.

7. The method of claim 2, wherein the primate embryonic stem cells are human embryonic stem cells.

8. A method of culturing primate embryonic stem cells in defined media without serum, the method comprising:

culturing the primate embryonic stem cells in a culture medium containing albumin, amino acids, vitamins minerals, at least one transferrin or transferrin substitute, and at least one in or insulin substitute, the culture medium being essentially free of mammalian fetal serum and containing exogenously supplied mammalian fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer, said culturing step being conducted for over one month with the embryonic stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.

9. A method of culturing primate embryonic stem cells in defined media without serum, the method comprising:

culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, and at least one insulin or insulin substitute, the culture medium being essentially free of mammalian fetal serum and in the presence of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is exogenously supplied to the culture from a source other than just a fibroblast feeder layer, so that the stem cells proliferate in culture and remain undifferentiated in the absence of serum in the medium.

10. The method of claim 9, wherein the culture is essentially free of any animal serum.

11. The method of claim 10, wherein the culture also comprises a fibroblast feeder layer.

12. The method of claim 10, wherein the primate embryonic stem cells are human embryonic stem cells.

13. A method of culturing primate embryonic stem cells in defined media without serum, the method comprising:

culturing the primate embryonic stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, and at least one insulin or insulin substitute, the culture medium being essentially free of mammalian fetal serum and in the presence of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is exogenously supplied to the culture from a source other than just a fibroblast feeder layer, said culturing step being conducted for over one month with the embryonic stem cells proliferating in culture while maintaining the potential of the stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the stem cells.

14. In a method of culturing primate embryonic stem cells without serum, the improvement comprising:

culturing the primate embryonic stem cells in a culture free of added mammalian fetal serum but including albumin, vitamins, minerals, insulin, and transferrin, and in the presence of fibroblast growth factor that is exogenously supplied to the culture from a source other than just a fibroblast feeder layer, so that the stem cells proliferate in culture and remain undifferentiated in the absence of serum in the medium.