IP Library › Granted Patent US 12,415,985
Granted Patent B2
US 12,415,985 · App. 17/097,023 · Granted Sep 16, 2025

Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state

Inventors: Michal Amit (Yuvalim, IL); Joseph Itskovitz-Eldor (Haifa, IL)
Assignee: Technion Research & Development Foundation Limited
C12N5/0602C12N5/0606C12N5/0696C12N2500/36C12N2500/90C12N2500/92C12N2500/98C12N2500/99C12N2501/115C12N2501/15C12N2501/23C12N2506/45
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Quick Facts
Patent No.
US 12,415,985
App. No.
17/097,023
Granted
Sep 16, 2025
Kind
B2
Abstract

Provided are novel serum-free culture media which comprise basic fibroblast growth factor (bFGF), transforming growth factor beta-3 and ascorbic acid at a concentration of at least about 50 microgram/ml; ascorbic acid at a concentration range of about 400-600 microgram/ml, bFGF at a concentration range of about 50-200 ng/ml, xeno-free serum replacement and a lipid mixture; the IL6RIL6 chimera at a concentration range of about 50-200 picogram per milliliter (pg/ml); or leukemia inhibitory factor (LIF) at a concentration of at least 2000 units/ml; cell cultures comprising same with pluripotent stem cells such as human embryonic stem cells and induced pluripotent stem (iPS) cells, and methods of using same for expanding pluripotent stem cells in an undifferentiated state using two-dimensional or three-dimensional culture systems; and methods of expanding iPS cells in a suspension culture devoid of substrate adherence and cell encapsulation.

Claims (37)

1. A culture medium comprising a cytokine consisting of an IL6RIL6 chimera at a concentration range of about 50-200 picogram per milliliter (pg/ml), wherein said IL6RIL6 chimera includes the amino acid sequence set forth by SEQ ID NO: 39, wherein the culture medium is serum-free and capable of maintaining pluripotent stem cells in an undifferentiated state in the absence of feeder cell support for at least 5 passages.

2. The culture medium of claim 1 , wherein said culture medium further comprises serum replacement.

3. The culture medium of claim 1 , wherein said concentration of said IL6RIL6 chimera is between about 90 pg/ml to about 120 pg/ml.

4. The culture medium of claim 1 , wherein said concentration of said IL6RIL6 chimera is about 100 pg/ml.

5. The culture medium of claim 1 , wherein said culture medium further comprises basic fibroblast growth factor (bFGF).

6. A cell culture comprising pluripotent stem cells and the culture medium of claim 1 .

7. The cell culture of claim 6 , wherein the cell culture is feeder cells free.

8. The cell culture of claim 6 , wherein said pluripotent stem cells are embryonic stem cells.

9. The cell culture of claim 6 , wherein said pluripotent stem cells are induced pluripotent stem (iPS) cells.

10. The cell culture of claim 6 , wherein said pluripotent stem cells are human pluripotent stem cells.

11. The cell culture of claim 6 , being a suspension culture.

12. The culture medium of claim 1 , wherein the culture medium is capable of maintaining a population of cells consisting of induced pluripotent stem cells having a normal karyotype for at least 15 passages.

13. A method of deriving an embryonic stem cell line, comprising

(a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and

(b) culturing said embryonic stem cell in the culture medium of claim 1 ;

thereby deriving the embryonic stem cell line.

14. A method of deriving an induced pluripotent stem cell line, comprising

(a) inducing a somatic cell to a pluripotent stem cell; and

(b) culturing said pluripotent stem cell in the culture medium of claim 1 ;

thereby deriving the induced pluripotent stem cell line.

15. A method of generating lineage-specific cells from pluripotent stem cells, the method comprising:

(a) culturing the pluripotent stem cells in the culture medium of claim 1 , to thereby obtain expanded, undifferentiated stem cells;

(b) subjecting said expanded, undifferentiated stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells;

thereby generating the lineage-specific cells from the pluripotent stem cells.

16. A method of generating embryoid bodies from pluripotent stem cells, the method comprising:

(a) culturing the pluripotent stem cells in the culture medium of claim 1 , to thereby obtain expanded, undifferentiated pluripotent stem cells; and

(b) subjecting said expanded, undifferentiated pluripotent stem cells to culturing conditions suitable for differentiating said stem cells to embryoid bodies;

thereby generating the embryoid bodies from the pluripotent stem cells.

17. A method of generating lineage-specific cells from pluripotent stem cells, the method comprising:

(a) culturing the pluripotent stem cells in the culture medium of claim 1 , to thereby obtain expanded, undifferentiated pluripotent stem cells;

(b) subjecting said expanded, undifferentiated pluripotent stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated stem cells to embryoid bodies; and

(c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells;

thereby generating the lineage-specific cells from the pluripotent stem cells.

18. A method of expanding and maintaining pluripotent stem cells in an undifferentiated state, the method comprising culturing the pluripotent stem cells in the culture medium of claim 1 , thereby expanding and maintaining the pluripotent stem cells in the undifferentiated state.

19. The method of claim 18 , wherein said culturing is effected on a matrix.

20. The method of claim 18 , wherein said culturing is effected in a suspension culture.

21. The method of claim 18 , wherein said culture medium is protein carrier-free.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 29, 2021
From: AMIT, MICHAL; ITSKOVITZ-ELDOR, JOSEPH
To: TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED
Reel/Frame 055072/0330 →
Continuity (4)
Division 14696520 · Apr 27, 2015
Continuation 13508991
Provisional Application 61272860 · Nov 12, 2009
Related Publication 20210079342A1 · Mar 18, 2021
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