Simplified basic media for human pluripotent cell culture
Fully defined media that support pluripotent cell viability, proliferation, cloning, and derivation, as well as methods and compositions including these media are described. Methods for deriving iPS cells from adult individuals under defined, xeno-free conditions are also described.
1. A defined, albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.
2. A method for culturing human pluripotent stem cells, the method comprising the steps of:
placing pluripotent stem cells on a matrix; and
contacting the cells with a defined, albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.
3. The method of claim 2 , wherein the matrix comprises laminin.
4. The method of claim 2 , wherein the matrix comprises vitronectin.
5. The method of claim 2 , wherein the cells are contacted with the medium under hypoxic conditions.
6. The method of claim 2 , wherein the pluripotent stem cells are embryonic stem cells.
7. The method of claim 2 , wherein the pluripotent stem cells are induced pluripotent stem cells.
8. A method for cloning a human pluripotent stem cell, the method comprising the step of:
plating human pluripotent stem cells at cloning density in a defined albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and NODAL, each in an amount sufficient to support human pluripotent stem cell cloning.
9. The method of claim 8 , wherein the medium further comprises a ROCK inhibitor.
10. The method of claim 9 , wherein the ROCK inhibitor is selected from the group consisting of HA100 and Y27632.
11. The method of claim 8 , wherein the medium further comprises blebbistatin.
12. A method of cryopreserving human pluripotent stem cells, the method comprising the step of:
freezing human pluripotent stem cells in a defined, albumin-free medium free of any component obtained from a non-human animal, the medium comprising water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and NODAL, each in an amount sufficient to support human pluripotent stem cell proliferation.
13. The defined, albumin-free medium of claim 1 , consisting essentially of: water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and NODAL.
14. The defined, albumin-free medium of claim 1 , wherein the medium comprises TGF-β.
15. The defined, albumin-free medium of claim 1 , wherein the medium comprises NODAL.
16. The defined, albumin-free medium of claim 1 , further comprising a ROCK inhibitor.
17. The defined, albumin-free medium of claim 16 , wherein the ROCK inhibitor is selected from the group consisting of HA100 and Y27632.
18. The defined, albumin-free medium of claim 1 , further comprising blebbistatin.
19. The defined, albumin-free medium of claim 1 , consisting of: water, salts, amino acids, vitamins, glucose, insulin, an FGF, selenium, transferrin, and one of TGF-β and NODAL.