Culture medium for pluripotent stem cells
The present invention relates to a chemically defined medium for eukaryotic cell culture, comprising water, at least one carbon source, one or more vitamins, one or more salts, one or more fatty acids, one or more buffer components, selenium, at least one substance of the group of Functional Inhibitors of Acid Sphingomyelinase (FIASMAs) and at least one polypeptide of the TGF-β superfamily with the ability to inhibit stem cell differentiation, its use in the culture of human pluripotent stem cells, a cell culture system comprising human pluripotent stem cells and the chemically defined medium, as well as a kit for proliferation and/or maintenance of human pluripotent stem cells.
1. A chemically defined medium for eukaryotic cell culture, comprising water, at least one carbon source, one or more vitamins, one or more salts, one or more fatty acids, one or more buffer components, selenium, at least one substance of the group of Functional Inhibitors of Acid Sphingomyelinase (FIASMAs) and at least one polypeptide of the TGF-β superfamily with the ability to inhibit stem cell differentiation,
wherein the chemically defined medium is free of FGF-2,
wherein the chemically defined medium is completely devoid of human- or animal-derived substances,
wherein the ratio between said FIASMA and said at least one polypeptide of the TGF-β superfamily is from 400:1 to 2000:1,
wherein the concentration of the at least one FIASMA is in the range of 0.1 mg/L to 10 mg/L, and
wherein the concentration of the at least one polypeptide of the TGF-β superfamily is in the range of 0.2 μg/L to 20 μg/L.
2. The chemically defined medium according to claim 1 , wherein the FIASMA is selected from the group consisting of Trimipramine, Mepacrine, Promethazine and Profenamine.
3. The chemically defined medium according to claim 1 , wherein the at least one polypeptide of the TGF-β superfamily is selected from the group consisting of activins, nodal, myostatin and GDF3.
4. The chemically defined medium according to claim 3 , wherein the at least one polypeptide of the TGF-β superfamily is selected from activins.
5. The chemically defined medium according to claim 1 , wherein the chemically defined medium is free of albumin and/or TGF-β1.
6. The chemically defined medium according to claim 1 , wherein the one or more buffer components are selected from inorganic bicarbonate, inorganic phosphate or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).
7. The chemically defined medium according to claim 1 , wherein the concentration of selenium is in the range of 1 μg/L to 100 μg/L.
8. The chemically defined medium according to claim 1 , further comprising insulin and/or transferrin, wherein the concentration of transferrin is in the range of 0.1 mg/L to 100 mg/L and/or the concentration of insulin is in the range of 0.1 mg/L to 100 mg/L.
9. The chemically defined medium according to claim 1 , further comprising ascorbic acid, wherein the concentration of ascorbic acid is in the range of 3 mg/L to 300 mg/L.
10. The chemically defined medium according to claim 1 , wherein one fatty acid is present at a concentration in the range of 4.6×10 −3 mg/L to 4.6×10 −2 mg/L.
11. A cell culture system, comprising human pluripotent stem cells, and a chemically defined medium according to claim 1 .
12. The cell culture system according to claim 11 , further comprising an extracellular matrix.
13. A method for culturing eukaryotic cells, the method comprising culturing eukaryotic cells in the chemically defined medium according to claim 1 .
14. The method according to claim 13 , wherein the eukaryotic cells are human pluripotent stem cells.
15. The method according to claim 13 , wherein the eukaryotic cells are human induced pluripotent stem cells.
16. The method according to claim 14 , wherein the culturing of human pluripotent cells is performed as an adherent cell culture.
17. The method according to claim 14 , wherein the culturing of human pluripotent cells is performed on an extracellular matrix.
18. The method according to claim 14 , wherein the culturing of human pluripotent cells is performed on a xeno-free extracellular matrix.