Single pluripotent stem cell culture
The present invention relates to the field of pluripotent stem cell culture and methods facilitate pluripotent stem cell culture at industrial levels.
1. A method for maintaining pluripotent stem cells, comprising the steps of:
a) obtaining clusters of pluripotent stem cells;
b) culturing the cluster of pluripotent stem cells in MEF-conditioned media;
c) releasing the pluripotent stem cells as single cells with an enzyme; and
d) plating the single pluripotent stem cells on a tissue culture substrate comprising an extracellular matrix, and either bFGF or fibroblast conditioned media;
wherein the enzyme: (1) is TrypLE Select™ or TrypLE Express™; (2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;
wherein the enzyme is used to culture the cells for two minutes; and
wherein there is no gain of chromosome abnormalities after releasing the cells.
2. The method of claim 1 , wherein the pluripotent stem cells are released as single cells by treatment with the enzyme at a concentration of about 2.5 g/l.
3. The method of claim 1 , wherein the pluripotent stem cells released as single cells are subsequently cultured with MEF-conditioned media.
4. The method of claim 1 , wherein the tissue culture substrate is selected from the group consisting of matrigel, growth factor-reduced matrigel, fibronectin, laminin, human serum and collagen.
5. The method of claim 4 , wherein the tissue culture substrate is growth factor-reduced matrigel.
6. The method of claim 5 , wherein the growth factor-reduced matrigel is used at a dilution from about 1:30 to about 1:10.
7. The method of claim 6 , wherein the growth factor-reduced matrigel is used at a dilution of 1:30.
8. The method of claim 1 , wherein the pluripotent stem cells are embryonic stem cells.
9. The method of claim 8 , wherein the embryonic stem cells are human.
10. A method for passaging pluripotent stem cells, comprising the steps of:
a) obtaining clusters of pluripotent stem cells;
b) culturing the cluster of pluripotent stem cells in MEF-conditioned media;
c) releasing the pluripotent stem cells as single cells with an enzyme;
d) plating the single pluripotent stem cells on a tissue culture substrate;
e) allowing the single pluripotent stem cells to expand;
f) releasing the single pluripotent stem cells; and
g) plating the single pluripotent stem cells on a new tissue culture substrate comprising an extracellular matrix, and either bFGF or fibroblast conditioned media;
wherein the enzyme: (1) is TrypLE Select™ or TrypLE Express™; (2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;
wherein the enzyme is used to culture the cells for two minutes; and
wherein there is no gain of chromosome abnormalities after releasing the cells.
11. The method of claim 10 , wherein the pluripotent stem cells are released as single cells by treatment with the enzyme at a concentration of 2.5 g/l.
12. The method of claim 10 , wherein the pluripotent stem cells released as single cells are subsequently cultured in MEF-conditioned media.
13. The method of claim 10 , wherein the tissue culture substrate is selected from the group consisting of matrigel, growth factor-reduced matrigel, fibronectin, laminin, human serum and collagen.
14. The method of claim 13 , wherein the tissue culture substrate is growth factor-reduced matrigel.
15. The method of claim 14 , wherein the growth factor-reduced matrigel is used at a dilution from about 1:30 to about 1:10.
16. The method of claim 15 , wherein the growth factor-reduced matrigel is used at a dilution of 1:30.
17. The method of claim 10 , wherein the pluripotent stem cells are embryonic stem cells.
18. The method of claim 17 , wherein the embryonic stem cells are human.
19. The method of claim 10 , wherein the single pluripotent stem cells on a new tissue culture substrate are passaged onto another tissue culture substrate by releasing the cells and plating the released cells onto another tissue culture substrate.
20. A method for maintaining pluripotent stem cells, comprising the steps of:
a) obtaining clusters of pluripotent stem cells;
b) culturing the cluster of pluripotent stem cells in MEF conditioned media;
c) releasing the pluripotent stem cells as single cells with an enzyme; and
d) plating the single pluripotent stem cells on a tissue culture substrate comprising an extracellular matrix, and MEF-conditioned media;
wherein the enzyme: (1) is TrypLE Select™ or TrypLE Express™; (2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;
wherein the enzyme is used to culture the cells from two to five minutes; and
wherein there is no gain of chromosome abnormalities after releasing the cells.
21. A method for passaging pluripotent stem cells, comprising the steps of:
a) obtaining clusters of pluripotent stem cells;
b) culturing the cluster of pluripotent stem cells in MEF conditioned media;
c) releasing the pluripotent stem cells as single cells with an enzyme; and
d) plating the single pluripotent stem cells on a tissue culture substrate;
e) allowing the single pluripotent stem cells to expand;
f) releasing the single pluripotent stem cells; and
g) plating the single pluripotent stem cells on a new tissue culture substrate comprising an extracellular matrix, and MEF-conditioned media;
wherein the enzyme: (1) is TrypLE Select™ or TrypLE Express™; (2) is present in a concentration from about 0.5 g/l to about 2.5 g/l;
wherein the enzyme is used to culture the cells from two to five minutes; and
wherein there is no gain of chromosome abnormalities after releasing the cells.
22. The method of claim 1 , wherein cell viability is at least 98% after releasing the cells.
23. The method of claim 10 , wherein cell viability is at least 98% after releasing the cells.
24. The method of claim 20 , wherein cell viability is at least 98% after releasing the cells.
25. The method of claim 21 , wherein cell viability is at least 98% after releasing the cells.