Single pluripotent stem cell culture
First Claim
Patent Images
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.
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Abstract
The present invention relates to the field of pluripotent stem cell culture and methods facilitate pluripotent stem cell culture at industrial levels.
139 Citations
25 Claims
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1. A method for maintaining pluripotent stem cells, comprising the steps of:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 22)
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10. A method for passaging pluripotent stem cells, comprising the steps of:
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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. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 23)
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20. A method for maintaining pluripotent stem cells, comprising the steps of:
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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. - View Dependent Claims (24)
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21. A method for passaging pluripotent stem cells, comprising the steps of:
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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. - View Dependent Claims (25)
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Specification