Methods for suspension cultures of human pluripotent stem cells
First Claim
Patent Images
1. An in vitro method of producing three-dimensional cell clusters of pluripotent stem cells comprising the steps of:
- a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture;
b. suspending the cell aggregates from the planar adherent culture in a culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells;
c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml;
d. transferring the suspension of cell aggregates to a dynamic suspension culture; and
e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate cell clusters, wherein the cell clusters maintain pluripotency.
1 Assignment
0 Petitions
Accused Products
Abstract
The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation.
182 Citations
31 Claims
-
1. An in vitro method of producing three-dimensional cell clusters of pluripotent stem cells comprising the steps of:
-
a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture; b. suspending the cell aggregates from the planar adherent culture in a culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells; c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml; d. transferring the suspension of cell aggregates to a dynamic suspension culture; and e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate cell clusters, wherein the cell clusters maintain pluripotency. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 23)
-
-
9. A method of differentiating cell clusters of pluripotent stem cells in a dynamically agitated suspension culture system comprising:
-
a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture; b. suspending the cell aggregates from the planar adherent culture in culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells; c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml; d. transferring the suspension of cell aggregates to a dynamic agitated suspension culture; e. expanding the suspension of cell aggregates in the dynamic agitated suspension culture to generate pluripotent cell clusters, wherein the cell clusters express CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of CXCR4; and f. differentiating the pluripotent cell clusters in the dynamic agitated suspension culture system to generate a gut tube cell population, a neural precursor cell population or a cardiomyocyte precursor population. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24)
-
-
25. An in vitro method of producing pluripotent stem cell clusters comprising the steps of:
-
a. treating pluripotent stem cells cultured in a planar adherent culture with an enzymatic or chelating agent at room temperature for a time sufficient to release the pluripotent stem cells as cell aggregates; b. removing said enzymatic or chelating agent; c. adding culture media supplemented with a Rho-kinase inhibitor to the cell aggregates and suspending the cell aggregates in culture without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells; d. transferring the suspension of cell aggregates to a dynamic suspension culture system; and e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate cell clusters, wherein the cells in the cell clusters are pluripotent. - View Dependent Claims (26, 27, 28, 29, 30, 31)
-
Specification