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US 10,377,989 B2Granted
Methods for suspension cultures of human pluripotent stem cells
AssigneeJanssen Biotech, Inc.
InventorsBenjamin Fryer, Daina Laniauskas, Marcia Blackmoore, Haiyun Wang, Kostadinka LiLova, Shelley Nelson, Elizabeth Rosocha
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Abstract
The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation.
Claims31
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.
3 more elements
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.
4 more elements
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.
3 more elements
Dependencies are inferred from the claim text.