Automated method and apparatus for embryonic stem cell culture
First Claim
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1. A method for automated expansion of pluripotent stem cells comprising:
- (a) obtaining a first population of human pluripotent cells in a growth media;
(b) separating the pluripotent cells with an automated separation system;
(c) delivering the separated cells in fresh growth media into a plurality of culture wells;
(d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and
(e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells;
wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein the automated separation system comprises contacting the first population of pluripotent cells with a proteolytic enzyme, wherein the proteolytic enzyme is trypsin, recombinant trypsin, a trypsin-like protease or TRYPLE.
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Abstract
The invention concerns methods for automated culture of embryonic stem cells (ESCs) such as human ESCs. In some aspects, methods of the invention employ optimized culture media and limited proteolytic treatment of cells to separate cell clusters for expansion. Automated systems for passage and expansion of ESCs are also provided.
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Citations
58 Claims
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1. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein the automated separation system comprises contacting the first population of pluripotent cells with a proteolytic enzyme, wherein the proteolytic enzyme is trypsin, recombinant trypsin, a trypsin-like protease or TRYPLE. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein the fresh growth media comprises an inhibitor of a proteolytic enzyme, wherein the fresh growth media comprises an inhibitor of the proteolytic enzyme used for cell separation, wherein the proteolytic enzyme inhibitor is a trypsin inhibitor. - View Dependent Claims (18, 19)
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20. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum, wherein the growth media comprises a effective amount of a Rho-associated kinase (ROCK) inhibitor, wherein the Rho-associated kinase (ROCK) inhibitor is HA-100 or H-1135. - View Dependent Claims (21, 22, 23, 24)
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25. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein no or essentially no differentiation occurs in at least 97% of the expanded population of pluripotent cells, and wherein the pluripotent cells are ES cells. - View Dependent Claims (26, 27, 28)
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29. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum wherein the first population of pluripotent cells is comprised on a cell culture plate and further wherein the first population of pluripotent cells is between about 50% and about 99% confluent at the time of cell separation, and wherein the pluripotent cells are ES cells. - View Dependent Claims (30, 31, 32, 33)
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34. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein the automated separation system comprises contacting the first population of pluripotent cells with a proteolytic enzyme, and wherein the pluripotent cells are ES cells. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein the fresh growth media comprises an inhibitor of a proteolytic enzyme, and wherein the pluripotent cells are ES cells. - View Dependent Claims (47, 48, 49, 50)
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51. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum and further wherein the separation system is automated by a liquid handler robot, and wherein the pluripotent cells are ES cells. - View Dependent Claims (52)
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53. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum, wherein the population of pluripotent cells is free or essentially free from non-pluripotent cells and further wherein the population of pluripotent cells is human induced pluripotent stem cells.
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54. A method for automated expansion of pluripotent stem cells comprising:
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(a) obtaining a first population of human pluripotent cells in a growth media; (b) separating the pluripotent cells with an automated separation system; (c) delivering the separated cells in fresh growth media into a plurality of culture wells; (d) culturing the pluripotent cells in the growth media in the plurality of culture wells; and (e) repeating steps b-d one or more times to provide an expanded population of pluripotent cells; wherein said culturing does not comprise culturing the pluripotent cells in the presence of fibroblast feeder cells or serum, wherein the growth media comprises a effective amount of a Rho-associated kinase (ROCK) inhibitor, and wherein the pluripotent cells are ES cells. - View Dependent Claims (55, 56, 57, 58)
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Specification