Methods of expanding embryonic stem cells in a suspension culture
AssigneeTechnion Research & Development Foundation Limited
InventorsMichal Amit, Joseph Itskovitz-Eldor
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Abstract
A method of expanding and maintaining human embryonic stem cells (ESCs) in an undifferentiated state by culturing the ESCs in a suspension culture under culturing conditions devoid of substrate adherence is provided. Also provided are a method of deriving ESC lines in the suspension culture and methods of generating lineage-specific cells from ESCs which were expanded in the suspension culture of the present invention.
Claims48
A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, and wherein said medium comprises a TGFβ isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state.
A method of deriving an embryonic stem cell line, the method comprising:
- (a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus;
- and
- (b) culturing said embryonic stem cell in a suspension culture, under culturing conditions which allow expansion of said embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, wherein said medium comprises a TGFβ isoform,
- thereby deriving the embryonic stem cell line.
A culture medium comprising serum replacement, a soluble interleukin-6 receptor (sIL6R), a soluble interleukin-6 (IL6) and basic fibroblast growth factor (bFGF), wherein the culture medium is suitable for expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages.
A culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), wherein the culture medium is suitable for expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages.
A cell culture comprising human embryonic stem cells and a culture medium which comprises at least 2000 u/ml of leukemia inhibitor factor (LIF), wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.
A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a culture medium, which comprises a soluble interleukin-6 receptor (sIL6R), wherein said sIL6R is present at a concentration of 15-30 ng/ml, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages.
A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a culture medium which comprises leukemia inhibitor factor (LIF), wherein said LIF is present at a concentration of at least 2000 units per milliliter (u/ml), thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state for at least 5 passages.
A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages, the method comprising culturing the human pluripotent stem cells in a suspension culture without adherence of said cells to a substrate or to feeder cells under conditions comprising a serum-free culture medium selected from the group consisting of:
- a culture medium comprising a soluble interleukin-6 receptor (sIL6R) at a concentration of at least 10 nanogram per milliliter (ng/ml) and soluble interleukin-6 (IL6), a culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), and a culture medium which comprises a TGFβ isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages.
A culture medium comprising serum replacement, a soluble interleukin-6 receptor (sIL6R) at a concentration of 15-30 nanogram per milliliter (ng/ml), a soluble interleukin-6 (IL6) and basic fibroblast growth factor (bFGF) at a concentration of at least 2 ng/ml, wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.
A culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF) and basic fibroblast growth factor (bFGF) at a concentration of at least 2 ng/ml, wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.
A method of expanding and maintaining human pluripotent stem cells in an undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions devoid of substrate adherence, and which allow expansion of the human pluripotent stem cells in the undifferentiated state for at least 5 passages, thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state.
Dependencies are inferred from the claim text.