Method for culture of cells
AssigneesKyoto University, Takeda Pharmaceutical Company Limited
InventorsMakoto Ikeya, Yayoi Kamiya
Life of this patent
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Abstract
As a technique for proliferating neural crest cells without reducing differentiation capacity, provided is a method for producing neural crest cells, comprising the steps of: (1) obtaining neural crest cells; and (2) suspension-culturing the neural crest cells in a medium comprising a GSK3β inhibitor and a basic fibroblast growth factor (bFGF), wherein the medium comprises the GSK3β inhibitor with a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 with a concentration of higher than 1 μM and lower than 5 μM.
Claims17
A method for producing neural crest cells and maintaining their multipotency, comprising the steps of:
- (1) obtaining neural crest cells by inducing the differentiation of stem cells into the neural crest cells;
- and
- (2) suspension-culturing the neural crest cells in a medium comprising a GSK3β inhibitor and a basic fibroblast growth factor, wherein the medium comprises the GSK3β inhibitor with a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 with a concentration of 3.0 μM to 4.5 μM, thereby maintaining multipotent neural crest cells over 63 days or longer, wherein the cultured neural crest cells comprise a ratio of at least 75% or more of neural crest cells that maintain multipotency to cultured cell population.
A method for proliferating neural crest cells and maintaining their multipotency, comprising the step of:
- (I) suspension-culturing the neural crest cells in a medium comprising a GSK3β inhibitor and a basic fibroblast growth factor, wherein the medium comprises the GSK3β inhibitor with a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 with a concentration of 3.0 μM to 4.5 μM higher than 1 μM and lower that 5 μM, thereby maintaining multipotent neural crest cells over 63 days or longer, wherein the cultured neural crest cells comprise a ratio of at least 75% or more of neural crest cells that maintain multipotency to cultured cell population.
A method for producing nerve cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells or pigment cells, comprising the steps of:
- (i) suspension-culturing neural crest cells in a medium comprising a GSK3β inhibitor and a basic fibroblast growth factor, wherein the medium comprises the GSK3β inhibitor with a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 with a concentration 3.0 μM to 4.5 μM higher than 1 μM and lower that 5 μM, thereby maintaining multipotent neural crest cells over 63 days or longer, wherein the cultured neural crest cells comprise a ratio of at least 75% or more of neural crest cells that maintain multipotency to cultured cell population;
- and
- (ii) differentiating the neural crest cells obtained in the step (i) into cells of at least one lineage selected from the group consisting of nerve cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells.
A method for culturing neural crest cells and extending their multipotency 63 days or longer, comprising the steps of:
- (1) obtaining neural crest cell by inducing the differentiation of stem cells into the neural crest cells;
- and
- (2) suspension-culturing the neural crest cells in a medium comprising a GSK3β inhibitor and a basic fibroblast growth factor, wherein the medium comprises the GSK3β inhibitor with a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 with a concentration 3.0 μM to 4.5 μM higher than 1 μM and lower that 5 μM, thereby maintaining multipotent neural crest cells over 63 days or longer, wherein the cultured neural crest cells comprise a ratio of at least 75% or more of neural crest cells that maintain multipotency to cultured cell population.
Dependencies are inferred from the claim text.