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Culture medium and culture method for differentiating mammal pluripotent stem cells into definitive endoderm cells

A technology for pluripotent stem cells and definitive endoderm, applied in the field of stem cells and regenerative medicine, can solve the problems of high price and high cost of definitive endoderm cells, achieve low price, promote research and application, and shorten the effect of differentiation time

Active Publication Date: 2021-11-02
深圳市北科源细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a growth factor, Activin A is expensive, so the cost of obtaining definitive endoderm cells in vitro is relatively high

Method used

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  • Culture medium and culture method for differentiating mammal pluripotent stem cells into definitive endoderm cells
  • Culture medium and culture method for differentiating mammal pluripotent stem cells into definitive endoderm cells
  • Culture medium and culture method for differentiating mammal pluripotent stem cells into definitive endoderm cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. A culture medium for the differentiation of human pluripotent stem cells into qualitative endoderm cells

[0053] The medium is based on DMEM / F12, and also includes the following components: 100ng / ml Activin A, 2.5uM CHIR99021, 1% B27, 0.2% bovine serum albumin, 1% double antibody, ATP.

[0054] 2. A culture method for differentiating human pluripotent stem cells into definitive endoderm cells, using the above medium for culture, specifically comprising the following steps:

[0055] (1) Using mTeSR+1% double antibody as the growth medium, human embryonic stem cells were cultured on Matrigel at a dilution ratio of 1:100.

[0056] (2) After the human embryonic stem cells in step (1) were cultured for 4-5 days, the clones grew up. At this time, the cell density was about 80%-90% of the area of ​​the bottom of the culture plate. Set aside for 3 minutes, observed under the microscope that the cells were spherical, immediately added DMEM / F12 medium to stop digestion, gent...

Embodiment 2

[0062] 1. A culture medium for human pluripotent stem cells to differentiate into qualitative endoderm cells, the culture medium is based on DMEM / F12, and also includes the following components: 100ng / ml Activin A, 2.5uM CHIR99021, 1% B27, 0.2% bovine serum albumin, 1% double antibody, NAC.

[0063] 2. A culture method for differentiating human pluripotent stem cells into definitive endoderm cells, using the above medium for culture, specifically comprising the following steps:

[0064] (1) Using mTeSR+1% double antibody as the growth medium, human embryonic stem cells were cultured on Matrigel at a dilution ratio of 1:100.

[0065] (2) After the human embryonic stem cells in step (1) were cultured for 4-5 days, the clones grew up. At this time, the cell density was about 80%-90% of the area of ​​the bottom of the culture plate. Set aside for 3 minutes, observed under the microscope that the cells were spherical, immediately added DMEM / F12 medium to stop digestion, gently blo...

Embodiment 3

[0071] 1. A culture medium for human pluripotent stem cells to differentiate into qualitative endoderm cells. The culture medium is based on DMEM / F12 and also includes the following components: 10ng / ml Activin A, 1% B27, 0.2% bovine serum Albumin, 1% double antibody, ATP.

[0072] 2. A culture method for differentiating human pluripotent stem cells into definitive endoderm cells, using the above medium for culture, specifically comprising the following steps:

[0073] (1) Using mTeSR+1% double antibody as the growth medium, human embryonic stem cells were cultured on Matrigel at a dilution ratio of 1:100.

[0074] (2) After the human embryonic stem cells in step (1) were cultured for 4-5 days, the clones grew up. At this time, the cell density was about 80%-90% of the area of ​​the bottom of the culture plate. Set aside for 3 minutes, observed under the microscope that the cells were spherical, immediately added DMEM / F12 medium to stop digestion, gently blown and resuspended,...

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Abstract

The invention discloses a culture medium and a culture method for differentiating mammal pluripotent stem cells into definitive endoderm cells. The culture medium is prepared by adding at least one of ATP and NAC to a basic culture medium. The method comprises the following step: culturing the pluripotent stem cells of the mammals by adopting the culture medium, so that the pluripotent stem cells are differentiated into the typing endoderm cells. According to the method, at least one of ATP and NAC is added into the basic culture medium, so that the differentiation efficiency of the mammal pluripotent stem cells differentiated and cultured into the endoderm cells or / and the dosage of Activin A is reduced, and the cost is reduced; the culture medium provided by the invention is relatively low in cost and stable in performance, and can efficiently differentiate the human pluripotent stem cells into the definitive endoderm cells. A low-cost and high-efficiency research system is provided for in-vitro research on endoderm related development problems. Meanwhile, the application cost of regenerative medicine is also reduced.

Description

technical field [0001] The invention relates to the field of stem cells and regenerative medicine, in particular to a culture medium and a culture method for differentiating mammalian pluripotent stem cells into definitive endoderm cells. Background technique [0002] Pluripotent embryonic stem cells (embryonic stem cells, ESCs) and induced pluripotent stem cells (inducedpluripotent stem cells, iPSCs) are special types of cells. On the one hand, it has almost unlimited self-renewal ability, and on the other hand, it has the potential to differentiate into various cell types that make up the human body. Its in vitro differentiation system provides a fast and convenient means to elucidate the molecular basis of related diseases, and It provides a good technical system for regenerative medicine. Pluripotent stem cells can differentiate into the inner, middle, and outer three germ layer lineages, and related diseases caused by defects in tissues such as the liver, intestine, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2506/45C12N2506/02C12N2500/40C12N2500/32
Inventor 蒋卫吕静
Owner 深圳市北科源细胞科技有限公司
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