Preparation method of human feeder layer cells capable of supporting growth of human embryonic stem cells

Abstract

The present invention provides a method for preparing human-derived feeder cells that can support the growth of human-derived feeder cells that can support the growth of human embryonic stem cells, comprising the following steps: S1, placing human bone marrow mesenchymal stem cells in a culture medium for subculture ; S2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retroviral particles containing the hygromycin resistance gene to obtain human bone marrow mesenchymal stem cells; S3, infecting the human bone marrow mesenchymal stem cells containing the Wnt3a gene and the neomycin resistance gene The retroviral particles infected human bone marrow mesenchymal stem cells to obtain TW2R cells; S4, the lentiviral particles containing E-cadherin gene and puromycin resistance gene were transfected into TW2R cells to obtain TWE3R cells. The human feeder layer cells prepared by the present invention can stably express the E-cadherin protein, and the stable expression of the E-cadherin gene will not affect the proliferation ability of the TWE3R cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of human feeder layer cells that can support the growth of human embryonic stem cells. Background technique [0002] Human embryonic stem cells have broad application prospects in translational medicine, regenerative medicine, and new drug screening. At present, the culture of human embryonic stem cells (hESC) generally requires mouse embryonic fibroblasts as feeder cells, but the use of these animal sources Sexual materials will bring certain biosafety issues to future clinical applications. In addition, since human embryonic stem cells can only be cultured with MEF within 5 generations as a feeder layer, there are differences between different batches of MEFs derived from fetal mice, and the ability to maintain the pluripotent growth of human embryonic stem cells is also different. Both will bring instability to the long-term culture of human embry...

AI Technical Summary

Problems solved by technology

[0002] Human embryonic stem cells have broad application prospects in translational medicine, regenerative medicine, and new drug screening. At present, the culture of human embryonic stem cells (hESC) generally requires mouse embryonic fibroblasts as feeder cells, but the use of these animal sources Sexual materials will bring certain biosafety issues to future clinical applications

In addition, since human embryonic stem cells can only be cultured with MEF within 5 generations as a feeder layer, there are differences between different batches of MEFs derived from fetal mice, and the ability to maintain the pluripotent growth of human embryonic stem cells is also different. Both will bring instability to the long-term culture of human embryonic stem cells in vitro

Although the TeSR recently developed by the famous Canadian STEMCELL company TM -E8 TM It is a highly defined, feeder-free medium suitable for the cultivation of human embryonic stem cells. Although TeSR-E8 medium can support the growth of stem cells without animal protein, it adds human serum albumin and human matrix proteins, which makes culture conditions extremely expensive and unsuitable for routine use

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