Preparation method of human embryonic stem cell growth-supporting humanized feeder layer cell

Abstract

The invention provides a preparation method of a human embryonic stem cell growth-supporting humanized feeder layer cell. The preparation method comprises the following steps of S1, subculturing humanmesenchymal stem cells in a culture medium; S2, infecting the subcultured human mesenchymal stem cells in S1 through retrovirus particles containing hygromycin resistance gene to obtain infected human mesenchymal stem cells; S3, inflecting the infected human mesenchymal stem cells through retrovirus particles containing Wnt3a genes and neomycin resistance genes to obtain TW2R cells; S4, infectingthe TW2R cells through lentivirus particles containing E-cadherin genes and puromycin resistance genes to obtain TWE3R cells. The prepared human embryonic stem cell growth-supporting humanized feederlayer cell can stably express E-cadherin and avoid affecting the proliferation capacity of the TWE3R cells after stably expressing the E-cadherin genes.

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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