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Method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as culture layer

A technology of pluripotent stem cells and amniotic mesenchyme, applied in artificially induced pluripotent cells, non-embryonic pluripotent stem cells, microorganism-based methods, etc.

Inactive Publication Date: 2013-08-07
李凌松 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the culture of iPS cells, like hESCs, requires animal-derived MEFs as feeder cells to keep them in an undifferentiated state. This problem has become a major obstacle and problem that needs to be overcome in the clinical application of iPS cells.

Method used

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  • Method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as culture layer
  • Method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as culture layer
  • Method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as culture layer

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Embodiment Construction

[0021] The method for culturing human-derived artificially induced pluripotent stem cells using human amniotic mesenchymal cells as a culture layer of the present invention includes the following steps:

[0022] (1) Place the in vitro human amniotic mesenchymal cells in a 15cm cell culture flask containing human amniotic mesenchymal cell culture medium. When the cell density reaches 90% or more confluence, remove the culture medium and use 5ml D-Hanks' balanced salt The solution (Hanks'balanced salt solution without calcium and magnesium, manufactured by Invitrogen) was washed 3 times, and then 2ml 0.25% trypsin (Trypsin, manufactured by Invitrogen) was added and placed in CO at 36.5-37.5°C. 2 In the cell incubator for 2-3 minutes, observe under the microscope, when most of the cell edges are bright, tap the culture flask to make the cells in suspension, add 5ml containing DMEM / F12 (Invitrogen) and 10% fetal cattle The stop solution of serum (produced by Invitrogen), transfer it t...

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Abstract

The invention relates to a method for culturing human artificially induced pluripotent stem cells by using human amnion mesenchyme cells as a culture layer, which comprises the steps that: a human amnion mesenchyme cell culture medium is used, and exosomatic human amnion mesenchyme cells are cultured in a 15cm cell culture bottle for reaching 80 to 90 percent fusion; then, a human artificially induced pluripotent stem cell culture medium is added for use; and human artificially induced pluripotent stem cells put in the 15cm cell culture bottle are blown and beaten to be crushed into smaller stem cell masses, the stem cell masses are put into a human amnion mesenchyme cell culture bottle for culture, when human embryo stem cells grow to 4mm to 6mm, the stem cells are repeatedly blown and beaten to be crushed into smaller stem cell masses, and the stem cell masses are put into a new amnion mesenchyme cell culture bottle for continuous culture or are used for other purposes. By the method, cells with the same form as the human artificially induced pluripotent stem cells cultured on mouse embryonic fibroblast (MEF) are obtained, and in addition, higher safety is realized through beingcompared with the MEF culture.

Description

Technical field [0001] The invention relates to a method for culturing human-derived artificially induced pluripotent stem cells, in particular to a method for culturing human-derived artificially induced pluripotent stem cells by using human amniotic membrane mesenchymal cells as a culture layer. Background technique [0002] Human embryonic stem cells (hESC) were originally isolated from early embryos in the blastocyst stage by Thomson in 1998. By separating the inner cell mass of human blastocysts, mouse embryonic fibroblast (MEF) irradiated with 35Gy gamma rays are cultured in vitro as a culture layer. After about 9-15 days, the cell mass is blown out and then cultured on MEF. , Select the monoclonal cell clusters with uniform undifferentiated morphology for culture, and repeat the culture to form a line. hESC has the characteristics of totipotency of self-renewal and differentiation. When injected into severe combined immunodeficiency mice (SCID), teratomas containing three...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074C12N5/077C12R1/91
Inventor 李凌松蔡哲张可华
Owner 李凌松
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