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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, animal cells, etc.

Inactive Publication Date: 2012-04-11
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] A method of using human amniotic mesenchymal cells as a culture layer to cultivate human-derived artificially induced pluripotent stem cells comprises the following steps:

[0022](1) Place human amniotic mesenchymal cells in vitro in a 15cm cell culture flask filled with human amniotic mesenchymal cell culture medium. When the cell density reaches 90% confluence, remove the medium, and use 5ml D-Hanks'balanced salt Solution (Hanks'balanced salt solution without calcium and magnesium, produced by Invitrogen Company) was washed 3 times, and then 2ml of 0.25% trypsin (Trypsin, produced by Invitrogen Company) was added and placed in CO at 36.5-37.5°C 2 In the cell culture incubator for 2-3 minutes, observe under the microscope, when most of the cell edges are bright, tap the culture bottle to make the cells in a suspended state, add 5ml of DMEM / F12 (manufactured by Invitrogen) and 10% fetal calf The stop solution of serum (produced by Invitrogen Company) was transferred to...

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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 cultivating human-derived artificially induced pluripotent stem cells, in particular to a method for cultivating human-derived artificially induced pluripotent stem cells by using human amniotic mesenchymal cells as a culture layer. Background technique [0002] Human embryonic stem cells (human embryonic stem cells, hESC) were initially isolated from early embryos at the blastocyst stage by Thomson et al. in 1998. By separating the inner cell mass of human blastocysts, mouse embryonic fibroblast (MEF) irradiated with 35Gy γ-rays was used as a culture layer for in vitro culture, and the cell mass was blown away after about 9-15 days, and then continued to be cultured on MEF , select monoclonal cell clusters with uniform undifferentiated morphology for culture, and repeat culture until lines are formed. hESC has the characteristics of self-renewal and differentiation totipotency. When injected into severe combined ...

Claims

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Application Information

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IPC IPC(8): C12N5/074
Inventor 李凌松蔡哲张可华
Owner 李凌松
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