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A kind of totipotent stem cell culture method

A technology of totipotent stem cells and cell culture, applied in the field of induced pluripotent stem cells (iPSCs) culture, can solve the problem of high cost of recombinant laminin, and achieve the effect of simplifying basement membrane components

Active Publication Date: 2016-02-17
HANGZHOU WEIJIZHI BIOTECH
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  • Abstract
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Problems solved by technology

However, the cost of recombinant laminin is still extremely high; and two tested peptides derived from Vitronectin are suggested to specifically provide integrin alphavbeta3 adhesion, thereby enabling stem cell growth

Method used

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  • A kind of totipotent stem cell culture method
  • A kind of totipotent stem cell culture method

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Embodiment 1

[0031] Example 1 : Growth, passage and differentiation of induced human totipotent stem cell lines in this culture system

[0032] The polypeptide sequence as the extracellular matrix is: NCKHQCTCIDGAVGCIPLCP (SEQ ID NO: 1). The concentration of the polypeptide aqueous solution is 2mg / ml. Draw 1mL of polypeptide aqueous solution with a pipette gun, and the area of ​​transfer is 10cm 2 In the petri dish, shake slowly to make it evenly distributed on the bottom of the petri dish, and then place the petri dish covered with aqueous peptide solution at 37°C±1°C for 12 hours. Polypeptides are immobilized on the surface for culture through adsorption methods such as covalent bonds, electrostatics, and hydrophobic interactions.

[0033] In step 2), the induced pluripotent stem cells before inoculation undergo the following processing steps: 1) thawing. Induced pluripotent stem cells already in continuous culture do not need to be thawed. 2) Digestion. Use cleavage enzyme or E...

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Abstract

The invention provides a method for cultivating totipotent stem cells. Artificially synthesized polypeptide is used as the extracellular matrix, spread on the surface of the cell culture dish, inoculated totipotent stem cells on the surface of the aforementioned culture dish, injected with feeder-free medium, and then cultured and passaged the inoculated totipotent stem cells. The method provided by the invention can support the expansion of totipotent stem cells and keep the content of Oct4+ cells above 80%. After at least 40 days of culture (6 passages, about 40 times of cell doubling), the cells still highly express various markers of totipotent stem cells, Oct4, and the karyotype remains consistent after expansion and culture.

Description

technical field [0001] The purpose of the present invention is to provide a method for culturing totipotent stem cells, in particular, to provide a method for culturing induced totipotent stem cells (iPSCs). Background technique [0002] Stem cells are cells capable of differentiating into various cell types. Stem cells from the embryonic stage and iPSCs produced by re-induction are capable of differentiating into most cell types of an individual. These two kinds of cells can be infinitely multiplied in in vitro culture, providing basic materials for regenerative medicine research and industry. However, both ESCs and iPSCs are prone to death in cell culture, especially stem cells from primates such as humans. And this kind of death occurs especially during the passage of cells, no matter whether the passage is in a mechanical block or in a way of enzymatic digestion and dispersion, it is unavoidable. [0003] On the other hand, Matrigel has been used as a plating material...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C12N5/07
Inventor 韩雪林慧盈王猛刘兴宇
Owner HANGZHOU WEIJIZHI BIOTECH
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