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Totipotent stem cell culture method

A technology of totipotent stem cells and cell culture, applied in the direction of embryonic cells, germ cells, animal cells, etc., can solve the problem of high cost of recombinant laminin, and achieve the effect of simplifying the composition of basement membrane

Active Publication Date: 2013-11-20
HANGZHOU WEIJIZHI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of recombinant laminin is still extremely high; and two tested peptides derived from Vitronectin were suggested to specifically provide integrin alpha v beta 3 binding, thereby enabling stem cell growth

Method used

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  • Totipotent stem cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Growth, passage and differentiation of human embryonic stem cell lines in this culture system

[0035] The polypeptide sequence as the extracellular matrix is: NCKHQCTCIDGAVGCIPLCP (SEQ ID NO: 1), and the concentration of the polypeptide aqueous solution is 1 mg / ml. Draw 1mL of polypeptide aqueous solution with a pipette gun, and the area of ​​transfer is 10cm 2In the petri dish, shake slowly, shake well, so that it is evenly distributed on the bottom of the petri dish, and then place the petri dish covered by the aqueous peptide solution at 37°C±1°C for 3 hours to allow it to fully absorb or complete the reaction. Polypeptides are immobilized on the surface for culture through adsorption methods such as covalent bonds, electrostatics, and hydrophobic interactions.

[0036] In step 2), the human embryonic stem cells before inoculation undergo the following processing steps: 1) thawing. Embryonic stem cells already in continuous culture do not need to be tha...

Embodiment 2

[0039] Example 2: Growth, passage and differentiation of induced human totipotent stem cell lines in this culture system

[0040] The polypeptide sequence as 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.

[0041] 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 EDTA to de...

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Abstract

The invention provides a totipotent stem cell culture method. A polypeptide which is artificially synthesized is taken as an extracellular matrix and spread on the surface of a cell culture dish, totipotent stem cells are inoculated to the surface of the culture dish and injected into a culture medium without a feeder layer, and the totipotent stem cells after inoculation are used for culture and passage. According to the method provided by the invention, the amplification of the totipotent stem cells can be supported, and the cell content of Oct4+ cells can be always kept above 80%. Through at least 40 days of culture (six passages and about 40 times of cell doubling), the cells can still realize high expression of various markers Oct4 of the totipotent stem cells, and after the amplification culture, the karyotype can still be kept consistent.

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 embryonic stem cells (ESCs) or 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 b...

Claims

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Application Information

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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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