Shortcut method for separation and purification of embryonic cranial neural stem cells

A technology for separation and purification of stem cells, applied in the field of purification of biological materials, can solve the problems of stem cell damage, expensive precision instruments, etc.

Inactive Publication Date: 2013-04-10
北京清美联创干细胞科技有限公司
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Problems solved by technology

Although this method can obtain stem cells with higher purity, it has certain damage to stem cells and requires expensive precision instruments (references: Muotri, R.A. et al: Proc Natl Acad Sci USA 97, 14720-14725, 2000)

Method used

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  • Shortcut method for separation and purification of embryonic cranial neural stem cells

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Embodiment Construction

[0016] The following examples describe the present invention in more detail:

[0017] 1. Preparation of thermal conversion polymer (N-isopropylacrylamide and polyethylene glycol): This polymer is an artificial glue that is in a gel state at high temperature and in a liquid state at low temperature. It changes from liquid to gel The conversion temperature is 25°C, it can be dissolved in the medium, and it can be liquidized in an ice bath.

[0018] 2. The embryonic brain tissue was repeatedly pipetted in low-temperature PBS buffer to disperse the cells and pass through a nylon mesh to form a single-cell suspension. The single cell suspension was centrifuged to remove the supernatant, and the cells were resuspended in cryogenic liquid medium.

[0019] 3. Mix 0.7ml of heat transfer polymer with 0.3ml of cell suspension, and the cell density can be adjusted according to the source of brain tissue.

[0020] 4. Take the cell mixture-heat conversion polymer mixture and drop it into ...

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Abstract

The invention provides a method able to realize separation, amplification and purification of embryonic cranial neural stem cells rapidly and simply. The invention uses the thermal conversion polymer N-isopropylacrylamide and polyethylene glycol to conduct three-dimensional culture on neural cells, and makes use of the characteristic that neural stem cells can proliferate in a gel environment and form neurospheres to achieve the purposes of separation and purification of the neural stem cells. The formed neurospheres are subjected to immunofluorescent labeling determination, which shows that about 93% of the cells are Nestin positive, so that the method is simple and effective for separation and purification of neural stem cells. The embryonic neural stem cells separated and purified by the method can differentiate in vitro to form neurons, oligodendrocytes and Glias.

Description

technical field [0001] The present invention relates to a method for purifying biological materials, in particular to a rapid method for purifying embryonic brain neural stem cells. Background technique [0002] The study of embryonic brain neural stem cells first requires the separation of brain neural stem cells. At present, there are two methods for isolating embryonic brain neural stem cells: (1) Suspension culture method: the dispersed brain nerve cells are suspended and cultured in a culture dish to form neurons. The brain neural stem cells were isolated by retrobulbar subculture several times. The purity of brain neural stem cells obtained by this method is not high, and the cycle is long (reference: Piper, D.R. et al.: Experimental Neurology 156, 71-83, 1999). (2) FACS method: Firstly, the specific antigen of neural stem cells is fluorescently labeled, and the stem cells are screened with precision instruments. Although this method can obtain stem cells with higher...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/0797
Inventor 田杰
Owner 北京清美联创干细胞科技有限公司
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