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Method for separating and subculturing primary embryo-derived neural stem cell

A neural stem cell and primary cell technology, applied in nervous system cells, animal cells, vertebrate cells, etc., can solve problems such as difficulty in culturing embryonic neural stem cells

Inactive Publication Date: 2013-04-03
陆华
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Problems solved by technology

[0003] In order to solve the problem that embryonic neural stem cells are difficult to cultivate, the object of the present invention is to provide a method for the isolation and subculture of embryo-derived neural stem cells, which is obtained from human embryonic cortex by using serum-free culture and single-cell cloning technology. Neural stem cells with self-renewal and multi-differentiation potential identified using immunofluorescence staining

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  • Method for separating and subculturing primary embryo-derived neural stem cell

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Embodiment Construction

[0012] like figure 1 Shown is a flow chart of the steps of the embryo-derived neural stem cell primary cell separation and subculture method of the present invention, the method comprising the following steps:

[0013] S1. Isolation and culture of primary cells: Fresh human embryos induced by water sac at 12 weeks of embryonic age were taken, and the cerebral cortex of the fetal brain was isolated under aseptic conditions, and the dura mater and surface blood vessels were peeled off. ) in the cell culture medium for three times, and the tissue fragments in the bottle were repeatedly blown with a fine pipette until completely scattered to make a cell suspension, which was filtered through a 100-mesh stainless steel mesh to make a single-cell suspension, which was stained and counted Live cells were cultured in DMEM / F12 (1:1) containing B27 (1:50), EGF (epidermal growth factor) (20ng / ml), bFGF (fibroblast growth factor) (20ng / ml) without serum Base, trypan blue staining count l...

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Abstract

The invention provides a method for separating and subculturing an embryo-derived primary neural stem cell, comprising the following steps: separating and culturing the primary neural stem cell, subculturing the primary neural stem cell, cloning and subculturing a single cell, carrying out induced differentiation, and carrying out indirect immunofluorescence detection. According to the method, a neural stem cell with the self-renewing and differentiating potential is separated from the fresh human embryonic cortex with an embryonic age of 12 weeks and cultured by adopting the serum-free culturing and single-cell cloning technology, and a large number of neural stem cells are formed three week later, wherein the neural stem cells have continuous cloning capacity and can subculature and express the neural nidogen antigen. The differentiated cells can express the specific antigens of the neuron cells, the colloid cells and oligodendroglia cells, and the problem that the embryo-derived neural stem cell is difficult to culture is solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for the isolation and subculture of primary cells of embryo-derived neural stem cells. Background technique [0002] The discovery of neural stem cells is a major breakthrough in the field of neuroscience, which has opened up broad prospects for research fields such as neural development and neural tissue transplantation. Neural stem cells are cells with self-renewal and extensive differentiation potential. They are in a non-terminal state of differentiation. They can divide symmetrically or asymmetrically into new stem cells or daughter cells with gradually decreasing differentiation potential, and finally produce the central nervous system. The three main types of cells are neurons, astrocytes, and oligodendrocytes. It is generally believed that neural stem cells mainly exist in the striatum, cerebellar hemispheres, and ventricle areas of the mammalian fetal brain, and in...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
Inventor 陆华
Owner 陆华
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