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Method for isolating and subculturing primary cell of human embryo cortex neural stem cell

A neural stem cell, subculture technology, applied in the direction of nervous system cells, animal cells, vertebrate cells, etc., can solve the problem of less research on neural stem cells

Inactive Publication Date: 2014-05-14
陆华
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AI Technical Summary

Problems solved by technology

The existence of neural stem cells in specific brain regions in mammalian embryonic stages and adult animals has been continuously confirmed, but there is little research on human neural stem cells

Method used

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  • Method for isolating and subculturing primary cell of human embryo cortex neural stem cell

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Embodiment Construction

[0012] Such as figure 1 The step flow diagram of the separation and subculture method of neural stem cell primary cells shown in the present invention, which is applied to the isolation and culture process of neural stem cell single cell clone cell line, said method comprises the following steps:

[0013] S1: Isolation and culture of primary cells: Fresh human embryos induced by water sac at embryonic age of 12 weeks were taken, the cerebral cortex of the fetal brain was isolated under aseptic conditions, the dura mater and surface blood vessels were peeled off, and the cells were cultured in DMEM / F12 (1:1 ) 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 added with B 27 and bFGF (fibroblast growth factor) in DMEM / F...

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Abstract

The invention provides a method for cloning and subculturing a neural stem cell single cell, and the method is applied to the processes of the isolated culture, single cell cloning and subculture of a neural stem cell primary cell. The isolated culture method comprises the following steps of: isolation and culture of the neural stem cell primary cell, the subculture, single cell cloning and subculture, cloning induced differentiation and indirect immunofluorescence detection. The method provided by the invention can be used for successfully establishing a human embryo neural stem cell from a fresh human embryo cortex at the embryo age of 12 weeks by adopting a serum-free culture and single cell cloning technology; the human embryo neural stem cell has continuous cloning capacity, can be subjected to the subculture and expresses the neural nidogen antigen; the differentiated human embryo neural stem cell expresses the specific antigens of a neuronal cell, a colloid cell and an oligodendroglia cell.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for the isolation and culture of primary cells of neural stem cells, single cell cloning and subculture, which is applied to the cloning culture process of cell lines of 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 e...

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Application Information

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IPC IPC(8): C12N5/0797C12N5/0793C12N5/079
Inventor 陆华
Owner 陆华
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