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In-vitro separation culture and differentiation method for human fetal midbrain nerve stem cells

A technology for separating and culturing brain nerves, which can be applied in the field of biomedicine and can solve the problem of insufficient research on neural stem cells.

Inactive Publication Date: 2012-05-09
江苏迈健生物科技发展股份有限公司
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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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  • In-vitro separation culture and differentiation method for human fetal midbrain nerve stem cells
  • In-vitro separation culture and differentiation method for human fetal midbrain nerve stem cells

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Embodiment Construction

[0014] Such as figure 1 Shown is a schematic flow chart of the steps of the method for in vitro isolation, culture and differentiation of human embryonic midbrain neural stem cells of the present invention, and the method is specifically realized by the following steps:

[0015] S1: Isolation and culture of primary midbrain neural stem cells: Take fresh human embryos induced by water sac at embryonic age 10-13 weeks, aseptically separate the midbrain tissue of the embryos under a microscope, peel off the meninges and surface blood vessels, and put them in PBS solution Rinse three times, repeatedly beat the tissue fragments with a fine pipette until they are completely scattered, centrifuge and wash, blow and beat to make a cell suspension, filter through a 100-mesh stainless steel filter to make a single-cell suspension, add B27 (1:50) and EGF (20ng / ml), bFGF (20ng / ml) DMEM / F12 (1:1) serum-free medium, trypan blue staining and counting viable cells, adjust the concentration of...

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Abstract

The invention provides an in-vitro separation culture and differentiation method for human fetal midbrain nerve stem cells, which is used for separation culture and identification of nerve stem cells from the human fetal midbrain and comprises the steps of: original generation midbrain nerve stem cell separation culture, original generation cell subculture, cell induction differentiation, immune cell chemical dyeing and cell counting. In the method, the serum-free culture and the single-cell clone technology is adopted for separating the cells with the single-cell clone capability in the embryonic cortex, in addition, the culture and the transfer culture are carried out, the wall pasting differentiation observation is carried out, and the indirect immunofluorescence is adopted for detecting the expression of the Nestin antigen of the clone cells and the antigen of the differentiated specific mature nerve cells.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for in vitro isolation, culture and differentiation of human embryo midbrain 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 fe...

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Application Information

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IPC IPC(8): C12N5/0797C12N5/0793
Inventor 陆华
Owner 江苏迈健生物科技发展股份有限公司
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