Method for separating and subculturing primary neural stem cell of human embryo midbrain

A technology of primary cells and subculture, which is applied in the field of biomedicine and can solve the problem of less research on neural stem cells

Inactive Publication Date: 2013-04-03
陆华
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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

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  • Method for separating and subculturing primary neural stem cell of human embryo midbrain

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Embodiment Construction

[0013] Such as figure 1 Shown is a schematic flow chart of the steps of the method for the isolation and subculture of primary cells of human embryonic midbrain neural stem cells of the present invention, and the method is specifically realized by the following steps:

[0014] S1: Isolation and culture of primary midbrain neural stem cells: take fresh human embryos induced by water sac at 10-13 weeks of embryonic age, aseptically isolate the midbrain tissue of the embryos under a microscope, peel off the meninges and surface blood vessels, and put them in PBS ( Phosphate buffer solution) for three times, blow and beat the tissue fragments repeatedly with a fine pipette until they are completely dispersed, 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 1 :50 B27 (human leukocyte antigen), 20ng / ml EGF (epidermal growth factor), 20ng / ml bFGF (fibroblast growth factor) in DMEM / F12...

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Abstract

The invention provides a method for separating and subculturing the primary neural stem cell of the human embryo midbrain. The method is used for separating, culturing and differentiating the primary neural stem cell of the human embryo midbrain in vitro by separating, culturing and identifying the neural stem cell of the human embryo midbrain. The method comprises the following steps: separating and culturing the primary neural stem cell of the human embryo midbrain, subculturing the primary cell, carrying out induced differentiation on the cell, carrying out immunocytochemical staining, and counting the number of cells. According to the method, a neural stem cell is separated from human embryo midbrain successfully by adopting the serum-free culturing and single-cell cloning technology, and the neural stem cell is cultured, subcultured and subjected to adherent differentiation observation, wherein the cell has the continuous cloning capacity and can subculture and express the neural nidogen antigen. The differentiated cell can express the specific antigens of the neuron cells, the colloid cells and oligodendroglia cells.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a method for separating and subcultureing primary cells of human embryonic midbrain neural stem cells, which is applied to the in vitro isolation, culture and differentiation process of human embryonic 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/0793
Inventor 陆华
Owner 陆华
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