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Astrocyte separating and cultivating method

An astrocyte and culture method technology, which is applied in the field of in vitro separation and culture of astrocytes, can solve the problems of few passages, cell contamination, difficult separation of fibroblast hybrid cells, etc., and achieves short operation time, Low cost of reagents and the effect of promoting cell adhesion

Inactive Publication Date: 2012-10-03
黄柏胜
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Problems solved by technology

[0003] At present, there is no patent literature on the method of in vitro isolation or culture of astrocytes. Although astrocytes can achieve the purpose of culture to a certain extent by using conventional cell culture techniques, there are serious cell pollution and the number of passages less, fibroblasts and other miscellaneous cells are difficult to separate, and cannot meet the requirements of cell experiments

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Examples

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Embodiment 1

[0025] Example 1: Isolation and culture of rat astrocytes

[0026] Experimental animals: SD1-2 newborn 1-3 days old (provided by the Experimental Animal Center of Xiangya School of Medicine, Central South University), male or female.

[0027] Main reagents and instruments: DMEM (Hyclone Company), trypsin (Ameresco Company), newborn bovine serum (Hangzhou Sijiqing Company), rabbit anti-GFAP polyclonal antibody (Sigma Company), goat anti-rabbit IgG kit (Vector Company), D-Hank's solution (homemade according to recipe), rat tail collagen (homemade), CO 2 Incubator (FormaScientific Company), inverted microscope (Olympus Company).

[0028]Primary culture: take newborn SD suckling mice 1 to 3 days old, soak the skin in 75% ethanol for 5 to 10 minutes to disinfect the skin, decapitate, carefully cut the skull with ophthalmic scissors under sterile conditions, and then take out both sides with ophthalmic forceps Place the cerebral cortex in a petri dish filled with D-Hank's solution...

Embodiment 2

[0036] Example 2: Isolation and culture of astrocytes from other animals

[0037] Experiments have proved that this method can be used for the cultivation of relatively fragile tissues such as fetal rats, newborn (1-3 days after birth) rats, mice, and guinea pigs, especially astrocytes in the cerebral cortex, and the experimental protocol is the same as above. But for adult humans, it is difficult to achieve the goal with this method because the cerebral cortex tissue is relatively tough. However, experiments have shown that the same effect can be achieved by using this method to obtain the cerebral cortex tissue of induced fetuses. The gestational age of 5 months is the most suitable, and the experimental results will be affected if the gestational age is too small or too large. The purity of the astrocytes isolated and cultured by the method is over 95%.

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Abstract

The invention relates to an astrocyte separating and cultivating method, which is appropriate for various experimental animals and human. The method mainly aims at the problems in the prior art, and improves the method for separating and cultivating astrocyte in vitro at present. The method comprises five experimental steps: cerebral cortex tissue separation, filtering by a seive after tissue cell trituration, cell primary culture, continuous cell culture, and astrocyte identification. The whole method is simple and practicable. Purity of the astrocyte cultivated and obtained by the method reaches more than 95% and the astrocyte grows well, is rapid in cell proliferation, and can be continuously cultured for many times and meets the demand of various experiments of cytobiology.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a method for separating and culturing astrocytes in vitro. Background technique [0002] In the central nervous system, the number of glial cells accounts for almost 90%, and astrocytes are the largest type of glial cells, and together with oligodendrocytes, they are called macroglia. Traditional studies mostly focus on neurons, thinking that astrocytes only provide nutritional support for neurons, but do not play a role in the transmission and processing of nerve signals. It has been confirmed that astrocytes play a role in the central nervous system. It also plays an important role in the development and pathophysiological process of the brain. It also has the functions of uptake, inactivation and supply of neurotransmitters. Understanding the function of astrocytes. With the understanding of its function, it has gradually become a research hotspot in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
Inventor 黄柏胜邬力祥刘发益
Owner 黄柏胜
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