Method for separation and primary culture of cerebral cortex neurons

Abstract

The invention belongs to the field of cell biology and relates to a method and reagent for the separation and culture of neurons. The method and the reagent are used for solving problems in the prior art, the current in-vitro hippocampal neuron separation and culture methods are improved, and the problem that the proliferation and activity of cortex nerve cells during primary culture can not be maintained is solved. According to the method and the reagent, a relatively mature in-vitro cortex nerve cell culture method is established, the number of obtained nerve cells is sufficient, the growth status is relatively good, and a large number of high-activity cortex nerve cells can be separated from mammalian hippocampal tissue, so that the requirements for the primary culture of the cortex nerve cells are met, and the demands on experiments of cell biology during neuroscience research can be met.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method and a reagent for the separation and primary culture of cerebral cortex cells. Background technique [0002] Neuron is the basic unit that constitutes the structure and function of the nervous system. It is a highly differentiated cell that rarely divides after birth in mammals. Therefore, it is very difficult to extract neurons. The primary culture of neurons is a method in which parts of the central nervous system of embryonic mammals, such as the cerebral cortex, cerebral cortex, cerebellum, hypothalamus, hippocampus, spinal cord and nerve plexus, are directly removed from the body, and then inoculated and cultured. At present, there are many methods for culturing neurons at home and abroad, but there are still some urgent problems to be solved in terms of the purity and yield of neurons in primary culture. Because neurons are highly differentiated cells that rarel...

AI Technical Summary

Problems solved by technology

[0003] At present, the factors that make it difficult to obtain sufficient quantity and vitality of primary cerebral cortex cells are as follows: (1) During the separation of cerebral cortex cell tissue, most people use D-hank's or hank's as a rinse solution, In the isolated mammalian cerebral cortex tissue, impurities such as red blood cells, capsule and connective tissue are removed. The separation process takes a long time, sometimes more than 2 hours, and the time in the rinsing solution will be longer. False-negative culture results

It is impossible to carry out routine culture of cerebral cortex cells in experiments, mainly because there is no suitable rinsing solution / dissecting solution for neuron culture in cerebral cortex tissue specimens

In the process of tissue separation, neurons still metabolize to a large extent even in the ice bath. The sugar-free environment of Hank's solution is not good for neuron separation. Add DMEM or high sugar to supply the brain in Hank's solution. Metabolism, but the concentration of glucose is too high, it is easy to increase the chance of bacterial contamination; adding DMEM medium will make it alkaline, which is not conducive to the survival of neuron cells

Chinese patent CN102978162A "neuron separation and culture method and reagent", Chinese patent CN102994452A "neuron separation and culture method with high efficiency" and Chinese patent CN102994451A "neuron separation and culture improved method", taken Put the brain tissue into a petri dish filled with 1×PBS, the cleaning solution used is PBS solution, DMEM-high glucose or DMEM-F12 and horse serum to soak the brain during dissection, to supply the metabolism of the brain, taking into account Needed for energy metabolism of neurons, but no antibacterial substances have been added. If the concentration of glucose is too high, it is easy to increase the chance of cell contamination

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