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Method for extracting and culturing embryonic neural stem cells in vitro, and preparation of culture medium

A technology of neural stem cells and complete medium, applied in the field of extracting and culturing embryonic neural stem cells in vitro, can solve the problems of inability to optimize the biological characteristics of embryonic neural stem cells, unfavorable cell culture and experimental research, and poor viability of embryonic neural stem cells. Effects of promoting in vitro viability and proliferation, reducing the probability of contamination, and maintaining pluripotent differentiation potential

Active Publication Date: 2019-12-31
QINGDAO MUNICIPAL HOSPITAL
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, how to effectively extract a large number of high-purity and high-viability embryonic neural stem cells and culture them in vitro to maintain their cell viability and pluripotent differentiation potential has always been a difficult problem
[0003] At present, the existing technology for extracting neural stem cells mostly extracts neural stem cells from the hippocampal region of young rat brains, which can lead to a large number of other cells and non-cellular components such as blood vessels and meninges mixed in the extracted neural stem cells, resulting in low purity of the extracted neural stem cells. It is not conducive to later cell culture and experimental research; in addition, most of the commonly used culture media for in vitro culture of neural stem cells are commercial media, which cannot be optimized for the cellular biological characteristics of embryonic neural stem cells, resulting in embryonic neural stem cells cultured in vitro Poor viability, slow cell proliferation, differentiation and even massive death

Method used

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  • Method for extracting and culturing embryonic neural stem cells in vitro, and preparation of culture medium
  • Method for extracting and culturing embryonic neural stem cells in vitro, and preparation of culture medium
  • Method for extracting and culturing embryonic neural stem cells in vitro, and preparation of culture medium

Examples

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Effect test

Embodiment 1

[0021] Example 1 Extraction of embryonic neural stem cells and preparation of medium

[0022] Take out the newborn rats within 24 hours, kill them by decapitation, quickly immerse them in 75v / v% ethanol for disinfection, rinse with 0.9w / v% sodium chloride solution, peel off the skin, expose the spine, and use sterile scissors on the upper cervical and sacral vertebrae. Cut off the spine and take out the cadre of the spine; use the "toothpaste squeeze" method to squeeze out the spinal cord in the spine with wide-mouth tweezers, put it in 0.9w / v% sodium chloride solution, peel off the meninges under the microscope, and cut the spinal cord into 1mm 3 After filtering, put it into a centrifuge tube; add 0.25w / v% trypsin at a volume ratio of 1:1 and digest for 20 minutes while pipetting, add 10mL of DMEM / F12 medium to stop digestion, and filter with a cell strainer Centrifuge, discard the supernatant, and collect the cells. Prepare complete culture medium for embryonic stem cells, ...

Embodiment 2

[0023] Example 2 Cell Culture

[0024] The culture medium prepared above was added to the collected cells for culturing. cells in 3 x 10 9 The density is planted at 75cm 2 Add 3 mL of complete embryonic neural stem cell culture medium to a cell culture dish, and incubate at 37°C with 5% CO 2 cultured in a cell culture incubator. After 3 days of culture, a large number of embryonic neural stem cells can be seen, and some cells aggregate into balls and proliferate rapidly ( figure 1 ), after 7 days of culture, a large number of embryonic neural stem cells can be seen to aggregate into suspended balls, proliferate rapidly, and the cell shape has not changed, which conforms to the biological characteristics of embryonic neural stem cells, undifferentiated ( figure 2 ).

[0025] The CCK8 cell proliferation kit was used to detect the cell proliferation rate after 3 days, 6 days, 9 days and 12 days of cell culture, and it was found that the proliferation rate of the embryonic n...

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Abstract

The invention discloses a method for extracting and culturing embryonic neural stem cells in vitro. The method comprises the following steps: newborn mice born within 24 hours are taken, after being killed in a cervical dislocation mode, the newborn mice are soaked into ethyl alcohol to be disinfected, flushing is conducted with a sodium chloride solution, spines are shorn at upper cervical spinesand sacral vertebrae through sterile scissors, and the spine trunks are taken out; spinal cord in the spines are extruded out through wide-mouth tweezers and put into a sodium chloride solution, spinal membranes are stripped under a microscope, and the spinal cord is filtered and then put into a centrifuge tube; and trypsin is added for digestion while blowing and beating, a DMEM / F12 culture medium is added for stopping digestion, after filtering through a cell filter screen, centrifuging is conducted to collect the cells, and an embryonic neural stem cell complete culture medium is added forculture. The formula of the embryonic neural stem cell complete culture medium comprises DMEM / F12, glutamine, lysine, a B-27 additive, an N2 additive, penicillin, streptomycin, bFGF, EGF, insulin anda small molecule inhibitor CHIR99021.

Description

technical field [0001] The invention belongs to the technical field of embryonic neural stem cells, and in particular relates to a method for extracting and culturing embryonic neural stem cells in vitro and preparation of a culture medium. Background technique [0002] Embryonic neural stem cells are considered as a new technology for repairing nervous system damage because of their pluripotent differentiation potential. However, how to effectively extract a large number of high-purity and high-viability embryonic neural stem cells and culture them in vitro to maintain their cell viability and pluripotent differentiation potential has always been a difficult problem. [0003] At present, the existing technology for extracting neural stem cells mostly extracts neural stem cells from the hippocampal region of young rat brains, which can lead to the extraction of neural stem cells mixed with a large number of other cells and non-cellular components such as blood vessels and me...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/0797
CPCC12N5/0603C12N5/0623C12N2500/32C12N2501/11C12N2501/115C12N2501/33C12N2501/999
Inventor 范筱张英羽
Owner QINGDAO MUNICIPAL HOSPITAL
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