Method for separating and purifying oligodendrocyte precursor cells of cerebral cortex of tupaia belangeri in vitro

A technology of cerebral cortex and precursor cells, applied in cell dissociation methods, biochemical equipment and methods, animal cells, etc., can solve the problems of tree shrew OPCs culture methods and studies that have not yet been reported, and achieve the goal of avoiding growth and proliferation Interference, good stability, and high reliability effects

Active Publication Date: 2019-04-23
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many reports on the cultivation of rat and mouse OPCs at home and abroad, but there is no report on the cultivation method and research of tree shrew OPCs.

Method used

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  • Method for separating and purifying oligodendrocyte precursor cells of cerebral cortex of tupaia belangeri in vitro
  • Method for separating and purifying oligodendrocyte precursor cells of cerebral cortex of tupaia belangeri in vitro
  • Method for separating and purifying oligodendrocyte precursor cells of cerebral cortex of tupaia belangeri in vitro

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Embodiment 1

[0049] A method for separating and purifying tree shrew cerebral cortex oligodendrocyte precursor cells in vitro, comprising the following steps:

[0050] Step (1), oligodendrocyte precursor cell isolation:

[0051] Cut the cerebral cortex of the isolated brain of a newborn tree shrew within 48 hours into fuzzy shapes, then add 3mL 0.25% trypsin and 1mL 500U / mL DNase Ⅰ to digest, pipette into a cell suspension, and put it in a 37℃, 5% CO 2 Incubate in an incubator for 5 minutes; add 6 mL of mixed culture medium to stop digestion, pipette and mix well, filter through a 70-mesh cell sieve, centrifuge the filtrate, discard the supernatant, add mixed culture medium to the precipitate to resuspend the cell pellet, and inoculate the obtained cell suspension Place in a culture flask at 37°C, 5% CO 2 Cultivate in the incubator for 1 hour, suck out the cell suspension and inoculate it into a cell culture flask that has been coated with PDL beforehand, and then place it at 37°C, 5% CO...

Embodiment 2

[0064] A method for separating and purifying tree shrew cerebral cortex oligodendrocyte precursor cells in vitro, comprising the following steps:

[0065] Step (1), oligodendrocyte precursor cell isolation:

[0066] Cut the cerebral cortex of the isolated brain of a newborn tree shrew within 48 hours into fuzzy shapes, then add 3mL 0.25% trypsin and 1mL 1000U / mL DNase Ⅰ to digest, pipette into a cell suspension, and put it in a 37℃, 5% CO 2Incubate in an incubator for 10 minutes; add 6 mL of mixed culture medium to stop digestion, pipette and mix well, filter through a 70-mesh cell mesh, centrifuge the filtrate, discard the supernatant, add mixed culture medium to the precipitate to resuspend the cell pellet, and inoculate the obtained cell suspension Place in a culture flask at 37°C, 5% CO 2 Cultivate in the incubator for 2 hours, suck out the cell suspension and inoculate it into a cell culture flask that has been coated with PDL beforehand, and then place it at 37°C, 5% C...

Embodiment 3

[0080] A method for separating and purifying tree shrew cerebral cortex oligodendrocyte precursor cells in vitro, comprising the following steps:

[0081] Step (1), oligodendrocyte precursor cell isolation:

[0082] Cut the cerebral cortex of the isolated brain of a newborn tree shrew within 48 hours into fuzzy shapes, then add 3mL 0.25% trypsin and 1mL 700U / mL DNase Ⅰ to digest, pipette into a cell suspension, and put it in a 37℃, 5% CO 2 Incubate in an incubator for 8 minutes; add 6 mL of mixed culture medium to stop digestion, pipette and mix well, filter through a 70-mesh cell sieve, centrifuge the filtrate, discard the supernatant, add mixed culture medium to the pellet to resuspend the cell pellet, and inoculate the obtained cell suspension Place in a culture flask at 37°C, 5% CO 2 Cultivate in the incubator for 1.5h, suck out the cell suspension and inoculate it into a cell culture flask pre-coated with PDL, and then place it at 37°C, 5% CO 2 incubator cultivation; ...

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Abstract

The invention relates to a method for separating and purifying oligodendrocyte precursor cells of the cerebral cortex of tupaia belangeri in vitro, and belongs to the technical field of cell separation and purification. According to the method, trypsin and DNase I are combined to digest the cerebral cortex, and the OPCs of tupaia belangeri are well separated and purified by conducting differentialadherence purification twice. The method is simple in operation step, has the advantages of being quick in cell adherence, high in survival rate and the like, has the extremely high cell purity in asubsequent identification experiment and the high reliability of an identification result, can meet the requirements for relevant experimental research on tupaia belangeri in various research fields of medicine, biology, pharmacy and life science, and is suitable for application and popularization in general laboratories.

Description

technical field [0001] The invention belongs to the technical field of cell separation and purification, in particular to a method for the in vitro separation and purification of tree shrew cerebral cortex oligodendrocyte precursor cells. Background technique [0002] Oligodendrocytes (OL) are the only cells that form myelin in the central nervous system (CNS), originating from the neuroepithelial cells of the ventral neural tube of the embryo. Its main function is to produce myelin to surround the axons and maintain the jumping conduction of nerve impulses along the axons. In addition, it can also secrete a variety of neurotrophic factors to support the growth, development and survival of neurons and glial cells. During the development process of oligodendrocytes (OL), they roughly experienced oligodendrocyte progenitor cells, oligodendrocyte precursor cells (OPC), immature oligodendrocytes (immature oligodendrocyte) and mature Oligodendrocytes (mature oligodendrocyte) ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2501/395C12N2501/33C12N2501/73C12N2509/00C12N2533/32C12N2501/13C12N2501/115
Inventor 孙晓梅李明学陆彩霞代解杰仝品芬黎晓慧王璇袁圆
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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