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Culture method of oligodendrocyte precursor cells

A technology of precursor cells and culturing method is applied in the field of culturing oligodendrocyte precursor cells, which can solve the problems of difficulty in popularization, large physiological and psychological damage of donors, etc., and achieve the effect of reducing the cost of cultivation

Active Publication Date: 2017-11-21
北京再生生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, bone marrow collection is an invasive collection, which causes great physical and psychological damage to the donor, and is difficult to popularize

Method used

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  • Culture method of oligodendrocyte precursor cells
  • Culture method of oligodendrocyte precursor cells
  • Culture method of oligodendrocyte precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: UCB source culture and harvest MSCs

[0072] UCB is derived from UCB mononuclear cells frozen in cord blood banks.

[0073] Resuscitate UCB-MNCs, wash once with normal saline, resuspend cells with 25mL normal saline, carefully pour into 15mL lymphocyte separation medium, 800g, and centrifuge horizontally for 20 minutes; take the middle cloud layer, wash twice with normal saline; culture with MSCs Resuspend the cells and adjust the cell density to 2×10 5 / cm 2 , inoculated to 175cm 2 Culture in tissue culture flasks (product number: EasyFlask, manufacturer: NUNC), change the medium every other day, and harvest when it reaches 70-80% confluence; discard the culture supernatant when harvesting, wash the culture surface once with normal saline, and add 0.25% pancreatic Protease solution, digest at room temperature for 5 minutes, add 1mL aprotinin solution to stop the digestion; centrifuge at 400g for 5min, discard the supernatant, and harvest the precipitate; ...

Embodiment 2

[0075] Example 2: UCB-MSCs source cultured and harvested neurosphere cells

[0076] Resuspend P1 passage UCB-MSCs in neurosphere medium, adjust the cell density to 2×10 5 / mL, inoculated into bacterial culture dishes (product number: 4021, manufacturer: NUNC), 20mL per dish, 37°C, 5% CO 2 , cultured under saturated humidity conditions; add 10mL of new neurosphere medium every other day, continue to culture for 7 days, and subculture; when subculture, gently pipette the culture medium, centrifuge at 300g for 10 minutes, resuspend the pellet with neurosphere medium, and gently Pipette 10 times to make the neurospheres form 3-5 cell clusters or single cells, subculture in a 1:5 split plate; harvest P3 generation neurosphere cells.

[0077] Wherein, the neurosphere culture medium is neurobasal medium comprising the following substances:

[0078] 0.5 mM L-Glutamine;

[0079] 2% B-27™ Additive;

[0080] 20ng / mL recombinant human basic fibroblast growth factor;

[0081] 20ng / mL ...

Embodiment 3

[0082] Example 3: Cultivate and harvest OPCs

[0083] Suspend P3 neurosphere cells in neurosphere medium, adjust the cell density to about 2×10 5 / mL, inoculate 20mL cell suspension to recombinant human laminin, recombinant human vitronectin-coated 175cm 2 In tissue culture flasks (article number: EasyFlask, manufacturer: NUNC), add 20mL of LOPCs medium at the 24th hour of culture; at 72 hours of culture, replace all OPCs medium, continue to culture for 4 days, and passage. When subcultured, discard the culture supernatant, wash the culture surface once with normal saline, and add Accutase TM Digest 5 mL of the digestion solution at room temperature for 5 minutes, add 1 mL of aprotinin solution to terminate the digestion; centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend the pellet in OPCs medium and adjust the cell density to 1X10 4 / cm 2 Inoculated into tissue culture flasks coated with recombinant human laminin and recombinant human vitronectin, and ...

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Abstract

The invention discloses a culture method of oligodendrocyte precursor cells, which comprises the following steps of: S1, culturing mononuclear cells of cord blood in an MSCs (mesenchymal stem cells) culture medium to form P1 generation cord blood MSCs, S2, culturing the P1 generation cord blood MSCs in a neurosphere culture medium to form P3 generation neurosphere cells, and S3, culturing the P3 generation neurosphere cells in an OPCs (oligodendrocyte precursor cells) culture medium to form the OPCs. The method has the benefits that the culture method of the oligodendrocyte precursor cells adopts residual blood in obstetrical wastes, namely neonatal placentae and umbilical cords for culture to obtain the oligodendrocyte precursor cells; the traditional bone marrow MSCs are substituted for the preparation of the OPCs; and a new transplant cell resource is found for central nervous system injury repair. The culture method of the oligodendrocyte precursor cells adopts the P1 generation cord blood MSCs to induce neurosphere formation, and a neurosphere formation rate is up to 41%.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing oligodendrocyte precursor cells. Background technique [0002] The main function of oligodendrocytes (OCs) is to surround axons in the central nervous system to form an insulating myelin sheath structure, maintain and protect the normal physiological functions of neurons, and the myelin sheath formed by OCs is the most specialized And one of the complex animal membranes. [0003] Oligodendrocyte precursor cells (OPCs) differentiate in the central nervous system to produce oligodendrocytes to form myelin sheath structures, which are endogenous cells for the repair of myelin sheath damage. OPCs cell transplantation can improve the transmission of action potential and restore nerve function after spinal cord injury. [0004] However, the clinical collection of adult OPCs is extremely difficult, and finding more suitable sample resources is one of the ...

Claims

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Application Information

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IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2500/32C12N2500/38C12N2501/11C12N2501/115C12N2501/13C12N2501/135C12N2501/999C12N2506/1369C12N2533/52
Inventor 张洪钿苑春慧
Owner 北京再生生物科技研究院有限公司
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