Culture method for Schwann cells

A technology for Schwann cells and culture methods, applied in the directions of cell culture active agents, cell culture supports/coatings, animal cells, etc., which can solve problems such as the limitation of collection volume, improve yield and purity, promote adherence and Effects of migration, prevention of agglomeration and multilayer growth

Active Publication Date: 2017-12-26
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, these tissue samples need to be collected invasively, and the amount of collection is also limited.

Method used

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  • Culture method for Schwann cells
  • Culture method for Schwann cells
  • Culture method for Schwann cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: UCB source culture and harvest MSCs

[0067] UCB is derived from UCB mononuclear cells frozen in cord blood banks.

[0068] 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

[0070] Example 2: UCB-MSCs source culture and harvest neurospheres

[0071] 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 neurospheres.

[0072] Wherein, the neurosphere culture medium is neurobasal medium comprising the following substances:

[0073] 0.5 mM L-Glutamine;

[0074] 2% B-27™ Additive;

[0075] 20ng / mL recombinant human basic fibroblast growth factor;

[0076] 20ng / mL recombinant h...

Embodiment 3

[0077] Example 3: culturing and harvesting Schwann cells

[0078] Suspend P3 generation neurospheres with Schwann cell culture medium, inoculate them into tissue culture flasks coated with recombinant human laminin, recombinant human fibronectin, and recombinant human heregulin-β1, change the medium every other day, 8-12 When 60-80% confluence, discard the culture supernatant, wash the culture surface once with PBS, add 1mL of AccutaseTM digestion solution, digest at room temperature for 5 minutes, add 200uL aprotinin solution to stop the digestion; centrifuge at 400g for 5min, discard the supernatant, The precipitated cells were harvested and recorded as Schwann cells derived from UCB at generation P0. P0 generation Schwann cells were subcultured once.

[0079] Wherein, the Schwann cell culture medium is neurobasal medium comprising the following substances:

[0080] 0.5 mM L-Glutamine,

[0081] 2% B-27™ Additive,

[0082] 10% Animal Component Free Serum Replacement,

[...

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PUM

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Abstract

The invention discloses a culture method for Schwann cells. The culture method comprises the following steps: S1, culturing mononuclear cells in umbilical cord blood by virtue of an MSCs culture medium to obtain P1 generation umbilical cord blood MSCs; S2, culturing the P1 generation umbilical cord blood MSCs by virtue of a neural sphere culture medium, and harvesting P3 generation neural spheres; and S3, culturing the P3 generation neural spheres by virtue of a Schwann cell culture medium, so that the Schwann cells are obtained. The culture method disclosed by the invention has the beneficial effects that the Schwann cells sourced from umbilical cord blood MSCs are cultured by virtue of a nestin+ neural sphere stage; and formation of the neural spheres is induced by adopting the P1 generation umbilical cord blood MSCs, and neural sphere formation rate reaches up to 41%. The invention also provides a specific neural sphere culture medium, the umbilical cord blood MSCs can be sufficiently induced to be formed into nestin+ neural spheres; and a specific culture scheme that nestin+ neural precursor cells are differentiated into the Schwann cells is provided, adherence and migration of the nestin+ neural precursor cells can be obviously promoted, and agglomeration and multilayer growth in a cell proliferation process are prevented.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing Schwann cells. Background technique [0002] Schwann cells (Scs) are glial cells in the peripheral nervous system and have various physiological functions. When peripheral nerves are injured, Schwann cells undergo morphological changes, secrete growth factors, build axon growth microenvironment, and guide axon regeneration. As early as 1995, Gulati et al. found that transplanting Schwann cells and scaffold components could promote axon regeneration, but sufficient Schwann cells were required to provide neurotrophic substances and chemokines. The application of Schwann cells or Schwann cell-based tissue engineering products in nerve injury repair and neurodegenerative diseases has been paid attention to. During the 90 years from 1905 to 1995, many researchers studied how to obtain enough Schwann cells for transplantation, and verified the methods of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2500/32C12N2501/01C12N2501/11C12N2501/115C12N2501/13C12N2501/135C12N2501/392C12N2506/1369C12N2533/50C12N2533/52
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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