MSCs (Mesenchymal stem cells) sourced schwann cell trophoblast type axon growth method of sensory neurone

A technology of Schwann cells and neurons, applied in the field of cell culture, can solve the problems of complex induction system and cumbersome operation

Inactive Publication Date: 2018-01-05
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, its induction system is relatively complicated and the operation is cumbersome.

Method used

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  • MSCs (Mesenchymal stem cells) sourced schwann cell trophoblast type axon growth method of sensory neurone
  • MSCs (Mesenchymal stem cells) sourced schwann cell trophoblast type axon growth method of sensory neurone
  • MSCs (Mesenchymal stem cells) sourced schwann cell trophoblast type axon growth method of sensory neurone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Cultivation of MSCs and establishment of MSCs trophoblast

[0087] The umbilical cord of healthy full-term newborns was washed 3 times with normal saline to remove residual blood, aseptically dissected, peeled off the Wharton’s glue, and cut to 2-3mm 3 Suspended in 10mL MSCs medium, the MSCs medium was Ultra CULTURE containing 2% Ultroser G serum substitute.

[0088] Transfer to 25cm 2 Tissue culture flask (model: EasyFlask, manufacturer: NUNC), placed at 37 ℃, 5% CO 2 Cultivate in an incubator.

[0089] At the 14th day of incubation, aspirate the culture supernatant, wash the surface twice with physiological saline, add 5mL trypsin digestion solution, digest for 5 minutes at room temperature, stop the digestion with 1mL aprotinin solution, centrifuge at 400g, 5min, discard the supernatant, Harvest the sediment. The trypsin digestion solution contains 0.25% recombinant human trypsin and 0.08% EDTA.

[0090] The precipitate was washed twice with physiological saline,...

Embodiment 2

[0093] Example 2: Preparation of neuroepithelial pre-induced cells

[0094] Suspend P2 generation umbilical cord MSCs in MSCs medium, press 6000 / cm 2 Inoculate into recombinant human laminin and recombinant human vitronectin-coated 6-well plates (model: 174901, manufacturer: NUNC), culture to 60-70% confluence, change the neural differentiation medium, 37°C, 5 %CO 2 , Cultivate for 4 days under saturated humidity conditions.

[0095] Aspirate and discard the culture supernatant, wash the culture surface with PBS once, add 1 mL of 0.25% trypsin solution, digest for 5 minutes at room temperature, and add 100 uL of aprotinin solution to terminate the digestion.

[0096] Centrifuge at 400g for 5min, discard the supernatant, and harvest the pelleted cells, which are neuroepithelial pre-induced cells.

[0097] Among them, the neural differentiation medium is DMEM / F12 containing the following components:

[0098] 1×B27,

[0099] 10% non-animal-derived serum substitute,

[0100] 5nM human dihydr...

Embodiment 3

[0104] Example 3: Cultivation of SCs and establishment of SCs trophoblast

[0105] Resuspend the neuroepithelial pre-induced cells in SCs medium, press 2×10 4 / cm 2 Inoculate into a 6-well plate coated with recombinant human laminin, recombinant human fibronectin, and recombinant human regulatory protein-β1. Change the medium every other day. When 60-80% confluence is 8-12 days, aspirate and discard Wash the culture surface once with PBS, add 1mL AccutaseTM digestion solution, digest for 5 minutes at room temperature, add 200uL aprotinin solution to terminate the digestion; centrifuge at 400g for 5min, discard the supernatant, and harvest the pelleted cells, which are recorded as P0 generation SCs.

[0106] The P0 generation SCs were subcultured once. Resuspend the P1 generation SCs in SCs medium, press 2×10 4 / cm 2 Inoculate into a 96-well plate coated with recombinant human laminin, recombinant human fibronectin, and recombinant human modulin-β1, culture for up to 24 hours, and c...

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Abstract

The invention discloses an MSCs (Mesenchymal stem cells) sourced schwann cell trophoblast type axon growth method of sensory neurone. The method comprises the following steps: S1, culturing P2-generation umbilical cord MSCs with umbilical cord wharton jelly of a newborn through an MSCs culture medium; S2, suspending the P2-generation umbilical cord MSCs through the MSCs culture medium; inoculatingto a recombinant human laminin and recombinant human vitronectin coated cell culture plate for culturing until the confluence reaches 60-70%; and preparing neuroepithelium pre-induced cell through aneural differentiation culture medium; S3, culturing P0-generation SCs with the neuroepithelium pre-induced cell through an SCs culture medium; and performing a subculture until P1 generation is obtained; S4, preparing an SCs trophoblast through P1-geneation SCs; and S5, preparing DRG (Dorsal root ganglion) sensory neurone cells; and performing co-culture through the SCs trophoblast and the DRG sensory neurone cells. According to the method, a new ideal is provided for cell therapy on nerve injury and repair of central nervous system.

Description

Technical field [0001] The present invention relates to the technical field of cell culture, in particular to a method for MSCs-derived Schwann cells to nourish sensory neuron axon growth. Background technique [0002] When nerve injury occurs, it is very difficult to regenerate neurons, but the axon regeneration, growth and synaptic reconnection of residual neurons are the key to the recovery of nerve function. Nerve injury repair involves three stages: axon sprouting, regenerated axon growth and extension, and nerve function recovery. Previous studies have found that extracellular matrix molecules such as adhesion molecules of extracellular matrix, NGF, BDGF, NT-3, FGF, neurotransmitters, and bioactive phospholipids can regulate the formation, guidance, extension, branching, and turning of neuronal axons , Pause, retract, etc., affect the morphological structure of neurons. However, local injection of these components in the body has a good effect on injury repair, but the e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793C12N5/079
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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