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Method for culturing sciatic nerve Schwann cells

A technology of Schwann cells and sciatic nerves, applied in the field of cell biology, can solve the problems of difficult time control, unsatisfactory purity, and delayed treatment timing, etc., and achieve the effect of broad application prospects

Inactive Publication Date: 2013-01-30
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Some studies have obtained a large number of SCs with strong proliferative ability through in vivo pre-denaturation (Waller denaturation) for one week and then cultured in vitro (Keilhoff et al. in 1999), which confirmed that a large number of SCs with strong proliferative ability can be obtained through pre-denaturation and then cell culture; However, the in vivo pre-denaturation requires two operations, which obviously takes a long time, so that the timing of treatment is delayed; at the same time, due to individual differences, it is difficult to control the time point at which the in vivo pre-denaturation achieves the desired effect. App is restricted
Therefore, some studies have taken out the sciatic nerves of adult mice and placed them in in vitro culture medium (DMEM+10% FBS) to pre-culture for a period of time to simulate the microenvironment in the body, and obtain Schwann cells SC after culture, but their purity is not ideal.
[0005] It is well known in the art that as the seed cells of tissue engineered artificial nerves, high purity is required, and there is no report on the method of obtaining high-purity Schwann cells

Method used

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  • Method for culturing sciatic nerve Schwann cells
  • Method for culturing sciatic nerve Schwann cells
  • Method for culturing sciatic nerve Schwann cells

Examples

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Effect test

Embodiment 1

[0052] Example 1 Cell culture under the sciatic nerve without pre-degeneration

[0053] Experimental materials: 15ml centrifuge tube, C57 mouse, 50ml culture bottle, compound collagenase, dispasell rabbit anti-mouse polyclonal S100 antibody (primary antibody A), rabbit anti-mouse P75 NTR Antibody (primary antibody), goat anti-rabbit PE-labeled antibody (secondary antibody), etc. Unless otherwise specified, the reagents of the present invention were all purchased from Sigma Company.

[0054] The bilateral sciatic nerves of adult C57 mice were obtained under routine sterile conditions.

[0055] (1) Mince the obtained sciatic nerve, and digest the nerve fragments with 0.2% collagenase NB4 and 0.2% dispase mixed enzyme DMEM solution with a mass volume ratio of 3-20 times the tissue amount for 60-90 minutes;

[0056] (2) repeatedly blowing the suspension obtained in step (1) with a straw for 3 to 5 minutes;

[0057] (3) The cells dissociated in step (2) were centrifuged at 600 g...

Embodiment 2

[0062] The pre-denatured subsciatic nerve cell culture in embodiment 2DMEM+10%FBS

[0063] Experimental materials: 15ml centrifuge tube, C57 mouse, 50ml culture bottle, compound collagenase, dispasell rabbit anti-mouse polyclonal S100 antibody (primary antibody A), rabbit anti-mouse P75 NTR Antibody (primary antibody), goat anti-rabbit PE-labeled antibody (secondary antibody), etc. Unless otherwise specified, the reagents of the present invention were all purchased from Sigma Company.

[0064] The bilateral sciatic nerves of adult C57 mice were obtained under routine sterile conditions.

[0065] (1) Place the obtained nerve segments in DMEM+10% FBS, 5% CO2, in a 37°C incubator, and incubate for one week to undergo pre-denaturation;

[0066] (2) Change the liquid every 3 days;

[0067] (3) Shred the obtained sciatic nerve, and digest the nerve fragments with 0.2% collagenase NB4 and 0.2% dispase mixed enzyme DMEM solution with a mass volume ratio of 3-20 times the tissue amo...

Embodiment 3

[0074] Subsciatic nerve cell culture of pre-denaturation in embodiment 3 SCCM

[0075] Experimental materials: 15ml centrifuge tube, C57 mouse, 50ml culture bottle, compound collagenase, dispasell rabbit anti-mouse polyclonal S100 antibody (primary antibody A), rabbit anti-mouse P75 NTR Antibody (primary antibody), goat anti-rabbit PE-labeled antibody (secondary antibody), etc. Unless otherwise specified, the reagents of the present invention were all purchased from Sigma Company.

[0076] The bilateral sciatic nerves of adult C57 mice were obtained under routine sterile conditions.

[0077] (1) Place the obtained nerve segment in SCCM, 5% CO2, in a 37°C incubator, and incubate for one week to undergo pre-denaturation;

[0078] (2) Change the liquid every 3 days;

[0079] (3) Shred the sciatic nerve, and digest the nerve fragments with 0.2% collagenase NB4 and 0.2% dispase mixed enzyme DMEM solution with a mass volume ratio of 3-20 times the tissue amount for 60-90 minutes;...

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Abstract

The invention belongs to the field of cell biology, and relates to a method for culturing sciatic nerve Schwann cells of adult mouse. The invention also comprises a method for acquiring Schwann cells with high purity through a purification process. The obtained Schwann cells with purity reaching 91% are purified to reach a purity of 98%. Results show that in vitro predegeneration promotes proliferation of the Schwann cells and inhibits fibroblast proliferation, so as to obtain Schwann cells with high purity. The method provided by the invention employs steps of culture and purification of in vitro predegeneration sciatic nerve to simply and effectively obtain a large amount of Schwann cells with high purity, so as to provide abundant high-quality Schwann cells for restoration of injured nerve, and the Schwann cells can be used as seed cells in tissue engineering.

Description

technical field [0001] The invention belongs to the field of cell biology and relates to a method for cultivating sciatic nerve Schwann cells, in particular to a method for cultivating and purifying Schwann cells through in vitro pre-denatured sciatic nerve. Background technique [0002] Studies have reported that the development of tissue-engineered artificial nerves provides a new approach for the repair of peripheral nerve injuries, in which Schwann cells (Schwann cells, SCs) are used as seed cells for tissue-engineered artificial nerves to play a role in the repair of peripheral nerve injuries played an important role in. The existence of the Schwann cells provides a good microenvironment for the regeneration of nerve axons, which guides the axons to grow distally by secreting corresponding nerve growth factors and extracellular matrix; but since SC is a terminal cell, The Schwann cell culture is directly obtained, the cells are difficult to adhere to the wall, the expa...

Claims

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Application Information

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IPC IPC(8): C12N5/079A61L27/38
Inventor 王晓盼沈尊理秦金保金羽青
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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