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Method for separating and purifying schwann cells

A technology of Schwann cells and cells, which is applied in the field of cell biology, can solve the problems of cumbersome steps, expensive reagents, and difficulty in peeling off the outer membrane, and achieve obvious effects, simple steps, and economic benefits

Inactive Publication Date: 2011-04-20
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nerves from newborn suckling mice and embryonic mice within 3-5 days are small, tender, brittle and easy to break, and it is difficult to peel off the outer membrane, especially the perineurium.
However, due to the non-specific effect of complement can damage cells, and the reagent is expensive, it is not suitable for the cultivation of a large number of cells. In addition, there are immunomagnetic beads, flow cytometry, etc., but the above methods are common, time-consuming and laborious. , the steps are cumbersome or require large-scale equipment and the purification effect is not ideal, etc.

Method used

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  • Method for separating and purifying schwann cells
  • Method for separating and purifying schwann cells
  • Method for separating and purifying schwann cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Separation and Purification of Schwann Cells

[0038] Experimental materials: 15ml centrifuge tube, SD mouse, 50ml culture bottle, compound collagenase, dispaseII rabbit anti-mouse polyclonal S100 antibody (primary antibody A), rabbit anti-mouse P75 antibody (primary antibody), goat anti-rabbit PE-labeled antibody (secondary antibody) Anti) etc. Unless otherwise specified, the reagents of the present invention were all purchased from Sigma Company.

[0039] Newborn 6-day-old SD mice were killed by dislocation, put into 75% alcohol, and soaked for 5-10 minutes. Under sterile conditions, the bilateral sciatic nerves with a length of about 1 cm were harvested.

[0040] (1) Under aseptic conditions, the nerve segments of humans or animals are taken, and the epineurium is stripped and removed under a microscope.

[0041] (2) Digest the nerve bundles with a DMEM solution of 0.1-0.2% complex collagenase NB4 and 0.05-0.1% dispase mixed enzyme in a mass volume ratio...

Embodiment 2

[0051] Embodiment 2 Two kinds of digestive enzyme system digestion results comparison

[0052] experimental method:

[0053] 1) Take primary cells

[0054] 1 Six newborn 6-day-old C57BL6 mice were killed by dislocation, put in 75% alcohol and soaked for 5-10 minutes

[0055] 2 Under sterile conditions, take bilateral sciatic nerves with a length of about 1 cm

[0056] 3. Put 12 segments of nerves into 15ml centrifuge tube A containing 3ml 2% compound collagenase NB4 and 15ml centrifuge tube B containing 2ml 2% compound collagenase NB4 and 1ml 0.1% dispaseII respectively.

[0057] 4 Put it in a 37°C incubator containing 5% CO2, shake it every five minutes, and digest for about 1.5 hours.

[0058] 5 Centrifuge tube A and tube B at 600g for 5 minutes, collect cells, count and plant them into culture flasks, 0.5min per bottle.

[0059] 2) Purification of Schwann cells

[0060] After the primary cells were cultured for 48 hours, the cells were close to confluence: then the obt...

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Abstract

The invention belongs to the field of cell biology and relates to a method for separating and purifying schwann cells. The method sequentially comprises the following steps of: firstly, taking a nerve segment under the aseptic condition and removing an epineurium; secondly, digesting a nerve tract by using a NB4 composite collagenase and dispase mixed digestive enzyme solution and discarding the solution; thirdly, placing the mixture in a trypsin and an ethylene diamine tetraacetic acid (EDTA) for 5 to 10 minutes to obtain a nerve fiber from which a perineurium is removed; fourthly, placing the obtained nerve fiber in the mixed enzyme solution, and oscillating the mixture to obtain a cell suspension; and finally, performing centrifugation and heavy suspension. By using the method, a greatquantity of high-purity schwann cells are obtained in a low-cost and high-efficiency mode so that the abundant and high-quality schwann cells are supplied for repairing the damaged nerve.

Description

technical field [0001] The invention belongs to the field of cell biology and relates to a method for separating and purifying Schwann cells. Background technique [0002] Schwann cells (Schwann cells, SCs) are an important type of glial cells in the peripheral nervous system, first discovered by Schwann (1939), and play an important role in the myelination of myelinated nerve fibers and the repair of damaged nerves. It is also an important source of neurotrophic factors and can secrete nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), ciliary nerve growth factor (CNTF), fibroblast growth factor (FGF), etc. The axons of the injured central nervous system in adult mammals lack regeneration ability, but after peripheral nerve injury, the axon regeneration ability is relatively strong. regeneration. Further studies have shown that after peripheral nerves are transplanted to the central nervous system, axons can re-grow into the peripheral nerves, and the ke...

Claims

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Application Information

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IPC IPC(8): C12N5/06
Inventor 祝加学沈尊理秦金保沈华张兆峰
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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