A kind of in vitro culture and purification method of perineurial cells

A purification method and in vitro culture technology, applied in the field of in vitro culture and purification of perineurial cells, can solve the problems of large damage to perineurial cells, difficulty in moving out of perineurial cells, and multiple suspended debris, so as to promote migration The effects of export and proliferation, promotion of migration ability, and shortening of culture time

Active Publication Date: 2021-04-20
THE NAVAL MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the problem that the perineurium cells are difficult to move out, the inventor once cut the tissue pieces of the perineurium into pieces, and then used the enzyme repeated digestion method to process the tissue pieces (0.2% collagenase+0.05% trypsin mixed solution in saturated humidity , 5%CO 2 , in a 37°C incubator to process the shredded tissue pieces, and after 5 to 10 minutes, use a glass pipette to absorb the viscous perineurial tissue and blow it in a centrifuge tube to obtain a cell suspension), but this method is more harmful to the perineurial cells. Large, the cells are not attached to the wall under the microscope, and there are many suspended debris

Method used

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  • A kind of in vitro culture and purification method of perineurial cells
  • A kind of in vitro culture and purification method of perineurial cells
  • A kind of in vitro culture and purification method of perineurial cells

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Isolation, culture, purification and identification of perineurial cells

[0040] Steps: Take about 100g of SD rats, take the sciatic nerve, carefully peel off the epineurium under a dissecting microscope, cut the nerve bundle into about 1mm segments and cut them in half longitudinally, remove the nerve fibers from the cut surface as much as possible, and separate them to obtain Sheet-shaped perineurium, washed with pre-cooled sterile PBS, quickly placed in a cell culture plate, in saturated humidity, 5% CO 2 , Cultured in a 37°C incubator. Thereafter, the medium was changed every 3 days, and cultured for about 10 days. When many cells crawled out around the tissue block, they were passaged, observed under an inverted phase-contrast microscope, and their growth and proliferation were recorded.

[0041] The complete medium of perineurial cells was DMEM / F12, containing 10% fetal bovine serum, 2 μmol / L Forskolin (Sigma) and 12.5ng / mL Heregulin-β1 (Peprotech), a...

Embodiment 2

[0044] Example 2: Purification and identification of "time-limited digestion-differential attachment" of perineurial cells

[0045] 1. Removal of Schwann cells by time-limited digestion

[0046] Principle: When the cells grow to confluence, according to the difference in the reaction time of the cells to trypsin, the time-limited digestion method is used to separate Schwann cells from perineurial cells and fibroblasts. Schwann cells are sensitive to trypsin, which takes about 10 seconds, while it takes a long time for trypsin to digest perineurial cells and fibroblasts, which takes about 2-3 minutes.

[0047] Steps: Add 0.25% trypsin to the culture plate and stay for 10 seconds, then quickly add the complete medium of perineurial cells to stop digestion. At this time, the Schwann cells become round, shake the cell plate gently, the Schwann cells fall off, and the perineurial cells Cells and fibroblasts remain attached to the bottom of the plate.

[0048] 2. Initial Fibroblas...

Embodiment 3

[0052] Example 3: Purification and identification of "time-limited digestion-differential attachment-chemical drugs" of perineurial cells

[0053]Principle: Cytarabine is a pyrimidine anti-metabolic drug that affects DNA synthesis and replication by inhibiting DNA polymerase, and mainly acts on the S proliferation phase of cells. It inhibits the growth of rapidly proliferating cells, such as fibroblasts, while having little effect on slow proliferating cells, such as perineurial cells. According to the speed of fibroblast adhesion and division and proliferation, it usually begins to proliferate 24 hours after inoculation, which is earlier than the division and proliferation speed of perineurial cells. Therefore, choosing to add appropriate concentration of cytarabine to act on the remaining fibroblasts after re-seeding the cells for 24 hours can fully improve the purity of the perineurial cells, and has little effect on the growth state of the perineurial cells. .

[0054] s...

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Abstract

The present invention relates to the field of biomedicine, and specifically relates to a method for culturing and purifying perineurial cells in vitro. The steps are as follows: (a) take the sciatic nerve of SD rats, separate and obtain the perineurium, and culture it as a patch to obtain mixed growth nerves Perinecular cells, fibroblasts and Schwann cells; (b) first remove Schwann cells by time-limited digestion, and then initially remove fibroblasts by differential adhesion method; (c) repeat the above operation once; (d) Fibroblasts were further removed with the chemical drug cytarabine. The invention establishes a fast and efficient method for purifying and culturing perineurial cells in vitro, and provides a stable cell source for in-depth research on the mechanism of peripheral nerve injury repair.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an in vitro culture and purification method of perineurial cells. Background technique [0002] The research on the repair of peripheral nerve injury is relatively extensive, but the treatment of long-distance peripheral nerve defects urgently needs to be overcome. Peripheral nerves are composed of several nerve fiber bundles, the surface of the nerve is wrapped by the epineurium, and the surface of the nerve fiber bundle is covered by the perineurium; the surface of each nerve fiber in the nerve fiber bundle is surrounded by the endoneurium; the outer nerve Fibroblasts are distributed in membrane, perineurium and endoneurium, and Schwann cells form myelin sheath to surround axons. Perineurial Glial Plasticity and the Role of TGF-β in the Development of the Blood-Nerve Barrier. J Neurosci,2017, 37(18):4790- 4807.], divided into an outer layer and an inner layer, the outer l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079
CPCC12N5/0618C12N2500/40C12N2501/13C12N2501/70C12N2509/00C12N2509/10
Inventor 刘芳于皓杰许家军杨向群张志英蔺海燕
Owner THE NAVAL MEDICAL UNIV OF PLA
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