Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

Summary

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

Abstract

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were >90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Introduction

In the peripheral nervous system, the nerve fibers mainly consists of axons, Schwann cells (SCs), and fibroblasts, and also contains a small number of macrophages, microvascular endothelial cells, and immune cells¹. SCs wrap the axons in a 1:1 ratio and are enclosed by a connective tissue layer called the endoneurium. The axons are then bundled together to form groups called fascicles, and each fascicle is wrapped in a connective tissue layer known as the perineurium. Finally, the whole nerve fiber is wrapped in a layer of connective tissue, which is termed as the epineurium. In the endoneurium, the whole cell population is comprised of 48% SCs....

Protocol

This study was carried out in accordance with the Institutional Animal Care Guidelines of Nantong University. All the procedures including the animal subjects were ethically approved by the Administration Committee of Experimental Animals, Jiangsu Province, China.

1. Isolation and culture of motor and sensory nerve fibroblasts and SCs

1. Use seven-day-old Sprague-Dawley (SD) rats (n=4) provided by the Experimental Animal Center of Nantong University of China. Place the rats in a tank .......

Discussion

The two major cell populations of peripheral nerves included SCs and fibroblasts. The primarily cultured fibroblasts and SCs can accurately assist in modeling the physiology of fibroblasts and SCs during peripheral nerve development and regeneration. The study showed that P7 rat sciatic nerve cells contained about 85% of S100-positive SCs, 13% of OX7-positive fibroblasts and only 1.5% of OX42-positive macrophages¹³. Although the number of fibroblasts is less than SCs, the initial proliferation rat.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 594 Goat Anti-Mouse IgG(H+L)	Life Technologies	A11005	Dilution: 1:400
CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG(H+L)	Proteintech	SA00013-1	Dilution: 1:400
Confocal laser scanning microscope	Leica Microsystems	TCS SP5
Cell Quest software	Becton Dickinson Biosciences
D-Hank's balanced salt solution	Gibco	14170112
DMEM	Corning	10-013-CV
Dissecting microscope	Olympus	SZ2-ILST
Fetal bovine serum (FBS)	Gibco	10099-141C
Forskolin	Sigma	F6886-10MG
Fluoroshield Mounting Medium	Abcam	ab104135
Fixation medium/Permeabilization medium	Multi Sciences (LIANKE) Biotech, Co., LTD	GAS005
Flow cytometry	Becton Dickinson Biosciences	FACS Calibur
Mouse IgG1 kappa [MOPC-21] (FITC) - Isotype Control	Abcam	ab106163	Dilution: 1:400
Mouse monoclonal anti-CD90 antibody	Abcam	ab225	Dilution: 1:1000 for ICC, 0.1 µg for 106 cells for Flow Cyt
Mouse anti-S100 antibody	Abcam	ab212816	Dilution: 1:400
Polylysine (PLL)	Sigma	P4832
Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain Protein	R&D Systems	396-HB-050
0.25% (w/v) trypsin	Gibco	25200-072