An improved method for isolating Schwann cells from postnatal rat sciatic nerves

Abstract

The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-B-arabinoside hydrochloride (Ara-C) action and differential cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-beta1 extracellular domain (HRG1-beta1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro.

A nanofibrous PHBV tube with Schwann cell as artificial nerve graft contributing to Rat sciatic nerve regeneration across a 30-mm defect bridge

“…Schwann cells (SCs) were obtained from sciatic nerves of adult wistar rats according to methods described earlier (Wei et al, 2009). In brief, the nerve segments were washed in DMEM/F-12 twice, then re-suspended in 0.3% collagenase type II solution (100 μ l per segment) and incubated for 30 min at 37 ° C. After incubation, the enzymatic solution was carefully removed, and an equal volume of 0.25% trypsin-EDTA was added.…”

Behavioral evaluation of regenerated rat sciatic nerve by a nanofibrous PHBV conduit filled with Schwann cells as artificial nerve graft

“…SCs were obtained from sciatic nerves of 8 day old rats according to earlier described methods (Ishikawa et al, 2009;Wei et al, 2009). In brief, the nerve segments were washed in DMEM/F-12 twice, then re-suspended in 0.3% collagenase Type II solution (100 μ l per segment) and incubated for 30 min at 37 ° C. After incubation, the enzymatic solution was carefully removed, and an equal volume of 0.25% trypsin -EDTA was added.…”

Design of Oriented Porous PHBV Scaffold as a Neural Guide

“…Schwann cells were obtained from sciatic nerves of 8-day-old rats according to previously described methods [32]. In brief: The nerve segments were washed in DMEM=F-12 twice, then re-suspended in 0.3% collagenase type II solution (100 mL per segment), and incubated for 30 min at 37 C. After incubation, the enzymatic solution was carefully removed, and an equal volume of 0.25% trypsin-EDTA was added.…”