Isolation, purification and expansion of myelination‐competent, neonatal mouse Schwann cells

Abstract

Most studies of peripheral nerve myelination using culture models are performed with dorsal root ganglion neurons and Schwann cells pre-purified from the rat. The potential of this model is severely compromised by the lack of rat myelin mutants and the published protocols work poorly with mouse cells, for which numerous myelin mutants are available. This is partly due to difficulties in obtaining sufficient quantities of myelination-competent mouse Schwann cells. Here, we describe the isolation, purification and expansion of wild-type, myelination-competent Schwann cells from the sciatic nerves of 4-day-old mouse pups. The method consistently yields 1.9-3.3 x 10(6) of approximately 95% pure Schwann cells from the sciatic nerves of 12-15 4-day-old mouse pups, within 14-20 days. The Schwann cell proliferation rate ranges from 2.7- to 4.30-fold growth/week. Proliferation ceases within 4 weeks, when the cells become quiescent. Growth is reinduced by the presence of neurons; neuregulin is not sufficient for this effect. The Schwann cells isolated by this protocol are able to form compact myelin in culture, as judged by the segregated expression patterns of early (myelin-associated glycoprotein) and late (myelin basic protein) myelination markers in a three-dimensional neuron/Schwann cell coculture model. The Schwann cell batch yields are sufficient to perform 100-150 individual myelinating coculture assays. Employing mixed phenotype/genotype mouse neuron/Schwann cell cocultures, it will be possible to analyse the cell specificity of a mutation, and the cumulative effects of different mutations, without having to cross-breed the animals.

Progressive Postnatal Motoneuron Loss in Mice Lacking GDF-15

“…P5 mouse sciatic nerve was used as a source for primary Schwann cells and dissected and cultured as described (Honkanen et al, 2007). For immunocytochemistry, cultures were fixed in 4% PFA for 10 min and blocked in 10% BSA and 0.1% Triton X-100 in 1 × PBS for 1 hr.…”

Lysosomal exocytosis in Schwann cells contributes to axon remyelination

“…Primary cultures of Schwann cells were prepared as described previously (Honkanen et al, 2007). In brief, Schwann cells were prepared from sciatic nerves of 1-to 3-day-old Sprague-Dawley rats.…”

Enhanced Femoral Nerve Regeneration After Tubulization with a Tyrosine-Derived Polycarbonate Terpolymer: Effects of Protein Adsorption and Independence of Conduit Porosity

“…Embryonic spinal cord neurons were isolated and purified for a motor rich population. Schwann cells were isolated and purified from the sciatic nerves of P2 neonates according to published protocols, 47 yielding cultures with > 95% of cells staining positive for S100b. Cells were seeded onto coverslips (1.5 · 10 4 cells/coverslip) and maintained for 48 h at 37°C with 5% CO 2 .…”