CONICET | Buscador de Institutos y Recursos Humanos

Available methods to procure large populations of purified adult nerve-derived Schwann cells (SCs) involve an in vitro degeneration step of the nerve explants prior to enzymatic dissociation. By allowing axonal and myelin breakdown, the predegeneration step both facilitates the fast removal of myelin debri and allows for an enrichment of the SC population over that of contaminating cells. Still, this method requires 3 to 5 weeks after tissue harvesting to obtain sufficient yields of purified SCs. Here, we describe an optimized cell culture protocol for the fast isolation, purification and expansion of SCs from adult rat nerves. The need of a predegeneration phase is overcome by implementing an immediate dissociation of the nerve tissue and the use of MACS microbead technology for the removal of contaminating cells. The same methodology can be adapted for the fast isolation and purification of SCs from early postnatal nerves. Essentially, the nerve tissue is subjected to dissociation with a combination of collagenase and dispase immediately after harvesting, removal of myelin debri by selective adhesion of the cells to a laminin substrate and expansion of the SC population in medium containing neuregulin and forskolin, a combination of mitogens that favors the proliferation of SCs over that of fibroblasts. We achieved effective and fast fibroblast elimination by indirect labeling with magnetic microbeads and Thy.1.1 antibodies followed by double passing through MACS columns. Even though the purification step could be implemented essentially at any time during the expansion phase, cell yields were maximized if fibroblast removal was delayed to at least 1 week after initial plating. Characterization of the cell products rendered high efficiency of purification (>95%) of S100 positive, GFAP positive, O4 positive SCs that attached to and proliferated in response to contact with sensory neurites. To conclude, adult and postnatal rat SCs obtained by this fast and easy to implement method retained their phenotypic and functional capabilities.