Integrated culture and purification of rat Schwann cells from freshly isolated adult tissue

Abstract

We describe a simple, rapid and highly selective protocol for the primary culture of Schwann cells in vitro from freshly dissociated adult rat nerve. The protocol is based on a selective culture medium comprising both mitogens (forskolin and optionally N2 supplement plus bovine pituitary extract), to stimulate growth of Schwann cells, plus an inhibitory substrate to simultaneously restrict fibroblast overgrowth (D-valine), contained in DMEM. This protocol differs from other available methods in that it uses the preferential capacity of Schwann cells to metabolize D-valine because of the difference in expression of a D-amino acid oxidase (DAAO) enzyme between Schwann cells and fibroblasts plus the presence of a selective mitogen to stimulate growth of Schwann cells. This permits derivation of highly pure Schwann cells directly from fresh adult nerve. Average Schwann cell purities of 97% can be achieved after 19 d without pre-degeneration, purification or antimitotic steps.

Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits

“…Primary Schwann cells were isolated from 10 to 12 week old male Wistar rats, killed according to the regulation of Animals (Scientific Procedure) Act 1986, using a schedule 1 method of cervical dislocation (Home Office, U.K.) The isolation, culture and purification methods followed were of those outlined by Kaewkhaw et al [28]. Briefly a 35 mm Petri dish coated with poly-L-lysine and laminin was initially used to culture the Schwann cells.…”

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves

“…Although the exact mechanism(s) responsible for this finding need(s) more investigation, the combination of above-mentioned components would enrich SCs in cultures in a way that over 99 % purity was obtained within 10 days of primary plating. The whole procedure, from sciatic nerve collection to desired enrichment, took 3 weeks which is comparable in simplicity of schedule and being user-friendly with respect to other available methods implying either traditionally in vitro/in vivo predegeneration (Mauritz et al 2004) or reports omitting predegeneration periods (Kaewkhaw et al 2012). In MTT assay, the stable tetrazolium salts are reduced to formazan dyes by dehydrogenases present in viable cells (Mosmann 1983).…”

“…Yet, several techniques have been introduced and implemented in different laboratories in order to separate SCs from contaminating FBs. These modifications include: repeated explantations method (Morrissey et al 1995), combination of antimitotic treatment and antibody-mediated cytolysis which employs the use of complement (Assouline et al 1983;Brockes et al 1979), serum tapering (Komiyama et al 2003), manipulating differential adhesion and detachment proprieties of SCs and FBs (Jin et al 2008;Niapour et al 2010), immunoselective methods through fluorescent activated cell sorting (FACS) or magnetically activated cell sorting (MACS; Vroemen and Weidner 2003), and exploiting specific biochemical diversity between SCs and EBs (Kaewkhaw et al 2012). Requiring multiple steps, technically demanding procedures and utilizing animal products for culture are the main bottlenecks of reported systems.…”

Immortalized Schwann cells IFRS1 as a new strategic tool for the study of myelination and demyelination

“…Because it is recognized that some biological properties of these cells change with maturation and aging (Horie et al, 1990;Mirsky et al, 2001) (Suzuki et al, 1999;Kaewkhaw et al, 2012), it needs a time-consuming process to obtain good In our recent study (Sango et al, 2011a), spontaneously immortalized et al, 1995, 2003).…”