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

• Published in: Nature protocols Vol. 7; no. 11; pp. 1996 - 2004
• Main Authors: Kaewkhaw, Rossukon, Scutt, Andy M, Haycock, John W
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2012; Nature Publishing Group
• Subjects: 13; 13/106; 13/12; 631/1647/1407/651; 631/378; Amino acids; Analytical Chemistry; Animals; Biological Techniques; Cell culture; Cell Culture Techniques; Cell separation; Computational Biology/Bioinformatics; Culture Media; Life Sciences; Male; Methods; Microarrays; Organic Chemistry; Physiological aspects; protocol; Rats; Rats, Wistar; Schwann cells; Schwann Cells - cytology; Schwann Cells - metabolism; Sciatic Nerve - cytology; Valine - metabolism; United Kingdom
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2012.118

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.