Protocol abstract
Nature Protocols 2, - 99 - 104 (2007)
Published online: 8 February 2007 | doi:10.1038/nprot.2006.486
Subject Categories: Cell and tissue culture | Isolation purification and separation
Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol
Kirsten Haastert1, Christina Mauritz1, Sukhada Chaturvedi1 & Claudia Grothe1
Abstract
We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90–95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.
- Department of Neuroanatomy, Center of Anatomy, OE 4140, Medical University Hannover, Carl-Neuberg-Strasse1, D-30625 Hannover, Germany and Center for Systems Neuroscience (ZSN) Hannover, Germany.
Correspondence to: Kirsten Haastert1 e-mail: haastert.kirsten@mh-hannover.de
nature-products
A-Z product listing
- 15 ml Falcon tubes(Becton Dickinson GmbH)
- 4 mm electroporation cuvettes(EquiBio)
- 4',6'-Diamidino-2-phenylindole (DAPI(Sigma-Aldrich Chemie GmbH)
- Aqua dest(Ampuwa Fresenius Kabi Deutschland GmbH)
- Basic nucleofector kit(Amaxa GmBH)
- Bovine pituitary extract (BPE-26(PromoCell GmbH)
