Journal of Neuroscience Methods 99 (2000) 111 – 117 Expansion of adult Schwann cells from mouse predegenerated peripheral nerves Enrique Verdu ´ a , Francisco J. Rodrı ´guez a , Graciela Gudin ˜ o-Cabrera b , Manuel Nieto-Sampedro b , Xavier Navarro a, * a Department of Cell Biology, Physiology and Immunology, Neuroplasticity Group, Uniersitat Auto `noma de Barcelona, E-08193 Bellaterra, Spain b Neural Plasticity Group, Instituto Cajal, Madrid, Spain Received 4 January 2000; received in revised form 7 April 2000; accepted 7 April 2000 Abstract We present an effective technique for culture and expansion of Schwann cells (SC) from adult peripheral nerves. Cultures from adult mouse sciatic nerves (one to six nerves per culture) in defined medium showed markedly higher purity and density of SC when the nerve was predegenerated in vivo for 7 days than when it was harvested fresh. SC from degenerated nerves were then cultured in defined media conditioned by primary cultures of adult SC. The best results were obtained with a conditioned medium supplemented with 1% fetal calf serum. In these conditions the purity of SC was about 90% and the density about 190 cell/mm 2 by 7–10 days in vitro. These findings indicate that adult SC can be expanded from small preinjured nerve fragments in a short time period to provide a source of SC for autologous cellular transplants. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Schwann cell; Defined medium; Conditioned medium; Sciatic nerve; Nerve injury www.elsevier.com/locate/jneumeth 1. Introduction After severe nerve injuries, when loss of substance results in a significant gap between the nerve stumps, an autologous graft is usually employed to repair the gap. However, autograft repair implies several problems, such as the need of a second surgical step, elimination of the donor nerve function, a limited supply of donor nerves, and the mismatch between nerve and graft dimensions. Cellular prostheses constructed with a nerve guide seeded with a large population of trans- planted Schwann cells (SC) have been used experimen- tally to promote axonal regeneration, in attempts to design an alternative to autografts (Gue ´nard et al., 1992; Levi et al., 1997). The main shortcome of cellular prostheses for clinical or experimental applications is the immunological reaction promoted in the host by implanted foreign cells (Gue ´nard et al., 1992; Ro- drı ´guez et al., 2000). Successful nerve regeneration was reported when cellular prostheses containing human SC were transplanted in immune-deficient rats (Levi et al., 1994). Frequently, during surgical nerve repair, small nerve fragments are resected from the nerve stumps at the injury site. These nerve fragments might be a source of SC to construct autologous cellular prostheses. For this purpose efficient expansion of SC should be at- tained from small amounts of adult nerves within a short period of time. In this study we assessed options to improve the culture and expansion of SC from mouse adult nerves, using three types of culture media: serum-containing medium, serum-free medium (defined medium), and conditioned medium obtained from SC cultured in defined medium. 2. Materials and methods Sciatic nerves from adult (2–3 months old) female Swiss OF-1 mice under pentobarbital anesthesia (60 mg/kg, i.p.) were exposed and transected at the midthigh. Seven days later, the animals were re-anes- * Corresponding author. Tel.: +34-9-35811966; fax: +34-9- 35812986. E-mail address: xavier.navarro@uab.es (X. Navarro). 0165-0270/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0165-0270(00)00221-1