Isolation and characterization of neonatal Schwann cells from cryopreserved rat sciatic nerves

Much of our knowledge about the development and maintenance of the peripheral nervous system has been learned through studying the interaction of neurons, or their isolated membranes, with Schwann cells (SC), in tissue culture. Numerous approaches have been employed to obtain an adequate quantity of SC, but all have been limited by either the uncertainty of obtaining a sufficient amount of starting material, the time and expertise required to isolate the SC, or by the limited number of SC that can be generated. We have developed a procedure to isolate SC from cryopreserved sciatic nerves. This procedure allows for sciatic nerves to be pooled until adequate numbers of nerves are obtained, yet still produces cells that retain the functional abilities of SC isolated from fresh nerves. Sciatic nerves were isolated from 2 day old rat pups, placed in either DME media and used fresh or placed in a freezing solution containing DME media (25%), DMSO (25%), fetal calf serum (50%), frozen at -70 degrees C and stored in liquid nitrogen. The frozen nerves were rapidly thawed to 37 degrees C and single cells were prepared from both fresh and frozen nerves using enzymatic and mechanical disruption as previously described (Brockes et al., Brain Res 165: 105-118, 1979). Comparable cell yields were obtained for SC isolated from both frozen and fresh nerves. Immunohistochemical staining of both fresh and frozen SC produced similar staining patterns with antibodies to GFAP, laminin, CNPase, S100, MBP, and P0 protein. Addition of axolemmal enriched membrane fractions to both the frozen and fresh SC gave a similar dose response curve of 3H-thymidine incorporation, with SC from frozen sciatic nerves responding even better than fresh sciatic nerves at higher doses (50 micrograms and 100 micrograms of protein/ml). As demonstrated by the cell yield, immunohistochemical staining and responses to axolemmal mitogens, this procedure produces SC from frozen sciatic nerves with similar characteristics to those isolated from fresh nerves. This procedure will allow the production and utilization of a large number of SC, which will be critical in further studies on the development and maintenance of the peripheral nervous system.