Purification of mouse Schwann cells using neurite-induced proliferation in serum-free monolayer culture.

We have recently reported that neonatal mouse dorsal root ganglionic Schwann cells will (i) survive and assume characteristic morphologies in a serum-free, fully defined cultured medium (N1 medium), (ii) proliferate extensively in the same N1 medium if neurons are also present and maintained by nerve growth factor, and (iii) display a strong proliferative response to serum even in the absence of neuronal elements, while also undergoing marked changes in their morphology and their associative behavior toward neurites. In this report, we present a detailed procedure, based upon these earlier observations, which yields purified cultures of either neurons plus associated Schwann cells or Schwann cells in the absence of neurons. The procedure utilizes the neuritic mitogen for selective expansion of Schwann cell numbers in serum-free primary cultures, and a secondary culture step involving neuronal removal and additional Schwann cell expansion using the serum mitogen. The procedure requires 9 days for the generation of 3-4 X 10(6) Schwann cells from 12 newborn mice (with a Schwann cell to neuron ration of 10) and an additional 6-7 days for the generation of a neuron-free secondary population of 40 X 10(6) Schwann cells with less than 3% contamination by identifiable ganglionic fibroblasts.