Cell-based therapies have become a major focus in preclinical research that leads to clinical application of a therapeutic product. Since 1990, scientists at the Miami Project to Cure Paralysis have generated extensive data demonstrating that Schwann cell (SC) transplantation supports spinal cord repair in animals with spinal cord injury. After preclinical efforts in SC transplantation strategies, efficient methods for procuring large, essentially pure populations of SCs from the adult peripheral nerve were developed for rodent and pig studies. This chapter describes a series of simple procedures to obtain and cryopreserve large cultures of highly purified adult nerve-derived SCs without the need for additional purification steps. This protocol permits the derivation of ≥90% pure rodent and porcine SCs within 2–4 weeks of culture.