Schwann cells (SCs) are the principal glia of the peripheral nervous system, supporting neuronal function and regeneration. Healthy human SCs were derived from great auricular nerves (GANs). SCs isolated after enzymatic digestion were cultured and adhered onto coverslips in less than 24 h.
SC cultures demonstrated a mean 85% level of purity for up to 2 weeks, after which fibroblast-like cells predominated, as observed following immunostaining for cytoplasmic S100, a well-established SC marker.
Healthy human SCs were obtained from great auricular nerves (GANs). Nerve specimens measuring 1 cm (from parotidectomies) to 5 cm (from neck dissections) were processed within 20 min of GAN resection. GAN samples were washed with sterile PBS and transferred to DMEM/F12 medium supplemented with FBS (10%), penicillin/streptomycin mix (1%) and L-Glutamate (1%). The fascicles were isolated from the epineurium by tugging on the perineurium using no. 5 forceps, while clasping the epineurium with no. 3 forceps, under a dissecting microscope. The nerve was cut into 1- 2 mm segments, which were then incubated in an enzymatic mixture comprised of Hyaluronidase Type I-S (250 U/mL) and Collagenase Type I (160 U/mL) in DMEM/F12 medium. GAN pieces were incubated for 24 h at 370C with 5% CO2 levels. Following enzymatic incubation, the cell culture-containing medium was triturated using an 18-gauge needle and centrifuged at 1000 g for 5 min at room temperature. The pellet was resuspended in supplemented DMEM/F12 medium and plated on poly-L lysine and laminin pre-coated coverslips within the 12-well dishes coated with poly-L-ornithine and laminin. Culture medium was replaced after 24 h, and then every 3 days thereafter.