Abstract
This paper presents versatile protocols to prepare primary human Schwann cell (hSC) cultures from mature peripheral nervous system tissues, including fascicles from long spinal nerves, nerve roots, and ganglia. This protocol starts with a description of nerve tissue procurement, handling, and dissection to obtain tissue sections suitable for hSC isolation and culturing. A description follows on how to disintegrate the nerve tissue by delayed enzymatic dissociation, plate the initial cell suspensions on a two-dimensional substrate, and culture the primary hSCs. Each section contains detailed procedures, technical notes, and background information to aid investigators in understanding and managing all steps. Some general recommendations are made to optimize the recovery, growth, and purity of the hSC cultures irrespective of the tissue source. These recommendations include: (1) pre-culturing epineurium- and perineurium-free nerve fascicles under conditions of adherence or suspension depending on the size of the explants to facilitate the release of proliferative, in vitro-activated hSCs; (2) plating the initial cell suspensions as individual droplets on a laminin-coated substrate to expedite cell adhesion and thereby increase the recovery of viable cells; and (3) culturing the fascicles (pre-degeneration step) and the cells derived therefrom in mitogen- and serum-supplemented medium to accelerate hSC dedifferentiation and promote mitogenesis before and after tissue dissociation, respectively. The hSC cultures obtained as suggested in this protocol are suitable for assorted basic and translational research applications. With the appropriate adaptations, donor-relevant hSC cultures can be prepared using fresh or postmortem tissue biospecimens of a wide range of types and sizes.