A procedure for culturing rat neocortex explants in a serum-free nutrient medium

Abstract

A procedure is described for long-term culturing of rat neocortex explants in a serum-free growth medium. Slices spanning the entire cortical depth from pial to ventricular side are prepared from 6-day-old rat pups. After preincubation in Hanks' balanced salt solution with extra glucose, the explants are placed on polyamide gauze carriers in plastic culture dishes containing serum-free medium. The dishes are continuously rocked during the culture period. After 3 weeks in vitro the explants consist of a three-dimensional network of neural tissue with a mean thickness above the gauze of ca. 100 micron which corresponds with about 8 cell layers. Central necrosis is either fully absent (in one-third of the explants) or restricted to a minimal strip or patch located close to the gauze. From pial to ventricular side, 5 layers can be distinguished which, with respect to cell size and cell density, reflect a histiotypic architecture. The dense neuropil shows abundant axo-dendritic synapses (both on shafts and spines), myelinated fibers, and spontaneous bioelectric activity.