Cryopreserved neuronal cells in long-term cultures of dissociated rat cerebral cortex: survival and morphometric characteristics as revealed by immunocytochemistry.

Blocks of fresh tissue from embryonic rat cerebral cortex, dissociated either prior to or after freezing, were stored for up to 10 months in liquid nitrogen at -196 degrees C with 7% dimethylsulfoxide (DMSO) as cryoprotectant. Slow freezing and rapid thawing, a reduced medium volume and moderate elevation of both extracellular K+ (20 mM) and sera (20%) promoted survival of neurons (up to 7 weeks) on polylysine-coated glass coverslips. Although there was cell loss associated with freezing, the surviving cells developed morphological and immunocytochemical properties similar to those expressed by unfrozen cells when using anti-GFAP (glial fibrillary acidic protein), anti-NSE (neuron specific enolase) and anti-GABA (gamma-aminobutyric acid) antibodies. A comparative computer-aided analysis of the morphometric patterns of GABA-IR neurons allowed the individualization of two similar populations in both fresh and frozen controls. NSE- and GABA-immunoreactive (IR) cells were counted in frozen controls and thienyl-phencyclidine (TCP)-treated cultures. The latter yielded fewer cells whereas it was the opposite in fresh TCP-treated cultures. In view of these findings, a detailed analysis of the effects of both neuroprotective and neurotoxic agents on frozen cells is undertaken before considering that cryopreservation might be a suitable storage method for clinical trials of neuron grafting.