CRYOPRESERVED NEURONAL CELLS IN LONG-TERM CULTURES OF DISSOCIATED RATCEREBRAL-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 vol ume and moderate elevation of both extracellular KC (20 mM) and sera ( 20%) promoted survival of neurons (up to 7 weeks) on polylysine-coated grass coverslips. Although there was cell loss associated with freezi ng, the surviving cells developed morphological and immunocytochemical properties similar to those expressed by unfrozen cells when using an ti-GFAP (glial fibrillary acidic protein), anti-NSE (neuron specific e nolase) and anti-GABA (gamma-aminobutyric acid) antibodies. A comparat ive computer-aided analysis of the morphometric patterns of GABA-IR ne urons allowed the individualization of two similar populations in both fresh and frozen controls. NSE- and GABA-immunoreactive (IR) cells we re counted in frozen controls and thienyl-phencyclidine (TCP)-treated cultures. The latter yielded fewer cells whereas it was the opposite i n fresh TCP-treated cultures. In view of these findings, a detailed an alysis of the effects of both neuroprotective and neurotoxic agents on frozen cells is undertaken before considering that cryopreservation m ight be a suitable storage method for clinical trials of neuron grafti ng.