title: Cryopreserved neuronal cells in long-term cultures of dissociated rat cerebral cortex: survival and morphometric characteristics as revealed by immunocytochemistry.
abstract: 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.
authors.affiliation: Inserm U336, USTL Case Courrier 106, Montpellier, France.
authors.forename: D
authors.initials: D
authors.lastname: Petite
authors.forename: M C
authors.initials: MC
authors.lastname: Calvet
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000502:physiology / Q000648:ultrastructure*; D016380:Brain Tissue Transplantation; D002455:Cell Division; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000637:transplantation*; D007150:Immunohistochemistry; D009474:Neurons / Q000637:transplantation*; D051381:Rats; D014021:Tissue Preservation