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

Brain Res. 1995 Jan 16;669(2):263-74. doi: 10.1016/0006-8993(94)01269-n.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Astrocytes / physiology
  • Astrocytes / ultrastructure*
  • Brain Tissue Transplantation
  • Cell Division
  • Cells, Cultured
  • Cerebral Cortex / transplantation*
  • Immunohistochemistry
  • Neurons / transplantation*
  • Rats
  • Tissue Preservation*