Although much interest has attended the cryopreservation of immature neurons for subsequent therapeutic intracerebral
transplantation, there are no reports on the cryopreservation of organized adult cerebral tissue slices of potential interest for
pharmaceutical drug development. We report here the first experiments on cryopreservation of mature rat transverse hippocampal slices. Freezing at 1.2 degrees C/min to -20 degrees C or below using 10 or 30% v/v
glycerol or 20% v/v
dimethyl sulfoxide yielded extremely poor results. Hippocampal slices were also rapidly inactivated by simple exposure to a temperature of 0 degree C in artificial cerebrospinal fluid (aCSF). This effect was mitigated somewhat by 0.8 mM
vitamin C, the use of a more "intracellular" version of aCSF having reduced
sodium and
calcium levels and higher
potassium levels, and the presence of a 25% w/v mixture of
dimethyl sulfoxide,
formamide, and
ethylene glycol ("V(EG) solutes"; Cryobiology 48, pp. 22-35, 2004). It was not mitigated by
glycerol,
aspirin,
indomethacin, or
mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. 260-273, 1984) was used as a carrier
solution for up to 50% w/v V(EG) solutes, 0 degree C was more protective than 10 degrees C. Raising V(EG) concentration to 53% w/v allowed slice vitrification without injury from vitrification and
rewarming per se, but was much more damaging than exposure to 50% w/v V(EG). This problem was overcome by using the analogous 61% w/v VM3 vitrification
solution (Cryobiology 48, pp. 157-178, 2004) containing
polyvinylpyrrolidone and two extracellular "
ice blockers." With VM3, it was possible to attain a tissue K(+)/Na(+) ratio after vitrification ranging from 91 to 108% of that obtained with untreated control slices. Microscopic examination showed severe damage in frozen-thawed slices, but generally good to excellent ultrastructural and histological preservation after vitrification. Our results provide the first demonstration that both the viability and the structure of mature organized, complex neural networks can be well preserved by vitrification. These results may assist neuropsychiatric
drug evaluation and development and the
transplantation of integrated brain regions to correct
brain disease or injury.