CONICET | Buscador de Institutos y Recursos Humanos

Neural stem cells have become a great promise for tissue repairand regenerative therapies due to their potential to proliferate anddifferentiate into major neuronal cell lines and their ability to incorporateinto preexisting neuronal circuits. The advances achieved in theapplication and use of these cells have made their cryopreservationa great challenge.The goal of this work was to optimize a preservation protocol ofMurin ENSCs at subzero temperatures by the Slow Cooling technique.ENSCs were isolated from cerebral cortex of C57/BL mice embryos(13-15 days) and cultured as neurospheres (NE) in proliferationmedium. On day 7 of culture NE were disgregated to obtain singlecells, resuspended in DMEM/Ham?s F12/10% Fetal Bovine Serumplus 10% DMSO and incubated for 20 min at 30°C to allow DMSOdiffusion. After that, cryopreservation of ENSCs was performed usinga device to control cooling rate (3.4°C/min and 6.2°C/min) until-80ºC. ENSCs were finally store in liquid nitrogen for 60 days. After the set time, cells were rapidly thawed at 37°C andcultured in proliferation medium for 6-7 days; NE were disgregatedand single ENSCs were cultured in differentiation medium. Cellviability and viable cell yield were evaluated in all the proceduresby Trypan blue exclusion test. NE proliferation was determined bymeasure of NE diameter by light microscopy. The ENSCs ability todifferentiate into neurons or astrocytes was estimated by immunocytochemicalstains.Cryopreservation of ENSCs by slow cooling produced a significantdecrease in cell viability at both tested cooling rates; moreover theobserved viable cell yield decreased significantly with the cryopreservationtime. Additionally, NE proliferation declined with the raise ofthe cooling rate and the cryopreservation time. No differences wereobserved in ENSCs differentiation. However the best results wereobtained with a co