CRYOPRESERVATION OF EMBRYONIC NEURAL STEM CELLS (ENSCs) BY THE SLOW COOLING TECHNIQUE

BERCA, C; MONTANER A; RODRIGUEZ J.V.; BANCHIO C; ROBERT, MARIA CELESTE

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias- LII SAIB; 2017

Sociedad Argentina de Investigacion Bioquimica y Biología Molecular- SAIB

Neural stem cells have become a great promise for tissue repair and regenerative therapies due to their potential to proliferate and differentiate into major neuronal cell lines and their ability to incorporate into preexisting neuronal circuits. The advances achieved in the application and use of these cells have made their cryopreservation a great challenge.The goal of this work was to optimize a preservation protocol of Murin 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 proliferation medium. On day 7 of culture NE were disgregated to obtain single cells, resuspended in DMEM/Ham?s F12/10% Fetal Bovine Serum plus 10% DMSO and incubated for 20 min at 30°C to allow DMSO diffusion. After that, cryopreservation of ENSCs was performed using a 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 and cultured in proliferation medium for 6-7 days; NE were disgregated and single ENSCs were cultured in differentiation medium. Cell viability and viable cell yield were evaluated in all the procedures by Trypan blue exclusion test. NE proliferation was determined by measure of NE diameter by light microscopy. The ENSCs ability to differentiate into neurons or astrocytes was estimated by immunocytochemical stains.Cryopreservation of ENSCs by slow cooling produced a significant decrease in cell viability at both tested cooling rates; moreover the observed viable cell yield decreased significantly with the cryopreservation time. Additionally, NE proliferation declined with the raise of the cooling rate and the cryopreservation time. No differences were observed in ENSCs differentiation. However the best results were obtained with a cooling rate of 3.4ºC and a cryopreservation time less than 50 days.