C-2018: Human umbilical vein endothelial cell (HUVEC) response to interrupted cooling in the presence and absence of dimethyl sulfoxide

Abstract

HUVECs were first successfully cultured in vitro in 1973, initiating the growth of modern vascular biology. Study into HUVEC cryopreservation has made it readily available for purchase, however the cryobiological response of HUVECs has not been fully investigated. Viability by membrane integrity of HUVECs as received from the supplier was 50 ± 2%, indicating that cryopreservation of HUVECs can be improved. Membrane hydraulic conductivity is an osmotic property which is necessary to simulate cryobiological response. HUVEC membrane hydraulic conductivity was measured and found to be 0.147 μm 3 /μm 2 /min/atm at 20 °C, less than fibroblasts and TF-1 cells, therefore being the slowest osmotically-responding cells studied using interrupted cooling. HUVECs were subjected to interrupted slow cooling without hold time (graded freezing) and rapid cooling interrupted with a hold time (two-step freezing) in the presence or absence of 10% Me 2 SO. Preliminary results show that HUVECs have higher viabilities from graded freezing than from two-step freezing, similar to previously studied fibroblasts but different from TF-1 cells. Optimal cooling procedures cannot solely be explained by hydraulic conductivity. Graded freezing at 1 °C/min in the presence of 10% Me 2 SO resulted in the best viability, with almost no damage from direct thaw after cooling to between −5 °C and −40 °C and a maximum of 66% HUVEC membrane integrity after cooling to −35 °C, plunging into liquid nitrogen and then thawing (plunge-thaw). Two-step freezing in the presence of 10% Me 2 SO resulted in almost no damage from direct thaw from as low as −15 °C. Lower experimental hold temperatures resulted in damage after direct thaw and the highest membrane integrity after plunge-thaw was 42% for a hold temperature of −25 °C. Interrupted cooling in the absence of Me 2 SO resulted in no HUVEC membrane integrity after plunge-thaw. The understanding gained from this investigation may lead to improved cryopreservation of HUVECs. Funding: CIHR.