132 Viability of human umbilical vein endothelial cells ( HUVEC) in the presence of permeating and non-permeating cryoprotectants after interrupted cooling

Abstract

Graded freezing (interrupted slow cooling without hold time) and two-step freezing (interrupted rapid cooling with hold time) are two types of interrupted cooling protocols used to investigate freeze-thaw responses of cells and the actions of cryoprotectants. Graded freezing can be used to investigate progressive cell injury during slow cooling and two-step freezing can be used to examine cell injury during interrupted rapid cooling with hold time. Cell response using graded freezing has been studied using fast osmotically-responding cells such as fibroblasts or TF-1 cells but not with a slow osmotically-responding cell such as human umbilical vein endothelial cells (HUVEC). Cell response using two-step freezing has been studied for a variety of cell types in the presence or absence of permeating cryoprotectants, however, for only one cell type (fast osmotically-responding fibroblasts) has the effect of two-step freezing with both permeating dimethyl sulfoxide (Me 2 SO) and non-permeating hydroxyethyl starch (HES) been studied. In this study, HUVEC were subjected to graded and two-step freezing after which samples were rapidly thawed and assessed for cell viability using a dual fluorescence nucleic acid stain consisting of Syto®13 and ethidium bromide. HUVEC membrane integrity was measured throughout the interrupted-cooling profiles in (i) cryoprotectant-free endothelial growth medium (EGM), and measured throughout the two-step freezing protocol in the presence of (ii) permeating cryoprotectant (5% Me 2 S0), and (iii) non-permeating cryoprotectant (5% HES). Graded freezing resulted in higher viabilities of HUVEC after plunge-thaw compared to two-step freezing, opposite to what was observed with fast-osmotically responding TF-1 cells. Two-step freezing in the presence of 5% Me 2 SO resulted in higher viabilities of HUVEC after plunge-thaw as compared to the same cooling profile in 5% HES or EGM. Two-step freezing with HES did not result in higher viability of HUVEC than without cryoprotectant, whereas fibroblasts have higher reported viability with 5% HES than without cryoprotectant. Source of funding: University of Alberta, travel awards: Cryo2007 fund and Bell McLeod Education Fund; CIHR MOP 86492, and OGBF INO 126778 from the INMHA and NSERC of Canada; JAW Elliott holds a Canada Research Chair in Thermodynamics. Conflict of interest: None declared. asultani@ualberta.ca