51. A comparative assessment of mitochondria and membrane cryobiological responses of HUVEC to low temperatures

Abstract

The extent of cellular cryoinjury incurred during cryopreservation protocols is traditionally assessed with membrane integrity assays, often used as an upper limit in determining viability. However, exposure of cells to subzero temperatures and conditions may lead to changes in various aspects of cellular structure and function that would not be considered in studies that rely on membrane integrity assessments alone. Slower cooling rates in particular have been shown to adversely affect the metabolism (McGann et al., 1988; and Sherman, 1972) and structural properties (Tchir et al., 2010) of mitochondria in addition to the occurrence of damage to the plasma membrane during freeze–thaw stress. The objective of the study was to use a mitochondrial assay to investigate a secondary aspect of cell response to directly compare with a traditional assessment of membrane integrity under identical conditions. An interrupted slow cooling method was used to examine the occurrence of damage from slow cooling to intermediate subzero experimental temperatures, and damage during rapid cooling when plunged into liquid nitrogen. Human umbilical vein endothelial cells (HUVECs) were cooled at 0.2 °C/min to temperatures between −3 °C and −40 °C in the absence of cryoprotectant. Samples were then either thawed directly in a 37 °C water bath or plunged into liquid nitrogen before thawing. The membrane integrity of cells was determined using a combination of the nucleic fluorescent dyes Syto13 and ethidium bromide, and mitochondrial polarization was indicated with the cationic carbocyanine dye JC-1. The number of cells determined to be membrane intact and the number of cells containing polarized mitochondria decreased with decreasing temperature in directly-thawed samples. Depolarized mitochondria was found in 50% of cells at −15 °C, whereas 50% of cells were membrane compromised at a lower temperature (−30 °C). HUVEC plunged into liquid nitrogen from experimental temperatures showed an increase in the number of membrane intact cells with decreasing experimental temperature to a maximum of 40% at −20 °C, but This research was funded by the Canadian Institutes of Health Research (CIHR) MOP 86492, as well as the University of Alberta, and the Government of Alberta. J.A.W. Elliott holds a Canada Research Chair in Thermodynamics.