Investigating membrane and mitochondrial cryobiological responses of HUVEC using interrupted cooling protocols

The success of cryopreservation protocols is largely based on membrane integrity assessments after thawing, since membrane integrity can be considered to give an upper limit in assessment of cell viability and the plasma membrane is considered to be a primary site of cryoinjury. However, the exposure of cells to conditions associated with low temperatures can induce injury to cellular structure and function that may not be readily identified by membrane integrity alone. Interrupted cooling protocols (including interrupted slow cooling without a hold time (graded freezing), and interrupted rapid cooling with a hold time (two-step freezing)), can yield important information about cryoinjury by separating the damage that occurs upon cooling to (and possibly holding at) a critical intermediate temperature range from the damage that occurs upon plunging to the storage temperature (liquid nitrogen). In this study, we used interrupted cooling protocols in the absence of cryoprotectant to investigate the progression of damage to human umbilical vein endothelial cells (HUVEC), comparing an assessment of membrane integrity with a mitochondrial polarization assay. Additionally, the membrane integrity response of HUVEC to interrupted cooling was investigated as a function of cooling rate (for interrupted slow cooling) and hold time (for interrupted rapid cooling). A key finding of this work was that under slow cooling conditions which resulted in a large number of membrane intact cells immediately post thaw, mitochondria are predominantly in a non-functional depolarized state. This study, the first to look directly at mitochondrial polarization throughout interrupted cooling profiles and a detailed study of HUVEC response, highlights the complexity of the progression of cell damage, as the pattern and extent of cell injury throughout the preservation process differs by injury site.