Rational synthesis of total damage during cryoprotectant equilibration: modelling and experimental validation of osmomechanical, temperature, and cytotoxic damage in sea urchin (Paracentrotus lividus) oocytes.

Sea urchins (e.g., Paracentrotus lividus) are important for both aquaculture and as model species. Despite their importance, biobanking of urchin oocytes by cryopreservation is currently not possible. Optimized cryoprotectant loading may enable novel vitrification methods and thus successful cryopreservation of oocytes. One method for determining an optimized loading protocol uses membrane characteristics and models of damage, namely osmomechanical damage, temperature damage (e.g., chill injury) and cytotoxicity. Here we present and experimentally evaluate existing and novel models of these damage modalities as a function of time and temperature. In osmomechanical damage experiments, oocytes were exposed for 2 to 30 minutes in hypertonic NaCl or sucrose supplemented seawater or in hypotonic diluted seawater. In temperature damage experiments, oocytes were exposed to 1.7 °C, 10 °C, or 20 °C for 2 to 90 minutes. Cytotoxicity was investigated by exposing oocytes to solutions of Me2SO for 2 to 30 minutes. We identified a time-dependent osmotic damage model, a temperature-dependent damage model, and a temperature and time-dependent cytotoxicity model. We combined these models to estimate total damage during a cryoprotectant loading protocol and determined the optimal loading protocol for any given goal intracellular cryoprotectant concentration. Given our fitted models, we find sea urchin oocytes can only be loaded to 13% Me2SO v/v with about 50% survival. This synthesis of multiple damage modalities is the first of its kind and enables a novel approach to modelling cryoprotectant equilibration survival for cells in general.

Meta-analysis of the Boyle van 't Hoff relation: Turgor and leak models explain non-ideal volume equilibrium.

There has been much recent attention paid to the interaction of cell volume, its regulation, and the molecular biology of the cell. Cells are generally assumed to behave as linear osmometers, with their water volume linearly proportionate to the inverse of osmotic pressure as described by the Boyle van 't Hoff (BvH) relation. This study evaluates the generality of this and other long-standing assumptions about cell responses to anisotonic conditions. We present alternative models that account for osmoregulation including mechanical resistance to volumetric expansion (the turgor model) and ion-osmolyte leakage (the leak model). To evaluate the generality of the BvH relation and determine the suitability of alternative models, we performed a comprehensive survey of the literature and a careful analysis of the resulting data, and then we used these data to compare among models. We identified 137 articles published from 1964 to 2019 spanning 14 animal species and 26 cell types and determined the BvH relation is not an appropriate general model but is adequate when restricted to the hypertonic region. In contrast, models that account for either mechanical resistance or ion-osmolyte leakage fit well to almost all collected data. The leak model has fitted parameters that are in the same range as the current literature estimate, while the turgor model typically requires an elastic modulus value of one or multiple orders of magnitude larger than literature values. However, confirmation of the underlying mechanism of osmotic regulation is required at the cell-specific level and cannot be assumed a priori.