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

Abstract

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.

Cryopreservation Enables Long-Term Studies ofOctopus vulgarisHemocyte Immune Functions

“…The high diversity of marine species and their differences in form, complexity, size, or even in living/feeding/reproducing mechanisms, make this process extremely meticulous, requiring detailed studies of each stage, with a high risk of loss of cell viability if any step of the process is not fully optimized. Even in cells from the same organism, conditions can vary radically and the same CPA or freezing/thawing method can be toxic or lethal for some cells and not for others (Campos et al, 2024;Olver et al, 2023). For instance, in the field of marine biology, there are multiple studies on sperm cryopreservation in different species, while the process of preserving oocytes has not yet been optimized.…”