Time Dependent Osmotic Damage in Sea Urchin Oocytes

Most cryobiological protocols require loading and unloading of cryoprotective agents (CPAs) to mitigate ice damage during the freezing and thawing process. However, CPAs change the osmolality of the solution creating an osmotic gradient across the cell membrane, causing large volumetric changes. Classically, mechanical damages due to swelling or shrinking have been thought to have a constant osmotic tolerance limit (E.g. 20% reduction in survival of the population at a given hyper and hyposmolality), which are crucial in determining optimized cryoprotocols. Here we show that osmotic damage is not dependent solely on volume deviance for sea urchin (Paracentrotus lividus) oocytes, but instead osmotic damage is time-dependent. We exposed urchin oocytes (n >= 100 per treatment with 3 replicates) to two hypertonic treatments at differing osmolalities (1500, 2000, and 2500 mOsm/kg). The hypertonic solutions either had NaCl or Sucrose added to holding medium (Sea water 1000 mOsm/kg) to obtain the desired osmolality. The exposure duration periods were for 2, 6, 15, 30, 50, 75, and 90 minutes. We tested hypotonic damage by diluting the holding medium with DI water exposing them to osmolalities of 800, 700, 600, and 500 mOsm/kg. After exposure, oocytes were returned to isosmotic holding media, in vitro fertilization was performed, and development to the 4-arm-Pluteus stage was assessed at 48 h. We fit these data to a mathematical model of population cell death that is proportional to the integration of the absolute value of the isosmotic volume minus the cell volume throughout time. This model works well (Adjusted R2 values of 0.97, 0.96, and 0.76 for DI water, NaCl, and Sucrose respectively) to describe osmotic related damage across multiple concentrations and solution types. The next step is to include this novel model as a refined metric of mechanical or osmotic stress instead of standard osmotic tolerance limit models when determining optimal CPA equilibration protocols using cytotoxicity cost functions. This may result in more accurate models of cell damage, and better optimized protocols for loading and unloading of CPAs.