Water confinement effect on critical cooling and warming rates in tissue-CPA system

Understanding and controlling the phase transition of water is the foundation of cryopreservation. Water is an important constituent of all biological materials and is found naturally in confined spaces inside biological materials. Water in confined domains acts differently than in bulk domain, and has been studied in many porous materials, including biological materials. Physical and calorimetric properties of water in confined spaces deviate largely from in non-confined space. Past studies have shown the effect of confinement on mechanical, physical and chemical properties of a material containing confined water. Here we aim to characterize this effect in tissues loaded with cryoprotective agents (CPAs), which is important for the design of cooling and rewarming protocols for tissue and organ cryopreservation. Phase diagrams and critical rates for dilutions of cryoprotective agent (CPA) concentrations were measured in solutions and tissue equilibrated with CPA. Specifically, we report on water confinement effects on critical cooling and warming rates inside a biological material (rodent kidney tissue) equilibrated with a common CPA (VS55). Kidney biopsies (3 mm diameter) prepared from tissue slices (1.2 mm thick) are equilibrated (>1 week) in a range of CPA (VS55) concentrations (75- 100%) and then tested for CCR, CWR and transition temperatures (Tm, Tg, Td & Th). We found that there is a measurable reduction in CCR (max ∼88%) and CWR (max ∼27%) inside tissue equilibrated with CPA as compared to bulk CPA solution. This knowledge is critically important for appropriately choosing CPA concentrations and cooling rates in vitrification protocols for tissues and organs. While we currently report the effects for a single model system, these methods can be expanded to a wider range of CPAs and tissues and lay the groundwork for a more mechanistic understanding of confinement.