Pressure enhanced dual-solid-surface ultra-rapid cooling improves post-thaw recovery in hepatocytes and precision cut liver slices

Cell and tissue cryopreservation is essential in modern medicine for globally distributing biological materials; however, ice crystal formation hinders its efficacy. Non-equilibrium cryopreservation, which focuses on rapid cooling, shows promise in mitigating this issue and allows for reduced cryoprotective agent (CPA) concentrations, thereby minimizing cytotoxic effects. Increased pressure lowers both solution melting temperature (T_m) and solution homogeneous nucleation temperature (T_h), while simultaneously elevating solution glass transition temperature (T_g). These effects should improve vitrification outcomes by minimizing ice crystal formation. Here we designed, fabricated, and tested an apparatus capable of high-pressure dual-sided cooling for hermetically sealed tissue samples. Using this device, we demonstrated that dual-surface high-pressure solid-surface conductive cooling offers a more effective approach for increasing the cooling rate compared to convective methods like direct immersion in liquid nitrogen. The experiments were conducted on HepG2 cells and mouse precision-cut liver slices (PCLS, 300 μm thickness, 5 mm diameter) using dimethyl sulfoxide (DMSO) as the CPA at concentrations of 10 to 30% (v/v) and pressures ranging from 50 to 200 MPa. Optimal outcomes were achieved at 20% DMSO and 150 MPa, yielding the highest post-thaw tissue viability.