Enhanced Preservation of HEK293T Cells and Rat Kidneys by Optimizing Double-Phase Isochoric Freezing with Active Nucleation Management

Isochoric freezing offers a CPA-free technique for low-temperature storage of biological samples, enhancing their stability, transportability, and potential for clinical organ transplantation. Conventional isochoric systems exploit the pressure-temperature coupling in a rigid, sealed chamber to suppress uncontrolled ice growth but lack precise spatiotemporal control over ice-nucleation events, leading to stochastic freezing and reduced preservation efficiency. Here, we present a double-phasic isochoric system that physically separates the sample chamber (sample solution) from an external nucleation chamber (freezing solution) and verify its stability across diverse liquid media, including pure water, saline, HTK, and UW. By introducing cholesterol-crystal nucleators into the freezing solution, we achieve controlled ice nucleation that directs crystal formation away from the sample chamber and generates endogenous pressure to sustain supercooling (43.8 ± 3.7 MPa at -4 °C), reducing random freezing incidence by 50%. After optimizing the sample container, we performed 24 h of isochoric freezing of HEK293T cells in 7 mL PE tubes at -4 °C, retaining 92 ± 3.1% viability; following 72 h, the cells exhibited superior proliferation and lower ROS damage compared with conventional 4 °C static cold storage (SCS). Applied to rat kidneys, 72 h of isochoric preservation decreased tissue malondialdehyde by 64 ± 6.2%, preserved Na⁺/K⁺-ATPase activity, and maintained histological integrity relative to SCS. This study establishes critical methodology for clinical translation of isochoric freezing and demonstrates its promise for extending organ preservation times and improving transplant viability.