Ultrasonic ice seeding as an effective technique for Hep-G2 cell cryopreservation

BACKGROUND: Cryopreservation faces challenges from intracellular ice formation (IIF) and solution damage, influenced by cooling rates. Ice seeding mitigates supercooling risks, yet traditional methods like contact nucleation are impractical. Ultrasonic ice seeding emerges as a promising alternative, leveraging cavitation to induce nucleation without disrupting temperature stability. OBJECTIVE: To evaluate ultrasonic ice seeding’s efficacy in reducing IIF and enhancing post-thaw survival of Hep-G2 liver cancer cells. MATERIALS AND METHODS: A cryogenic microscopy platform and ultrasonic device (40 kHz, 500W) were constructed. Hep‐G2 cells were cryopreserved using three methods including 10% DMSO as cryoprotectant: cell freezing container (10%‐CFC), programmable controlled-rate freezing (10%‐PC), and programmable freezing with ultrasonic ice seeding (10%‐PC+UIC). Survival rates were assessed via AOPI staining, while intracellular ice formation and cell morphology were analyzed microscopically. RESULTS: The 10%‐PC+UIC group achieved the highest survival rate (93.29 ± 1.20%), surpassing 10%‐PC (90.32 ± 1.60%) and 10%‐CFC (80.33 ± 3.36%). Ultrasonic ice seeding reduced intracellular ice occurrence to 6.43% versus 20.71% in spontaneous crystallization. Microscopy revealed smaller, uniform ice crystals and controlled cell dehydration/rehydration dynamics, minimizing membrane damage. CONCLUSIONS: Ultrasonic ice seeding significantly improves HepG2 cell survival by reducing IIF through controlled nucleation. Its non‐contact, contamination‐free operation offers practical advantages for large-scale cryopreservation, outperforming traditional methods. This technique holds potential for broader applications in biological and medical storage protocols.