Ultrasonic rewarming of cryopreserved alginate encapsulated liver spheroids

Rapid volumetric rewarming methods are needed to enable the effective cryopreservation and recovery of large volumes of biological cells for therapy and banking of tissues and organs. Ultrasonic rewarming is currently under development, but its effect on cells and their post-rewarming viability has not yet been established. Here, we compare ultrasonic rewarming with the gold-standard [Formula: see text]C water bath using cryovials containing cryopreserved alginate encapsulated liver spheroids. Mean rewarming rates are used to establish the exposure time to rewarm to [Formula: see text]C for higher power (100 W) and lower power (20 W) ultrasonic rewarming. These electrical powers correspond to free-field pressures along the central cryovial axis of 2.8 MPa and 1.3 MPa, respectively. Ultrasonic rewarming is faster than the gold-standard (120±5 s), taking 88 s (36% faster) and 34 s (350% faster) to rewarm to [Formula: see text]C with the lower and higher powers. We measure post-rewarming liver spheroid viability and viable cell number across the 96-h recovery period. The lower power improves viability by 1% and the higher power reduces viability by 2% on average, relative to the gold-standard. There were no significant differences in viable cell number between rewarming methods. Our findings will serve as a foundation for ultrasonic cryovial rewarming and demonstrates potential for scaling to larger volumes.