Abstract

Cell therapies hold transformative potential for treating cancer, neurologic disorders, organ failure, diabetes, and other conditions, but their widespread clinical deployment is constrained by the lack of scalable cryopreservation methods that maintain high post-thaw viability. Current standard practice using slow freezing can lead to cell death and impaired cell function. Vitrification offers an alternative by cooling samples rapidly enough to bypass ice formation entirely, better preserving cell structure and function. The high cooling and warming rates required for vitrification have previously been achieved by quenching microliter-scale samples directly into convective cooling and warming baths. Here, we present a cryopreservation platform that overcomes the throughput limitations of existing systems by combining a vibrating orifice aerosol generator with an impinging conical nozzle to generate and confine micrometer-scale droplets mid-flight in liquid nitrogen. This approach mitigates cooling losses due to the inverse Leidenfrost effect, increasing cooling and warming rates by nearly an order of magnitude compared to conventional droplet vitrification, while improving throughput by two orders of magnitude. To test the efficacy of this system, we cryoaerosolized and rewarmed human induced pluripotent stem cells, porcine red blood cells, and human dermal fibroblasts using only 19-25 wt% (2.5-3.7 M) permeating cryoprotective agent, achieving >90% post-thaw viability for HDFs and hiPSCs and 94% recovery for RBCs, with retained colony-forming capacity additionally demonstrated in hiPSCs. This work demonstrates the first scalable vitrification-based cryopreservation method capable of achieving both high cooling (~200,000 K/min) and warming rates (~1,000,000 K/min) while maintaining the high-throughput processing required (>100 mL/h) for next-generation cell therapies.