Impact-induced droplet release thawing enables cryoprotectant-free cell cryopreservation of large inkjet droplets

Abstract

Inkjet-based superflash freezing (SFF) enables vitrification without cryoprotective agents (CPAs) by miniaturizing droplets. However, the insufficient cooling and warming rates of large droplets have restricted the applicable volume of SFF to 70 pL or less. The large thermal mass of the substrate in SFF has been a major factor limiting the thawing process. In this study, an impact-induced droplet release (IIDR) thawing method was developed, in which frozen droplets are mechanically separated from the substrate and directly thawed in prewarmed medium. This approach also improves cooling efficiency by direct freezing on an aluminum transporter with high thermal conductivity. Numerical analyses demonstrated that the IIDR thawing method improved both cooling and warming rates by approximately threefold for 200 pL droplets compared with the substrate-based method. Consequently, high cell viability of over 80 % was achieved in 200 pL droplets using NIH 3T3 fibroblasts (approximately 1600 droplets, corresponding to about 320 cells), extending the applicability of CPA-free SFF cryopreservation from the previous limit of 70 pL–200 pL. By contrast, in 70 pL droplets, viability decreased because the droplets remained at the medium interface, preventing sufficient warming, thereby indicating the need for further optimization of the thawing process. From the relationship with viability, the critical cooling and warming rates for cells were estimated to be approximately 1.5 × 10⁴ °C/s and 3 × 10⁴ °C/s, respectively. Overall, the IIDR thawing method provides a reproducible platform that overcomes key limitations of SFF and represents a promising basis for CPA-free inkjet-based cryopreservation.