On-chip loading and unloading of cryoprotectants facilitate cell cryopreservation by rapid freezing

Cryopreservation is a featured and unique technology widely used in the fields of cytotherapy, stem cells, tissue engineering and regenerative medicine, organ transplantation, and assisted reproduction. However, the routine loading/unloading of cryoprotectant agents (CPAs) prior to freezing/post-thaw often induce osmotic, mechanical and centrifugal injuries, and toxicity, reducing cell viability. This is especially true for high CPA concentration cryopreservation (e.g., vitrification). Optimization of CPA loading/unloading is thus needed for successful cryopreservation. Conventional multi-step loading/unloading methods can reduce cell volume changes, but centrifugation, time-consumption, and potential cross-contamination are their constant limitations. To this end, we developed a novel microfluidic approach for on-chip CPA loading/unloading. Compared with the traditional one- or multi-step method, the microfluidic method introduces less osmotic, toxic, mechanical and centrifugal injuries during CPA loading/unloading. Further analysis indicates that less centrifugation is the dominant reason for significantly improved post-cryopreservation cell viability. Additionally, compared with most of the microfluidic methods that can only handle one or several cells at one time, CPA loading/unloading of large amount of cells can be accomplished using the proposed approach in this study. Thus, we provide a universal, easy-to-use, high efficiency method to minimize osmotic, toxic, mechanical and centrifugal injuries, and to cryopreserve cells with high efficiency for modern cell-based medicine.