Cell cryopreservation stops the biological activity of cells by placing them in the frozen state, and can be used to preserve cells without subculturing, which can cause contamination and genetic drift. However, the freezing process used in cryopreservation can injure or damage the cells due to the cytotoxicity of cryoprotecting agents (CPAs). We have previously reported a CPA-free cryopreservation method based on inkjet technology. In this method, the vitrified cells were exposed to the room temperature atmosphere during the transport of the cells using tweezers, which caused devitrification due to the increased temperature and often lowered the cell viability. In the present study, we developed an automatic thawing apparatus that transports the vitrified cells rapidly into a prewarmed medium using a spring hinge. Observations with a high-speed camera revealed that the spring hinge drops the cells into the prewarmed medium within 20 ms. All heat-transfer simulations for the apparatuses with different designs and rotation speeds showed that the cells remained below the glass-transition temperature during the transport. Finally, the apparatus was evaluated using mouse fibroblast 3T3 cells. The cell viability was improved and its reproducibility was enhanced using this apparatus. The results indicate that the combination of superflash freezing with the rapid thawing process represents a promising approach to circumvent the problems typically associated with the addition of CPAs.
Keywords: Cell cryopreservation; Cryoprotectant-free; Inkjet printing; Rapid thawing; Superflash freezing.
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