Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing

Significance Cryopreservation is routinely used for the long-term storage of cells in various areas of academic, industrial, and clinical research. To keep frozen cells alive, it is necessary to vitrify (or nanocrystallize) water on the inside and outside of the cells. Vitrification is conventionally achieved by adding at least one cryoprotective agent (CPA) to the medium. However, CPAs should ideally be avoided due to their cytotoxicity and potential side effects on the cells. Herein, we demonstrate a method of CPA-free cryopreservation, in which cells are almost vitrified by ultrarapid cooling using inkjet printing. The freezing method should be generally suitable for all kinds of cells that are susceptible to CPAs, including stem cells. Cryopreservation is widely used to maintain backups of cells as it enables the semipermanent storage of cells. During the freezing process, ice crystals that are generated inside and outside the cells can lethally damage the cells. All conventional cryopreservation methods use at least one cryoprotective agent (CPA) to render water inside and outside the cells vitreous or nanocrystallized (near-vitrification) without forming damaging ice crystals. However, CPAs should ideally be avoided due to their cytotoxicity and potential side effects on the cellular state. Herein, we demonstrate the CPA-free cryopreservation of mammalian cells by ultrarapid cooling using inkjet cell printing, which we named superflash freezing (SFF). The SFF cooling rate, which was estimated by a heat-transfer stimulation, is sufficient to nearly vitrify the cells. The experimental results of Raman spectroscopy measurements, and observations with an ultrahigh-speed video camera support the near-vitrification of the droplets under these conditions. Initially, the practical utility of SFF was demonstrated on mouse fibroblast 3T3 cells, and the results were comparable to conventional CPA-assisted methods. Then, the general viability of this method was confirmed on mouse myoblast C2C12 cells and rat primary mesenchymal stem cells. In their entirety, the thus-obtained results unequivocally demonstrate that CPA-free cell cryopreservation is possible by SFF. Such a CPA-free cryopreservation method should be ideally suited for most cells and circumvent the problems typically associated with the addition of CPAs.