Kinetic (very rapid) vitrification (KVF) is a very promising approach in cryopreservation (CP) of biological materials as it is simple, avoids lethal intracellular ice formation (IIF) and minimizes damaging dehydration effects of extracellular crystallization. Moreover, achieving the ultra-high rates, which would prevent IIF during cooling and devitrification during resuscitation, and achieve KVF for practically any type of cells with one protocol of cooling and re-warming would be the “Holy Grail” of cell cryobiology [3]. However such hyperrapid rates currently require very small sample size which, however, is insufficient for many applications such as stem cells, blood or sperm. As the result, even smallest droplets of 0.25 microliters cannot be vitrified sufficiently fast to avoid the use of potentially toxic external vitrification agents such as DMSO or EG due to the Leidenfrost effect (LFE). In this presentation, we describe an entirely new system for hyperfast cooling of one-two order of magnitude larger samples that we call “KrioBlastTM”, which completely eliminates LFE. We have successfully vitrified up to 4,000 microliters of 15% glycerol solutions, which theoretically corresponds to the critical cooling rate of hundreds of thousands °C/min. We believe that such a system can revolutionize the future cryobiological paradigm.

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Human pluripotent stem cells (hPSCs) and their progeny such as hPSC-derived cardiomyocytes and neural cells hold great potential as a source for cell therapy and regenerative medicine, as well can be effectively used for high high content screening (HCS) of drug candidates and for toxicity tests. Cryopreservation (CP), storage, and shipment of the cells are key elements for eventual clinical, pharmaceutical and environmental applications, which will require large numbers of quality controlled and ready for use cells. Traditionally, the cells are frozen in suspensions of either fully dissociated cells) or loosely associated clusters such as clumps of hPSCs, clusters of beaters”of cardiomyocytes, (“or neurospheres of neural precursors. Beside logistical inconvenience for some applications such as HCS, additional manipulation with the cells (detachment, dissociation and centrifugation) can introduce substantial stress to the cells prior to freezing and after thawing, which per se may tremendously decrease the cell cryosurvival and functionality. Here, we are presenting ComfortFreezer™, a novel bench-top device specifically designed for cryopreservation in multi-well plates for cell-based high content screening (HCS), which combines a liquid-nitrogen (LN2) free programmable freezer This cryogenic equipemnt can bring serious advantage for HCS in drug screening, environmental toxicity evaluation, and other variety of HCS-based applications.

Kinetic (very rapid) vitrification (K-VF) is a promising approach for cryopreservation (CP) of cells but existing methods are not scalable due to the Liedenfrost effect (LFE), which substantially impedes the rate of cooling. Here, we compare 4 emerging approaches that discuss scalability and ultra-fast cooling, namely, cryogenic oscillating heat pipes (COHP), microstructured evaporation in thin films, K-VF in nanodroplets and thin film evaporation in microstructured with our methods of hyperfast cooling KrioBlastTM. We show that only KrioBlastTM can ensure hyper-fast rates of cooling by elimination of the LFE. The logistics and other aspects of practicality of 4 methods are also discussed.