Disaccharide-assisted inkjet freezing for improved cell viability

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing.