Influence of cooling and warming rates to cell viability in cryoprotectant-free cryopreservation

Abstract

Cryopreservation is widely used to maintain backups of cells as it enables the semipermanent storage of cells. In general, cell structures are damaged or destroyed by ice crystals during freezing method. Therefore, all conventional cryopreservation methods use at least one cryoprotectant agent (CPA) to suppress generation and growth of ice crystals, moreover, vitrify water inside and outside the cells. On the other hand, CPAs should be ideally avoided due to their cytotoxicity and potential side effects. We previously reported a novel cryopreservation method without a CPA by ultrarapid cooling. In this method, cells were ejected as tiny droplets by inkjet cell printing. The droplets were deposited on the liquid nitrogen (LN) cooled substrate and were vitrified, that enables cell cryopreservation. Remarkably, the cryopreserved cell viabilities were dependent on substrate and droplet size. We had 85.8 % cell viability when the cells were contained in 40 pL droplets and deposited on glass substrate 5 μm in thickness cooled by LN. Moreover, the cell viabilities were lower as the droplets size became larger or substrates became thicker. We found that the cell viabilities were influenced by both of cooling and warming rates in CPA-free cryopreservation.