Effective Cryopreservation and Recovery of Living Cells Encapsulated in Multiple Emulsions

The ability to preserve living cells or stem cells is critical for their use in cell therapy, especially for regenerative, reproductive, and transfusion medicine. This article addresses the low survival rates of cells and their loss of function during traditional freezing and banking (cells in a liquid medium with cryoprotectants). In this article, we developed multiple emulsions (water-in-oil-in-water type) for the effective encapsulation and cryopreservation of cells. In multiple emulsions, the oil drops, acting as a protective membrane, contain even smaller water droplets with encapsulated living cells, dispersed in the continuous water phase. The multiple emulsions with HEK293 cells encapsulated in the internal alginate droplets were successfully prepared in a Couette-Taylor flow biocontactor. The cryoprotectants (sucrose/dimethyl sulfoxide-DMSO) were located within the internal or external or both water phases of the emulsions. Encapsulated and non-encapsulated cells were frozen to -80°C (cooling rate: -1°C/min) and then transferred to liquid nitrogen (-196°C) for 24 hours. The standard rapid warming procedure was applied to thaw samples. Cell proliferation and viability were measured by using the AlamarBlue™ assay after recovery of cells. The results showed that the viability of cells encapsulated in the internal droplets of multiple emulsions, and then cryopreserved, was significantly higher, up to 27.9%, than that observed for cells conventionally cryopreserved (non-encapsulated cells in water). Moreover, the effective cell-loaded multiple emulsions-based banking method allowed DMSO-toxic cryoprotectant-to be eliminated from the cryopreservation system. The proposed approach of the cryoprotection of cells encapsulated in multiple emulsions could minimize cell damage, degradation, and their loss during freezing-thawing processes.