Controlled hydrogel-based encapsulation of macrophages determines cell survival and functionality upon cryopreservation.

The development of novel cell-based therapies has increased the necessity to improve the long-term storage of cells. The current method of cryopreservation is far from optimal, causing ice-associated mechanical and osmotic damage to sensitive cells. Cell encapsulation is emerging as a new strategy to overcome those current limitations; however, few data are applicable to slow freezing, with conflicting results and multiple experimental conditions. The objective of this research work was to evaluate the impact of capsule size and encapsulation method on cell survival and functionality after a conventional freezing protocol. To this end, cells were encapsulated in alginate beads of different sizes, spanning the range of 200-2000 µm thanks to multiple extrusion techniques and conditions, and further cryopreserved using a slow cooling rate (-1°C/min) and 10 % DMSO as cryoprotectant. Our data show that there is a strong correlation between bead size and cell survival after a slow cooling cryopreservation process, with cell viabilities ranging from 7 to 70 % depending on the capsule size, with the smallest capsules (230 µm) achieving the highest level of survival. The obtained results indicate that the beads' diameter, rather than their morphology or the technique used, plays a significant role in the post-thawing cell survival and functionality. These results show that a fine control of cell encapsulation in alginate hydrogels is required when it comes to overcoming the current limitations of long-term preservation techniques by slow cooling.