Nanofibrillated cellulose hydrogel is a cryoprotectant in conventional and high temperature cryopreservation

Cryopreservation of cells and cell products is essential for research, medical and agricultural applications but is limited by reliance on energy-intensive cryogenic infrastructure and toxic cryoprotective agents, principally dimethyl sulfoxide (DMSO). Here, we describe the successful use of nanofibrillated cellulose hydrogel (NFCh), a non-toxic, plant-derived material, as a novel non-permeating cryoprotectant in both conventional cryogenic and high-temperature cryopreservation. NFCh was combined with disaccharide excipients and glycine and tested with HepG2 and PC3 cell lines as both spheroids and cell suspensions. In high-temperature cryopreservation using a voltage freezing device operating at -7 °C, cells frozen as suspensions in NFCh retained greater viability than those frozen as spheroids, and addition of cellulase to degrade NFCh after thawing further improved recovery. Although cells survived phase transition, viability decreased with extended storage at high subzero temperatures, indicating that biological activity was not sufficiently arrested for long-term preservation without cryogenic cooling. Importantly, when used as a conventional cryoprotectant with standard controlled-rate freezing and liquid nitrogen storage, NFCh with excipients performed similarly to DMSO and outperformed medium-only controls in PC3 cells. Rapid freezing and lyophilisation of voltage-frozen cells were also attempted but did not yield viable cells under the conditions tested. These results demonstrate that NFCh is an effective, non-toxic and cost-effective replacement for DMSO in conventional cryopreservation. Combined with its previously reported lyoprotective properties, NFCh represents, to our knowledge, the first example of a 3D culture matrix which is also a combined cryo- and lyoprotectant, with potential applications in cryopreservation of cells, blood products and extracellular vesicles.