Inhibiting Ice Recrystallization by Nanocelluloses: Effects of Surface Charge Density and Fibril Length, and Applications in Cell Cryopreservation

Ice recrystallization (IR) is the change in size, number, and morphology of ice crystals during storage and thawing, which causes cell death in cryopreservation. Recent studies link a facial amphiphilic structure to the IR inhibition (IRI) activity in antifreeze (glyco) proteins (AF(G)Ps) and mimics. Recent studies also found that nanocelluloses are amphiphilic. We hypothesized that nanocelluloses are IRI active and can be used to improve the cell viability in cryopreservation. The first part of this research explored the IRI activity in nanocelluloses. At mg/mL level, Cellulose nanocrystals (CNCs) and 2, 2, 6, 6-tetramethylpiperidine-1oxyl oxidized cellulosenanofibrils (TEMPO-CNFs) inhibited IR in 0.01 M NaCl without thermal hysteresis or dynamic ice shaping activity. The IRI activity of nanocelluloses was not affected by the quenching temperature but decreased with the increase of annealing temperature. The second part investigated the relationship between surface charge density (SCD) and the IRI activity in nanocelluloses. Nanocelluloses with similar fibril lengths but varied SCDs were synthesized, characterized, and compared for IRI activity. Reducing SCD promotes the IRI activity until fibril aggregation occurred. The third part studied the effect of fibril length on the IRI activity of nanocelluloses. TEMPOCNFs with similar SCDs and fibril widths but with different fibril lengths were prepared, characterized, and compared for IRI activity. Longer nanocelluloses are more IRI active. The IRI activity was not correlated with the viscosity or gelling properties of nanocellulose dispersions. The fourth part examined the potential application of nanocelluloses in cryopreservation. A novel type of nanocelluloses - electrosterically stabilized cellulose nanocrystals (ECNCs) were synthesized. ECNCs remained dispersed and IRI-active at high ionic strengths. ECNCs improved the post-thaw viability of HCT-116 colorectal cancer cells in slow/fast freezing-slow thawing protocols in the presence of 1-20% dimethyl sulfoxide (DMSO), as well as in slow/fast freezingfast thawing protocols at reduced DMSO concentrations. The effectiveness in cryoprotection did not match the IRI activity in ECNCs, polyethylene glycol, and polyvinyl alcohol; and in ECNCs with different surface charge densities. Nanocelluloses are inexpensive, biocompatible, and sustainable materials, this dissertation has demonstrated their IRI activity, which have great potentials for frozen foods and cryopreservation applications.