Fucose is an essential feature in cryoprotective polysaccharides

Abstract

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Our current investigation studied 26 natural polysaccharides as potential CPA, probing correlations between post-thaw metabolic viability (PTV) of cryopreserved Vero cells and monomeric composition. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. To deconvolute multiple variable effects, principal component analysis (PCA) coupled to K-means clustering was performed. Two major mechanisms of action explained PTV variability: a charge-dependent effect of contrasting charged uronic acid and neutral monomer compositions, and a MW-scaled charge-independent mechanism exclusively attributed to fucose. Ultimately, our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection, highlighting the potential of fucose-rich polyanionic polysaccharides.