Development of a Cryoprotective Formula Based on the Fucose-Containing Polysaccharide FucoPol

Guerreiro, Bruno M.; Silva, Jorge; Torres, Cristiana A.V.; Alves, Vítor D.; Lima, João Carlos; Reis, Maria A.M.; Freitas, Filomena

doi:10.1021/acsabm.1c00007

Cited by 7 publications

(18 citation statements)

References 29 publications

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“…At 2.5 mg ml −1 , FucoPol increased the post-thaw viabilities of DMSO-cryopreserved cells by 70% (ref. 107 ). As with many emerging cryoprotectants, the exact mechanism is not yet clear and it has minimal effects on ice growth, but was suggested to reduce the extent of supercooling.…”

Section: Chemical Tools For Cryopreservationmentioning

confidence: 99%

Chemical approaches to cryopreservation

2022

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Cryopreservation of cells and biologics underpins all biomedical research from routine sample storage to emerging cell-based therapies, as well as ensuring cell banks provide authenticated, stable and consistent cell products. This field began with the discovery and wide adoption of glycerol and dimethyl sulfoxide as cryoprotectants over 60 years ago, but these tools do not work for all cells and are not ideal for all workflows. In this Review, we highlight and critically review the approaches to discover, and apply, new chemical tools for cryopreservation. We summarize the key (and complex) damage pathways during cellular cryopreservation and how each can be addressed. Bio-inspired approaches, such as those based on extremophiles, are also discussed. We describe both small-molecule-based and macromolecular-based strategies, including ice binders, ice nucleators, ice nucleation inhibitors and emerging materials whose exact mechanism has yet to be understood. Finally, looking towards the future of the field, the application of bottom-up molecular modelling, library-based discovery approaches and materials science tools, which are set to transform cryopreservation strategies, are also included.

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Section: Chemical Tools For Cryopreservationmentioning

confidence: 99%

Chemical approaches to cryopreservation

2022

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“…We have previously demonstrated that FucoPol can protect cells from freezing damage [19,20], but the absence of cell apoptosis after 6 h post-thaw was suggestive of FucoPol also enacting ROS scavenging. Some photolytic mechanisms also relate to oxidative damage when radical species are formed, and FucoPol has shown good photoprotective properties [21].…”

Section: Introductionmentioning

confidence: 96%

“…FucoPol is a high-molecular-weight (1.7-5.8 × 10 6 Da) fucose-containing polysaccharide secreted by the Gram-negative bacterium Enterobacter A47 (DSM 23139) [17,18], whose cryoprotective [19,20] and photoprotective [21] properties were recently demonstrated. FucoPol has a fucose, galactose, glucose, and glucuronic acid hexamer motif (2.0:1.9:0.9:0.5 M ratio), a main chain composed of a →4)-α-L-Fucp-(1→4)-α-L-Fucp-(1→3)β-D-Glcp(1→ trimer repeating unit, and a trimer branch α-D-4,6-pyruvyl-Galp-(1→4)-β-D-GlcAp-(1→3)-α-D-Galp(1→ in the C-3 of the first fucose [19] (Figure S1).…”

Section: Introductionmentioning

confidence: 99%

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Antioxidant Potential of the Bio-Based Fucose-Rich Polysaccharide FucoPol Supports Its Use in Oxidative Stress-Inducing Systems

et al. 2021

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Reactive oxygen species (ROS) are dangerous sources of macromolecular damage. While most derive from mitochondrial oxidative phosphorylation, their production can be triggered by exogenous stresses, surpassing the extinction capacity of intrinsic antioxidant defense systems of cells. Here, we report the antioxidant activity of FucoPol, a fucose-rich polyanionic polysaccharide produced by Enterobacter A47, containing ca. 17 wt% of negatively charged residues in its structure. Ferric reducing antioxidant power (FRAP) assays coupled to Hill binding kinetics fitting have shown FucoPol can neutralize ferricyanide and Fe3+-TPTZ species at an EC50 of 896 and 602 µg/mL, respectively, with positive binding cooperativity (2.52 ≤ H ≤ 4.85). This reducing power is greater than most polysaccharides reported. Moreover, an optimal 0.25% w/v FucoPol concentration shown previously to be cryo- and photoprotective was also demonstrated to protect Vero cells against H2O2-induced acute exposure not only by attenuating metabolic viability decay, but also by accentuating post-stress proliferation capacity, whilst preserving cell morphology. These results on antioxidant activity provide evidence for the biopolymer’s ability to prevent positive feedback cascades of the radical-producing Fenton reaction. Ultimately, FucoPol provides a biotechnological alternative for implementation in cryopreservation, food supplementation, and photoprotective sunscreen formula design, as all fields benefit from an antioxidant functionality.

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“…In this work, we explore the influence of the bio-based carbohydrate polymer FucoPol on aqueous supercooling under isochoric conditions. We show that a fucose-rich polysaccharide, with proven crystal size reduction and in vitro cryoprotective properties, can confine the randomness of ice nucleation to a narrow, well-defined, near-deterministic range of nucleation temperatures. This stochasticity is one of the most defining physical constraints in manipulating success/failure rates in supercooled biopreservation.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant

Guerreiro

Consiglio

Rubinsky

et al. 2022

ACS Biomater. Sci. Eng.

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Metastable supercooling has emerged as a transformative technique for ice-free biopreservation, but issues of stability inherent to the stochastic nature of ice formation have thus far limited its translation out of the laboratory. In this work, we explore the influence of the bio-based carbohydrate polymer FucoPol on aqueous supercooling using an isochoric nucleation detection technique. We show that FucoPol, a high-molecular-weight, fucose-rich polysaccharide, which has previously been shown to reduce average ice crystal sizes after nucleation, also induces a concentration-dependent stabilization of metastable supercooled water, as evidenced by both a significant reduction in nucleation stochasticity (i.e., the spread in temperatures over which the system will nucleate upon cooling) and a corresponding increase in the predicted induction time of nucleation. FucoPol is found to confine the stochasticity of ice nucleation to a narrow, well-defined band of temperatures roughly one-third as wide as that of pure water under identical conditions. Importantly, this substantial reduction in stochasticity is accompanied by only a minimal (<1 °C) change in the average nucleation temperature, suggesting that this effect is distinct from colligative freezing point depression. Reducing and characterizing the stochasticity of aqueous supercooling is essential to the engineering design of practical biopreservation protocols, and the results reported herein suggest that high-viscosity polymer systems may provide a powerful and largely unexplored lever by which to manipulate metastable-equilibrium phase change kinetics at subzero temperatures.

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Development of a Cryoprotective Formula Based on the Fucose-Containing Polysaccharide FucoPol

Cited by 7 publications

References 29 publications

Chemical approaches to cryopreservation

Chemical approaches to cryopreservation

Antioxidant Potential of the Bio-Based Fucose-Rich Polysaccharide FucoPol Supports Its Use in Oxidative Stress-Inducing Systems

Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant

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