UCIBIO - Applied Molecular Biosciences Unit, Department of Chemistry, School of Science and Technology, NOVA University Lisbon, Caparica, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy, School of Science and Technology, NOVA University Lisbon, Caparica, Portugal.
UCIBIO - Applied Molecular Biosciences Unit, Department of Chemistry, School of Science and Technology, NOVA University Lisbon, Caparica, Portugal.
Int J Biol Macromol. 2024 Mar;261(Pt 2):129577. doi: 10.1016/j.ijbiomac.2024.129577. Epub 2024 Jan 19.
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. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. 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. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. 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.
生物冷冻保存通常涉及使用冷冻保护剂 (CPA) 来减轻细胞在冷冻和解冻过程中承受的致命物理胁迫，但有效的 CPA 浓度具有细胞毒性。因此，天然多糖已被研究为具有生物相容性的替代品。在这里，研究了一组由 26 种不同化学组成的天然多糖，以探究它们在提高冷冻保存的 Vero 细胞代谢复苏活力 (PTV) 方面的潜力。表现最佳的冷冻保护多糖含有大量岩藻糖，与所有非冷冻保护和冷冻保护多糖相比，平均 PTV 提高了 2.8 倍（最高提高了 3.1 倍），分别比优化的商业 CryoStor™ CS5 配方（2.6 倍）高 2.8 倍。从化学计量学的角度来看，岩藻糖（18-35.7 mol%）、糖醛酸 (UA)（13.5-26 mol%）和高分子量 (MW>1 MDa) 之间的平衡产生了最佳的 PTV。主成分分析 (PCA) 表明，岩藻糖通过与电荷无关、MW 缩放机制（PC1）增强细胞存活，这与 UA 主导的冰生长破坏（PC2）截然不同。其中性性质和与其他中性单体不同的独特性质表明，岩藻糖可能在多糖对冰生长抑制的构象适应性中发挥被动作用，或者通过结合在细胞膜稳定中发挥主动作用。最终，富含岩藻糖的阴离子多糖可能会参与聚合物-冰和聚合物-细胞相互作用，这些相互作用积极破坏冰并最小化由于平衡的亲水性和疏水性而导致的致命体积波动。我们的研究表明，中性岩藻糖在增强细胞冷冻保存结果方面起着关键作用，这反驳了多糖的多阴离子性是冷冻保护的唯一主要预测因子的先前假设。
