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聚合物介导的噬菌体低温保存。

Polymer-Mediated Cryopreservation of Bacteriophages.

机构信息

Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.

School of Life Sciences, University of Warwick, Coventry CV4 7AL, U.K.

出版信息

Biomacromolecules. 2021 Dec 13;22(12):5281-5289. doi: 10.1021/acs.biomac.1c01187. Epub 2021 Nov 30.

Abstract

Bacteriophages (phages, bacteria-specific viruses) have biotechnological and therapeutic potential. To apply phages as pure or heterogeneous mixtures, it is essential to have a robust mechanism for transport and storage, with different phages having very different stability profiles across storage conditions. For many biologics, cryopreservation is employed for long-term storage and cryoprotectants are essential to mitigate cold-induced damage. Here, we report that poly(ethylene glycol) can be used to protect phages from cold damage, functioning at just 10 mg·mL (∼1 wt %) and outperforms glycerol in many cases, which is a currently used cryoprotectant. Protection is afforded at both -20 and -80 °C, the two most common temperatures for frozen storage in laboratory settings. Crucially, the concentration of the polymer required leads to frozen solutions at -20 °C, unlike 50% glycerol (which results in liquid solutions). Post-thaw recoveries close to 100% plaque-forming units were achieved even after 2 weeks of storage with this method and kill assays against their bacterial host confirmed the lytic function of the phages. Initial experiments with other hydrophilic polymers also showed cryoprotection, but at this stage, the exact mechanism of this protection cannot be concluded but does show that water-soluble polymers offer an alternative tool for phage storage. Ice recrystallization inhibiting polymers (poly(vinyl alcohol)) were found to provide no additional protection, in contrast to their ability to protect proteins and microorganisms which are damaged by recrystallization. PEG's low cost, solubility, well-established low toxicity/immunogenicity, and that it is fit for human consumption at the concentrations used make it ideal to help translate new approaches for phage therapy.

摘要

噬菌体(phages,细菌特异性病毒)具有生物技术和治疗潜力。为了将噬菌体作为纯物质或不均匀混合物应用,必须具有强大的运输和储存机制,不同的噬菌体在不同的储存条件下具有非常不同的稳定性。对于许多生物制品,冷冻保存用于长期储存,并且冷冻保护剂对于减轻冷诱导损伤是必不可少的。在这里,我们报告聚乙二醇(PEG)可以用于保护噬菌体免受冷损伤,其浓度仅为 10 mg·mL(约 1 wt %),在许多情况下优于甘油,后者是目前使用的冷冻保护剂。在 -20 和 -80°C 这两种实验室冷冻储存最常见的温度下,都可以提供保护。至关重要的是,所需聚合物的浓度导致在 -20°C 下形成冷冻溶液,而不像 50%甘油(导致液体溶液)那样。使用这种方法,即使在储存 2 周后,仍能实现接近 100%的噬菌斑形成单位的复苏,并且针对其细菌宿主的杀伤实验证实了噬菌体的溶菌功能。与其他亲水性聚合物的初步实验也显示出了冷冻保护作用,但在现阶段,无法得出这种保护的确切机制,但确实表明水溶性聚合物为噬菌体储存提供了一种替代工具。发现冰再结晶抑制剂聚合物(聚乙烯醇))不能提供额外的保护,与它们能够保护蛋白质和微生物不同,这些蛋白质和微生物会因再结晶而受损。PEG 的低成本、水溶性、已确立的低毒性/免疫原性,以及在使用浓度下适合人类食用,使其成为帮助转化噬菌体治疗新方法的理想选择。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1967/8672357/65476831ed3c/bm1c01187_0002.jpg

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