Polymer-Mediated Cryopreservation of Bacteriophages

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 (∼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.

Ice Recrystallization Inhibition Using l-Alanine/l-Lysine Copolymers

“…The size of the ice crystal formed in each solution was quantitatively analyzed by the dose-dependent curve of IRI activity, where the MLGS of ice crystals formed in PBS was assigned to a value of 100%. , The IRI activity was compared as a function of A/K composition (Figure c) and molecular weight (Figure d) of the PAK. The curves indicated that the IRI activities of the all polymers were very low at concentrations <0.01 mg/mL with a MLGS >70% of that of PBS.…”

Aptamers Enhance Oncolytic Viruses’ Antitumor Efficacy

“…Thus, delivery of the active viruses depends on the cold chain, a distribution network designed to maintain optimally low temperatures during transportation and storage. Several agents, such as metal ions, albumin, gelatin, and poly(ethylene glycol), have been used to increase the resistance of the viruses to low temperatures [ 105 , 106 , 107 ]. However, these compounds are characterized by low cryoprotection efficiency or high cellular toxicity.…”

Synthesis of Poly(allyl glycidyl ether)-Derived Polyampholytes and Their Application to the Cryopreservation of Living Cells

“…In this situation, polymeric materials have the advantage of filling volume that would otherwise be ice without exerting significant osmotic pressure across the cell membrane because of their high molecular weight. Some polymeric cryoprotectants, most notably poly­(vinyl alcohol) (PVA), inhibit the Ostwald ripening of ice, which is believed to impart mechanical stresses on cells during thawing. − Other polymers with little to no effect on Ostwald ripening have also been employed as cryoprotectants. − For example, hydroxyethyl starch, , poly­(glycerol monomethacrylate)- b -poly­(2-hydroxypropyl methacrylate), and polyvinylpyrrolidone have been used at 5–20 wt % to preserve red blood cells. Additionally, poly­(ethylene oxide) has been employed as a cryoprotectant for E. coli , alginate hydrogels have been used to encapsulate stem cells for frozen storage, and polymers with sulfoxide-appended backbones have been shown to promote superior recovery and viability in A549, 3T3, and NHDF cells …”