Abstract
All modern molecular biology and microbiology is underpinned by not only the tools to handle and manipulate microorganisms but also those to store, bank, and transport them. Glycerol is the current gold-standard cryoprotectant, but it is intrinsically toxic to most microorganisms: only a fraction of cells survive freezing and the presence of glycerol can impact downstream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of microorganisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, and successful cryopreservation shown as low as 1.1 wt % of additive. The mechanism of protection is demonstrated to be linked not only to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram-negative, Gram-positive, and mycobacteria strains. This represents a step-change in how microorganisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.
Highlights
Bacteria and their study underpin all research in infectious diseases, microbiology, and structural and molecular biology as well as being crucial in biotechnology and food processes
The primary aim of this study was to evaluate the role of ice recrystallization inhibiting (IRI) polymers to enable solventfree cryopreservation of bacteria
This study reports a disruptive approach to store bacteria in the frozen state through modulation of ice recrystallization with synthetic polymer formulations, which mimic antifreeze proteins used in nature to survive extreme environments
Summary
Bacteria and their study underpin all research in infectious diseases, microbiology, and structural and molecular biology as well as being crucial in biotechnology and food processes (notably probiotics). Cryopreservation traditionally requires the addition of organic solvents to mitigate the damage caused by ice formation and growth as well as membrane rupture and osmotic stress which would otherwise lead to cell death.[10,11] For mammalian cells, dimethyl sulfoxide (DMSO) is the most widely employed cryoprotectant for both slow freezing and vitrification (depending on the DMSO concentration), and for bacteria, glycerol is typically used. While very successful and used globally these are not perfect solutions They require high concentrations (10−25 wt %), the solvents can potentially have cytotoxic effects necessitating careful addition and rapid removal (post-thaw) to maintain viability,[12,13] and they do not lead to quantitative recovery of all cells, as such there is a need to investigate innovative cryopreservation methods
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