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Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Microorganisms

Cite this: Biomacromolecules 2018, 19, 8, 3371–3376
Publication Date (Web):June 22, 2018
https://doi.org/10.1021/acs.biomac.8b00660
Copyright © 2018 American Chemical Society
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Abstract

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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.

Introduction

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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). A key challenge with any biotechnology or cell-based processes is the logistics—the storage, transport, distribution, and recovery of intact and viable cells suitable for purpose, with no pheno/genotype alterations. Continuous culture is rarely practical; thus, lyophilization and cryopreservation are commonly employed in both academic and industrial settings.(1,2) Cryopreservation,(3,4) especially in research laboratories, is the most commonly used, as no infrastructure (other than a −80 °C freezer or liquid nitrogen (−196 °C)) is required, ensuring it is easy to use, whereas lyophilization requires optimization and more specialized equipment/formulations. The storage of bacteria for supplements such as probiotics is growing, and recent research into the microbiome points toward its involvement in a host of medical disorders including, but not limited to, irritable bowel syndrome, depression, cardiovascular disease, and obesity, showing the significant need for strategies to store bacteria,(5−8) especially if microbiome transplants become a reality for the treatment of drug-resistant infections, for example.(9)
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.
Extremophile organisms have evolved many strategies to enable them to survive in ice-rich environments, providing inspiration for new biomimetic strategies to improve cryopreservation.(14,15) For example, Arctic fish species produce a diverse range of antifreeze proteins (AFPs) which function to limit ice growth by ice recrystallization inhibition (IRI).(16−18) AFPs are not suitable for many applications though, as they cannot be produced readily on a large scale, and their secondary effect of dynamic ice shaping can lead to reduced cell recovery post-thaw.(19,20) Furthermore, biomedical applications are hampered due to lack of sufficient immunological/toxicological data. Therefore, synthetic macromolecular mimics of AFPs have emerged, which can reproduce their properties but benefit from the scalable and tunable synthesis of synthetic polymers.(18,21,22) These have been used to enhance the cryopreservation of mammalian cells including blood,(23−25) cell lines,(26) and primary cells.(27,28) Their primary function is ice recrystallization inhibition, where the rate of growth (not formation) of ice is slowed, leading to reduced cell death during thawing. The most active polymer mimics reported to date are based on not only poly(vinyl alcohol)(29−34) but also Safranin-O,(35) poly(ampholyte)s,(36,37) and glycopeptides.(38) However, there are no reports, to the best of our knowledge, on applying IRI-active materials to the challenge of bacterial cryopreservation, with only a report of nonfreezing stabilization due to freezing point depression, which is a distinct process for nonfreezing storage.(39)
This paper takes inspiration from extremophile organisms to demonstrate successful bacterial cryopreservation using a unique all-polymer, glycerol-free, formulation. This formulation benefits from a synergistic effect of an ice growth inhibiting polymer with a secondary hydrophilic polymer, and they outperform the current gold-standard glycerol, leading to increased cell yields at low cryoprotectant concentrations.

Experimental Section

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Bacteria Growth

Escherichia coli TOP10 cells, Bacillus subtilis (WT168), and Mycobacterium smegmatis (Mc2155) were chosen for freezing as three different types of bacteria with different cell structures. E. coli and B. subtilis were grown in Lysogeny broth (LB) medium containing 100 μg mL–1 ampicillin, and M. smegmatis was grown in 7H9 broth base at 37 °C with a stirring rate of 180 rpm.

Ice Recrystallization Inhibition (Splat) Assay

A 10 μL droplet of cryoprotectant in PBS solution is dropped from 1.4 m onto a glass microscope coverslip on top of an aluminum plate cooled to −78 °C using dry ice. Upon impact with the plate the droplet instantly freezes, spreading out and forming a thin wafer of ice. This wafer is then placed on a liquid nitrogen cooled cryostage cooled to −8 °C. The wafer is then left to anneal for 30 min at −8 °C. Three photographs are then taken of the wafer in different locations at 20× zoom under cross-polarizers. The longest grain crystals as well as the total number of crystals in the image are counted using ImageJ; the area of the field of view divided by this number of crystals gives the average crystal size per wafer, which is reported as a % of area compared to PBS control.

Freezing Protocols

Different molecular weight PEGs (100 mg mL–1; 200 Da, 400 Da, 1.5 kDa, 4 kDa, 6 kDa, and 8 kDa) and PVAs (1 mg mL–1; 10, 23, and 31 kDa) were dissolved in PBS for comparison in cryoprotective activity alongside poly(isopropenyl acetate-alt-maleic anhydride) functionalized with (dimethylamino)ethanol (poly(ampholyte)), AFPIII, and glycerol. After growing each of the bacteria until OD600, cells (150 μL) were added to separate solutions of the different cryoprotectants (150 μL) in 1.5 mL vials and snap frozen in liquid nitrogen before thawing at 25 °C in a water bath for 5 min. The freeze–thaw (FT) cycles were repeated seven times, and then the samples were added to LB in 96-well plates (200 μL). Serial dilutions took place, and the samples were plated on ampicillin plates. These were left to grow at 37 °C for subsequent colony counts. Confocal microscopy was performed to evaluate the number of live/dead cells obtained. When storage at −20 °C was to be assessed, samples were prepared similarly up until they were snap frozen in liquid nitrogen, after which they were directly stored at −20 °C.

Cryoprotectant Toxicity

Equal volumes of bacteria and cryoprotectant solutions were left shaking at 4 °C overnight. The cryoprotectant samples (20 μL) were then added to LB in wells (200 μL) in the first column of 96-well plates, and serial dilutions of the samples were produced by transferring 20 μL from column 1 to column 2, mixing, and then taking 20 μL from column 2 to column 3. This was repeated until column 7 was reached, giving dilutions up to 10–7. Dilutions 10–2–10–5 were plated on ampicillin plates, and colonies were grown at 37 °C for subsequent counting. Toxicity assays were repeated using three PEG concentrations (100, 50, and 10 mg mL–1) combined with a constant PVA concentration of 0, 1, or 10 mg mL–1.

Cellular Growth Profile

Toxicity and protection of cells in liquid culture were studied using absorbance spectroscopy. Samples were prepared as before and underwent seven FT cycles. The cells were then grown in LB (200 mL) over 14 h under continuous shaking at 37 °C, and their OD was measured at 600 nm at 10 min intervals and compared to a control sample that underwent no freeze–thaw cycles. This growth assay was performed also on cells combined with different AFP concentrations (0.1, 2.5, and 5 mg mL–1) for comparison as well as samples frozen at −20 °C as well as −196 °C.

Live/Dead Bacterial Viability Test

Following seven freeze–thaw cycles using the indicated conditions, samples were isolated at 10000g for 10 min, and the supernatant was discarded. An aliquot was taken prior to the freeze–thaw cycle as a live cell control, and a further aliquot was heat killed (incubated at 80 °C for 30 min) for a dead cell control. Cells were resuspended in 20 μL of 0.85% NaCl solution. 10 μL of this suspension was diluted in 200 μL of 0.85% NaCl solution, and the samples were incubated at room temperature for 1 h. Samples were pelleted at 10000g for 10 min, the supernatant was discarded, and the cells were resuspended in 100 μL of 0.85% NaCl solution. Next, the LIVE/DEAD bacterial viability staining mixture was prepared by mixing SYTO-9 and propidium iodide to final concentrations of 1.67 and 10 mM, respectively. The cells were stained by adding 0.3 μL of the staining solution to 100 μL of cell suspension and incubating in the dark for 15 min (at room temperature). Slides for microscopy were prepared by trapping 5 μL of the stained bacterial suspension between a slide and a coverslip. Samples were then analyzed by means of fluorescent microscopy (at either 100× or 60× magnification) using GFP (excitation 470/40 nm, emission 525/50 nm) and mCherry (excitation 560/40 nm, emission 630/75 nm) filter sets to visualize the SYTO-9 and propidium iodide staining, respectively.

Results and Discussion

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The primary aim of this study was to evaluate the role of ice recrystallization inhibiting (IRI) polymers to enable solvent-free cryopreservation of bacteria. A range of synthetic polymers were selected for this based on their previous use in mammalian cell cryopreservation: poly(vinyl alcohol), PVA, which is a potent IRI; poly(ethylene glycol), PEG, which has no IRI but was chosen to provide a hydrophilic “bulking” agent which has been shown to be important for blood cell cryopreservation;(40,41) a poly(ampholyte) (p(amph)), which has a weaker IRI activity than PVA;(25,42) and recombinant AFPIII, an antifreeze protein originally isolated from ocean pout(43) (Figure 1A). It is important to note that PVA and PEG were particularly of interest as they are low-cost (comparable to glycerol), are available in a range of molecular weights, and are produced to food/clinical grades, making them ideal for translational applications.(44) The polymers’ IRI activity was evaluated by a modified splat assay (Figure 1B–G). Briefly, ice wafers were nucleated to give small (<10 μm) ice crystals, which were allowed to grow for 30 min and then measured. Smaller ice crystals indicated more IRI activity, reported as the mean largest grain size (MGLS). The concentrations chosen for the IRI assays related to those used in cryopreservation experiments. Detailed studies on their IRI activity have been reported previously, and a comparison is shown in Figure S3 (Supporting Information).(29,36)

Figure 1

Figure 1. Cryoprotectants used and IRI activity at concentrations relevant for this work. (A) Chemical structures. Cryomicrographs of ice wafers grown in the presence of (B) 100 mg mL–1 4 kDa PEG + 1 mg mL–1 10 kDa PVA, (C) 1 mg mL–1 10 kDa PVA, (D) 100 mg mL–1 4 kDa PEG, (E) 1 mg mL–1 AFPIII, (F) 50 mg mL–1 poly(ampholyte), and (G) PBS control. Scale bar = 100 μm.

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To evaluate the performance of these IRI active polymers versus glycerol, a series of cryopreservation experiments were undertaken. E. coli was added to different cryoprotective formulations and then exposed to seven freeze–thaw cycles from liquid nitrogen (−196 °C) to room temperature (20 °C), and the number of colony forming units determined by growth on agar plates for 16 h was recorded (Figure 2A). This was chosen to mimic laboratory conditions where stocks are often frozen and thawed during routine use. 25% glycerol resulted in an average of 15 recovered colonies compared to 1 for no added cryoprotectant. PVA, AFP, and the poly(ampholyte) alone gave results identical to those with no cryoprotectant added. (It should be noted that the concentrations of each of the above were chosen based on their relative IRI activity to give similar effects, not at equal mass concentration to enable us to correlate the physical properties to the observed biological responses.) Our previous work using red blood cells (which also have no nucleus, like bacteria) has shown that IRI active polymers increased post-thaw recovery only when used in combination with a hydrating secondary cryoprotectant such as polymer gels or hydroxyethyl starch.(40,41) PEG was therefore added (due to biocompatibility and commercial availability), and the mixture PEG/PVA (100 mg mL–1 + 1 mg mL–1) was found to dramatically increase recovery to 69 colony forming units, which is a >4-fold increase compared to glycerol alone. PEG/AFP mixtures lead to similar results (52 colonies), supporting the hypothesis that controlling IRI is the key mechanism in protecting bacteria during cryopreservation, by reducing ice growth especially during thawing. The poly(ampholyte)s, however, showed no cryoprotective effect, despite them previously being used for mammalian cells, where they appear to function via cell membrane interactions.(37) Poly(ampholyte)s have far weaker IRI than PVA or AFPs, and hence this supports a mechanism of protection based on limiting ice recrystallization rather than membrane plasticization/stabilization. In order to test whether there is a significant difference between the recovery obtained from PEG/PVA versus that obtained from PVA alone, a two-sample t test was performed, and it was concluded that the difference between them was statistically significant (Tables S1 and S2, Supporting Information).

Figure 2

Figure 2. (A) Recovered colonies of E. coli after seven freeze (−196 °C)–thaw (20 °C) cycles. (B) Recovered colonies of E. coli after overnight incubation with cryoprotectants. Concentrations of cryoprotectants: [glycerol] = 25 wt %; [AFPIII] = 1 mg mL–1; [PVA] = 1 mg mL–1; [PEG/AFPIII] = 100 + 0.01 mg mL–1; [PEG/PVA] = 100 + 1 mg mL–1; [poly(ampholyte)] = 50 mg mL–1). Control is LB media alone.

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A key challenge associated with the use of glycerol is its intrinsic toxicity at cryopreservation concentrations, so the impact of incubating the polymers with E. coli compared to glycerol was evaluated. Each component (at the indicated cryopreservation concentration) was incubated with E. coli overnight at 4 °C, and subsequently the number of colony forming units was determined (Figure 2B). Glycerol at 15 or 25 wt % led to a significant reduction in recovered colonies. Conversely, none of the polymers showed toxicity to the E. coli. The increase in recovered colonies for PVA could be due to some metabolism of the polymer. This supports our hypothesis that biomimetic macromolecular antifreezes are “spectator additives” which only function when ice is present and are ignored by microorganisms (and indeed other cells) which is crucial for downstream applications.
To further optimize this formulation, the PEG concentration was varied from 100 to 10 mg mL–1, all with addition of 1 mg mL–1 PVA, and the number of recovered colonies after seven freeze–thaw cycles was counted (Figure 3A). Reducing the concentration of PEG to 50 and 10 mg mL–1 led to a significant reduction in the number of colonies recovered compared to 100 mg mL–1. However, it is important to note that 10 mg mL–1 PEG with 1 mg mL–1 PVA is just a 1.1 wt % solution but performs equally to 25 wt % glycerol, which represents a remarkable cryopreservation outcome with a 25-fold reduction in cryoprotectant. It shows that while there is an optimum formulation, there is scope to vary the components and hence supporting ease of use in a realistic laboratory situation. In some downstream applications (such as food) lowering cryoprotectants concentration, rather than maximizing total cell recovery, is desirable, and this new polymer-only formulation is clearly suitable.

Figure 3

Figure 3. (A) Effect of varying PEG concentration on number of recovered E. coli colonies after seven freeze (−196 °C)–thaw (20 °C) cycles. (B) Live/dead viability testing on E. coli immediately after freeze–thaw cycle, with percentage of green (intact membrane) bacteria determined by confocal microscopy. [PEG/PVA] = 100 + 1 mg mL–1. Error bars represent SD from six repeats.

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The above experiments relied on counting colony-forming units, which shows the application but does not give insight into the mechanisms of cell stress during freeze–thaw. To assess the bacteria immediately after thawing, confocal microscopy was employed with a live/dead viability assay which measures the integrity of cell membranes (Figure 3B and Figure S11). Bacterial cells with intact cell membranes exhibit green fluorescence (SYTO-9 dye) while those with compromized cell membranes exhibit red fluorescence (propidium iodide dye). Following freeze/thaw in PBS alone just 2.2% of the E. coli had intact membranes (green), demonstrating that ice growth causes significant mechanical damage. Post-freeze/thaw in either 25% glycerol or PEG/PVA resulted in 15–18% of the E. coli retaining intact membranes. These observations suggest that the mechanisms of cryoinjury of both glycerol and our macromolecular antifreezes are very similar. However, the colony counting results (Figure 2) confirmed the polymer approach to be superior, supporting a hypothesis that the reduced toxicity of the polymers means that more and healthier colonies can grow post-thaw, and reduced growth inhibition compared to glycerol.
Additional bacterial strains for cryopreservation were selected to cover a wide range of genera to ensure these effects are not unique to E. coli. Bacillus subtilis was chosen as a Gram-positive strain and Mycobacterium smegmatis as a mycobacteria (distinct cell wall compared to other Gram-positives) for further analysis. Using the same conditions as for E. coli, the cells were exposed to seven freeze–thaw cycles, and the recovered colonies were counted (Table 1). To enable comparison of the data and to account for the different growth rates of each bacterial strain, the recovered colonies were also normalized to the highest recovery (Figure 4).

Figure 4

Figure 4. Normalized cell recovery for three different bacteria upon addition of different cryoprotectants after seven freeze (−196 °C)–thaw (20 °C) cycles (red = control, blue = glycerol, black = PEG/PVA). Values obtained are normalized to themselves.

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Table 1. Mean Colonies Recovered after Seven Freeze–Thaw Cyclesa
 E. coliM. smegmatisB. subtilis
 totalnormalized (%)totalnormalized (%)totalnormalized (%)
control1 ± 0.414 ± 0.51229 ± 10.411
25% glycerol15 ± 2.82230 ± 6.08893 ± 18.935
AFPIII3 ± 0.942 ± 0.3626 ± 6.710
PVA0 ± 0.103 ± 1.595 ± 0.72
PEG53 ± 6.77728 ± 4.382150 ± 10.757
PEG/PVA69 ± 7.310034 ± 4.2100262 ± 39.3100
poly(ampholyte)5 ± 0.6728 ± 4.58220 ± 5.08
a

[Glycerol] = 25 wt %; [PEG] = 100 mg mL–1; [PEG/PVA] = 100 + 1 mg mL–1; [AFPIII] = 0.1 mg mL–1; poly(ampholyte) = 50 mg mL–1. Error represents the SD from six repeats. Note that the total colonies recovered for each organism vary based on their own growth rates; hence, normalized recovery (versus the highest recovery level) is also included.

In all cases, the PEG/PVA mixture gave equal or better levels of recovery than glycerol alone. It was noted that M. smegmatis (which is a slow-growing organism compared to other two) gave fewer colonies after a fixed period of growth in all conditions, but the PEG/PVA still matched the performance of glycerol. In some cases, the PEG alone gave favorable recovery levels also (as any uncharged solute will give some protection), but in all cases addition of PVA increased this recovery, showing it is an essential component to ensure recovery of viable cells, without resorting to multiple rounds of freezing controls. These results demonstrate the versatile nature of this approach and that replacing glycerol in laboratories with this polymer formulation is a reliable way to match or improve current storage methods.
This macromolecular cryoprotection solution using ice-inhibiting polymers is clearly suitable for bacteria storage, but there are many parameters which can be varied in this system including the molecular weight of the polymers and weight ratios. To enable a large number of conditions to be screened, the post-thaw growth rate of E. coli was also followed by OD600 (turbidity) measurements, which enable higher throughput measurements in 96-well plates. E. coli was frozen with the indicated formulations and post-thaw inoculated into LB media, and their growth was monitored. After seven freeze–thaw cycles in liquid nitrogen, cells cryopreserved in 25% glycerol and PEG/PVA (100 and 1 mg mL–1, respectively) had essentially identical growth rates (Figure 5), which were faster than all the other formulations used.

Figure 5

Figure 5. E. coli growth profiles after seven freeze (−196 °C)–thaw (20 °C) cycles and then inoculation into LB media.

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By monitoring growth profiles of E. coli, various formulations and molecular weights of PVA were also tested for their efficacy (Figures S5–S7). For PVA, 10 and 31 kDa gave approximately identical recovery levels. However, 10 kDa is easier to dissolve into buffer, making it preferable for real applications where concentrated stock solutions are required. Higher molecular weight PVA can also lead to dynamic ice-shaping which is known to reduce recovery of cells post-thaw.(23) Lower molecular weight PEGs (200–1500 Da) alone appeared to have a slightly greater cryoprotective effect than larger PEGs (4–8 kDa), as they reach a higher OD600 and display slower logarithmic decline phases, indicating improved cell health, but these differences were small (Figure S6). Various permutations of PEG/PVA were also screened (Figure S7), and in all cases addition of PVA increased the cryoprotective effect and lengthened the stationary phase of cells. The improvement was most significant in the case of larger PEGs. Considering the cryoprotective effect of each of the mixtures, and the solubility of each of the constituents, it was determined that 10 kDa PVA and 4 kDa PEG were the optimum molecular masses out of the 18 formulations we tested to use to ensure reliable and easy to use cryopreservation.
As a final test, the effect of storage at −20 °C in a standard laboratory freezer after first snap freezing (−80 °C) was also studied after 1 and 4 weeks of storage (Figure S4). It was found that bacterial recovery was similar for 25% glycerol and PEG/PVA after 1 week storage, which supports the use of this method for routine laboratory manipulations. For 4 week storage we noted that the glycerol formulation yielded more colonies, but this was not optimized, and significant recovery was still possible with the polymer-only mixture. It should be noted that the glycerol solution did not fully freeze at −20 °C, thus providing a carbon source to the E. coli.

Conclusions

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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. It is shown that PEG/PVA mixtures provide a synergistic effect with the number of recovered bacterial colonies after seven freeze–thaw cycles being up to 4-fold higher than the current gold standard of glycerol. Confocal microscopy showed that similar numbers of bacteria with intact membranes are recovered for both systems but that the polymer cryoprotectants lead to more viable colonies, suggesting their lower toxicity is crucial. This new cryopreservation approach is shown to be suitable for a range of bacterial genera including Gram-negative/positive and mycobacteria. It was found that the synergistic effect on cell survival was linked to PVAs ice recrystallization activity and the PEG bulking agent, as addition of type III antifreeze proteins (at similar total IRI activity) also enabled cryopreservation, although these proteins are significantly more expensive and not practical for routine cryopreservation compared to the polymer formulations. These results highlight the contrast with traditional solvent-based cryoprotectants which function by distinct mechanisms. Optimal conditions for use of this system in a laboratory or application-focused environment are presented, and as both PEG and PVA are commodity polymers available as food grade,(45,46) this system will have wide application across molecular biology, microbiome research, and for translation into the food or biotechnology industry, which are underpinned by the storage and transport of specific bacterial strains.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00660.

  • Additional growth curves and experimental details (PDF)

The authors declare the following competing financial interest(s): M.I.G., M.H., and A.F. are co-inventors on a patent filed relating to the cryopreservation of cells as described in this work.

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Acknowledgments

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M.I.G. holds an ERC starting grant (CRYOMAT 638661). The Royal Society is also thanked for funding the cryomicroscopes used in this study. The Midlands Integrative Biosciences Doctoral Training Partnership (MIBTP) is thanked for a studentship for Julia Lipecki (BB/M01116X/1) which provided assistance with confocal microscopy. We also thank Peter Davies (Queen’s University, Kingston, Canada) for providing the genetic construct encoding for AFPIII from ocean pout (rQAE isoform, M1.1HISPET20b), C. Stubbs for providing the poly(ampholyte), Dr. E. Denham for providing the Bacillus subtilis, Dr. E. Fullam for providing the Mycobacterium smegmatis, and Mohammed Ridwan Rahman for designing the image for the table of contents.

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    A review. Thermodn. and phys. chem. have played powerful roles the past 45 years in interpreting cryobiol. problems and in predicting cryobiol. outcomes. The author has been guided by a few core principles in using these concepts and tools and this paper discusses these core principles. They are (1) the importance of chem. potentials and of the difference between the chem. potentials of water and solutes inside the cell and outside in detg. the direction and rate of fluxes of water and solutes. (2) The influence of the curvature of an ice crystal on its chem. potential and on the ability of ice to pass through pores in cell membranes, on the nucleation temp. of supercooled water, and on the recrystn. of ice. (3) The use of Le Chatalier's Principle in qual. predicting the direction of a reaction in response to variables like pressure. (4) The fact that the energy differences between State A and State B are independent of the path taken to go from A to B. (5) The importance of being aware of the assumptions underlying thermodn. models of cryobiol. events. And (6), the difficulties in obtaining exptl. verification of thermodn. and phys.-chem. models.
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  5. 5
    Wang, Z.; Klipfell, E.; Bennett, B. J.; Koeth, R.; Levison, B. S.; DuGar, B.; Feldstein, A. E.; Britt, E. B.; Fu, X.; Chung, Y.-M.; Wu, Y.; Schauer, P.; Smith, J. D.; Allayee, H.; Tang, W. H. W.; DiDonato, J. A.; Lusis, A. J.; Hazen, S. L. Nature 2011, 472 (7341), 57– 63,  DOI: 10.1038/nature09922
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    Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease
    Wang, Zeneng; Klipfell, Elizabeth; Bennett, Brian J.; Koeth, Robert; Levison, Bruce S.; DuGar, Brandon; Feldstein, Ariel E.; Britt, Earl B.; Fu, Xiaoming; Chung, Yoon-Mi; Wu, Yuping; Schauer, Phil; Smith, Jonathan D.; Allayee, Hooman; Tang, W. H. Wilson; DiDonato, Joseph A.; Lusis, Aldons J.; Hazen, Stanley L.
    Nature (London, United Kingdom) (2011), 472 (7341), 57-63CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-mol. metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine - choline, trimethylamine N-oxide (TMAO) and betaine - were identified and then shown to predict risk for CVD in an independent large clin. cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a crit. role for dietary choline and gut flora in TMAO prodn., augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymic source of TMAO, segregated with atherosclerosis in hyperlipidemic mice. Discovery of a relationship between gut-flora-dependent metab. of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.
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    Sharon, G.; Sampson, T. R.; Geschwind, D. H.; Mazmanian, S. K. Cell 2016, 167 (4), 915– 932,  DOI: 10.1016/j.cell.2016.10.027
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    The Central Nervous System and the Gut Microbiome
    Sharon, Gil; Sampson, Timothy R.; Geschwind, Daniel H.; Mazmanian, Sarkis K.
    Cell (Cambridge, MA, United States) (2016), 167 (4), 915-932CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    A review. Neurodevelopment is a complex process governed by both intrinsic and extrinsic signals. While historically studied by researching the brain, inputs from the periphery impact many neurol. conditions. Indeed, emerging data suggest communication between the gut and the brain in anxiety, depression, cognition, and autism spectrum disorder (ASD). The development of a healthy, functional brain depends on key pre- and post-natal events that integrate environmental cues, such as mol. signals from the gut. These cues largely originate from the microbiome, the consortium of symbiotic bacteria that reside within all animals. Research over the past few years reveals that the gut microbiome plays a role in basic neurogenerative processes such as the formation of the blood-brain barrier, myelination, neurogenesis, and microglia maturation and also modulates many aspects of animal behavior. Herein, we discuss the biol. intersection of neurodevelopment and the microbiome and explore the hypothesis that gut bacteria are integral contributors to development and function of the nervous system and to the balance between mental health and disease.
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    Rea, K.; Dinan, T. G.; Cryan, J. F. Neurobiol. Stress 2016, 4, 23– 33,  DOI: 10.1016/j.ynstr.2016.03.001
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    The microbiome: A key regulator of stress and neuroinflammation
    Rea Kieran; Dinan Timothy G; Cryan John F
    Neurobiology of stress (2016), 4 (), 23-33 ISSN:2352-2895.
    There is a growing emphasis on the relationship between the complexity and diversity of the microorganisms that inhabit our gut (human gastrointestinal microbiota) and health/disease, including brain health and disorders of the central nervous system. The microbiota-gut-brain axis is a dynamic matrix of tissues and organs including the brain, glands, gut, immune cells and gastrointestinal microbiota that communicate in a complex multidirectional manner to maintain homeostasis. Changes in this environment can lead to a broad spectrum of physiological and behavioural effects including hypothalamic-pituitary-adrenal (HPA) axis activation, and altered activity of neurotransmitter systems and immune function. While an appropriate, co-ordinated physiological response, such as an immune or stress response are necessary for survival, a dysfunctional response can be detrimental to the host contributing to the development of a number of CNS disorders. In this review, the involvement of the gastrointestinal microbiota in stress-mediated and immune-mediated modulation of neuroendocrine, immune and neurotransmitter systems and the consequential behaviour is considered. We also focus on the mechanisms by which commensal gut microbiota can regulate neuroinflammation and further aim to exploit our understanding of their role in stress-related disorders as a consequence of neuroinflammatory processes.
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    Navaneetharaja, N.; Griffiths, V.; Wileman, T.; Carding, S. J. Clin. Med. 2016, 5 (6), 55,  DOI: 10.3390/jcm5060055
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    A role for the intestinal microbiota and virome in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)?
    Navaneetharaja, Navena; Griffiths, Verity; Wileman, Tom; Carding, Simon R.
    Journal of Clinical Medicine (2016), 5 (6), 55/1-55/22CODEN: JCMOHK; ISSN:2077-0383. (MDPI AG)
    Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous disorder of significant societal impact that is proposed to involve both host and environmentally derived etiologies that may be autoimmune in nature. Immune-related symptoms of at least moderate severity persisting for prolonged periods of time are common in ME/CFS patients and B cell depletion therapy is of significant therapeutic benefit. The origin of these symptoms and whether it is infectious or inflammatory in nature is not clear, with seeking evidence of acute or chronic virus infections contributing to the induction of autoimmune processes in ME/CFS being an area of recent interest. This article provides a comprehensive review of the current evidence supporting an infectious etiology for ME/CFS leading us to propose the novel concept that the intestinal microbiota and in particular members of the virome are a source of the "infectious" trigger of the disease. Such an approach has the potential to identify disease biomarkers and influence therapeutics, providing much-needed approaches in preventing and managing a disease desperately in need of confronting.
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    Manichanh, C.; Reeder, J.; Gibert, P.; Varela, E.; Llopis, M.; Antolin, M.; Guigo, R.; Knight, R.; Guarner, F. Genome Res. 2010, 20 (10), 1411– 1419,  DOI: 10.1101/gr.107987.110
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    Reshaping the gut microbiome with bacterial transplantation and antibiotic intake
    Manichanh, Chaysavanh; Reeder, Jens; Gibert, Prudence; Varela, Encarna; Llopis, Marta; Antolin, Maria; Guigo, Roderic; Knight, Rob; Guarner, Francisco
    Genome Research (2010), 20 (10), 1411-1419CODEN: GEREFS; ISSN:1088-9051. (Cold Spring Harbor Laboratory Press)
    The intestinal microbiota consists of over 1000 species, which play key roles in gut physiol. and homeostasis. Imbalances in the compn. of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were obsd. after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial compn. is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.
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    Ice Nucleation and Antinucleation in Nature
    Zachariassen, Karl Erik; Kristiansen, Erlend
    Cryobiology (2000), 41 (4), 257-279CODEN: CRYBAS; ISSN:0011-2240. (Academic Press)
    A review with 117 refs. Plants and ectothermic animals use a variety of substances and mechanisms to survive exposure to subfreezing temps. Proteinaceous ice nucleators trigger freezing at high subzero temps., either to provide cold protection from released heat of fusion or to establish a protective extracellular freezing in freeze-tolerant species. Freeze-avoiding species increase their supercooling potential by removing ice nucleators and accumulating polyols. Terrestrial invertebrates and polar marine fish stabilize their supercooled state by means of noncolligatively acting antifreeze proteins. Some organisms also depress their body fluid m.p. to ambient temp. by evapn. and/or solute accumulation. (c) 2000 Academic Press.
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    Shu, Z.; Heimfeld, S.; Gao, D. Bone Marrow Transplant. 2014, 49 (4), 469– 476,  DOI: 10.1038/bmt.2013.152
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    Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion
    Shu, Z.; Heimfeld, S.; Gao, D.
    Bone Marrow Transplantation (2014), 49 (4), 469-476CODEN: BMTRE9; ISSN:0268-3369. (Nature Publishing Group)
    A review. Transplantation of hematopoietic stem cells (HSCs) has been successfully developed as a part of treatment protocols for a large no. of clin. indications, and cryopreservation of both autologous and allogeneic sources of HSC grafts is increasingly being used to facilitate logistical challenges in coordinating the collection, processing, prepn., quality control testing and release of the final HSC product with delivery to the patient. Direct infusion of cryopreserved cell products into patients has been assocd. with the development of adverse reactions, ranging from relatively mild symptoms to much more serious, life-threatening complications, including allergic/gastrointestinal/cardiovascular/neurol. complications, renal/hepatic dysfunctions, and so on. In many cases, the cryoprotective agent (CPA) used-which is typically DMSO (DMSO)-is believed to be the main causal agent of these adverse reactions and thus many studies recommend depletion of DMSO before cell infusion. In this paper, we will briefly review the history of HSC cryopreservation, the side effects reported after transplantation, along with advances in strategies for reducing the adverse reactions, including methods and devices for removal of DMSO. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the DMSO concn. or using alternative CPAs for cryopreservation and removing DMSO before infusion. For DMSO removal, besides the traditional and widely applied method of centrifugation, new approaches have been explored in the past decade, such as filtration by spinning membrane, stepwise diln.-centrifugation using rotating syringe, diffusion-based DMSO extn. in microfluidic channels, dialysis and diln.-filtration through hollow-fiber dialyzers and some instruments (CytoMate, Sepax S-100, Cobe 2991, microfluidic channels, diln.-filtration system, etc.) as well. However, challenges still remain: development of the optimal (fast, safe, simple, automated, controllable, effective and low cost) methods and devices for CPA removal with min. cell loss and damage remains an unfilled need.
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    Capicciotti, C. J.; Kurach, J. D. R.; Turner, T. R.; Mancini, R. S.; Acker, J. P.; Ben, R. N. Sci. Rep. 2015, 5, 9692,  DOI: 10.1038/srep09692
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    Small Molecule Ice Recrystallization Inhibitors Enable Freezing of Human Red Blood Cells with Reduced Glycerol Concentrations
    Capicciotti, Chantelle J.; Kurach, Jayme D. R.; Turner, Tracey R.; Mancini, Ross S.; Acker, Jason P.; Ben, Robert N.
    Scientific Reports (2015), 5 (), 9692/1-9692/10CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    In North America, red blood cells (RBCs) are cryopreserved in a clin. setting using high glycerol concns. (40% w/v) with slow cooling rates (∼1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concns. with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small mol. ice recrystn. inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystn., a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addn. of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol soln. increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small mol. IRIs for the preservation of cells.
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    Microbial extremophiles at the limits of life
    Pikuta, Elena V.; Hoover, Richard B.; Tang, Jane
    Critical Reviews in Microbiology (2007), 33 (3), 183-209CODEN: CRVMAC; ISSN:1040-841X. (Informa Healthcare)
    A review. Prokaryotic extremophiles were the first representatives of life on Earth and they are responsible for the genesis of geol. structures during the evolution and creation of all currently known ecosystems. Flexibility of the genome probably allowed life to adapt to a wide spectrum of extreme environments. As a result, modern prokaryotic diversity formed in a framework of physico-chem. factors, and it is composed of: thermophilic, psychrophilic, acidophilic, alkalophilic, halophilic, barophilic, and radioresistant species. This artificial systematics cannot reflect the multiple actions of different environmental factors since one organism could unite characteristics of several extreme-groups. In this review we show the current status of studies in all fields of extremophiles and summarize the limits of life for different species of microbial extremophiles. We also discuss the finding of extremophiles from unusual places such as soils, and briefly review recent studies of microfossils in meteorites in the context of the significance of microbial extremophiles to Astrobiol.
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    Møbjerg, N.; Halberg, K. A.; Jørgensen, A.; Persson, D.; Bjørn, M.; Ramløv, H.; Kristensen, R. M. Acta Physiologica; Blackwell Publishing Ltd: Oxford, UK, 2011; pp 409– 420.
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    Phytochemistry: Heat-stable antifreeze protein from grass
    Sidebottom, Chris; Buckley, Sarah; Pudney, Paul; Twigg, Sarah; Jarman, Carl; Holt, Chris; Telford, Julia; McArthur, Andrew; Worrall, Dawn; Hubbard, Rod; Lillford, Peter
    Nature (London) (2000), 406 (6793), 256CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    There is no expanded citation for this reference.
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    Biggs, C. I.; Bailey, T. L.; Ben Graham; Stubbs, C.; Fayter, A.; Gibson, M. I. Nat. Commun. 2017, 8 (1), 1546,  DOI: 10.1038/s41467-017-01421-7
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    Polymer mimics of biomacromolecular antifreezes
    Biggs Caroline I; Bailey Trisha L; Ben Graham; Stubbs Christopher; Fayter Alice; Gibson Matthew I; Gibson Matthew I
    Nature communications (2017), 8 (1), 1546 ISSN:.
    Antifreeze proteins from polar fish species are remarkable biomacromolecules which prevent the growth of ice crystals. Ice crystal growth is a major problem in cell/tissue cryopreservation for transplantation, transfusion and basic biomedical research, as well as technological applications such as icing of aircraft wings. This review will introduce the rapidly emerging field of synthetic macromolecular (polymer) mimics of antifreeze proteins. Particular focus is placed on designing polymers which have no structural similarities to antifreeze proteins but reproduce the same macroscopic properties, potentially by different molecular-level mechanisms. The application of these polymers to the cryopreservation of donor cells is also introduced.
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    Chao, Heman; Davies, Peter L.; Carpenter, John F.
    Journal of Experimental Biology (1996), 199 (9), 2071-2076CODEN: JEBIAM; ISSN:0022-0949. (Company of Biologists)
    Antifreeze protein (AFP) types I, II and III were tested for their ability to protect red blood cells from lysis during warming, after cryopreservation in hydroxyethyl starch. All 3 types reduced hemolysis to 25% of control values at similar micromolar concns. but enhanced lysis as the AFP concn. approached millimolar levels. Site-directed mutants of type III AFP with different thermal hysteresis activities were tested for their ability to protect the cryopreserved cells from lysis. Their relative efficacy in protecting the cells correlated closely with their thermal hysteresis activity. Cryomicroscopy indicated that the protection of red cells by type III AFP and the mutant forms was due to inhibition of ice recrystn.
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    Carpenter, J. F.; Hansen, T. N. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (19), 8953– 8957,  DOI: 10.1073/pnas.89.19.8953
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    Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth
    Carpenter, John F.; Hansen, Thomas N.
    Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (19), 8953-7CODEN: PNASA6; ISSN:0027-8424.
    The influence of antifreeze proteins (AFPs) was detd. on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during warming. Relatively low concns. (e.g., 5-150 μg/mL) of winter flounder (Pseudopleuronectes americanus) AFP enhance survival of human red blood cells cryopreserved in hydroxyethyl starch solns. This effect is most apparent in samples warmed at suboptimal rates, i.e., where ice recrystn. would be exaggerated. Cryomicroscopy demonstrates that AFP inhibits ice recrystn. in the extracellular regions during the latter stages of the warming cycle. AFP concns. that enhance survival of red cells confer partial inhibition of recrystn. Relatively high concns. of AFP (e.g., 1.54 mg/mL) are much more effective at inhibiting extracellular recrystn. However, extensive growth of ice around the cell, and concomitant cell damage, is noted. The mechanism for this AFP-induced ice growth is unknown. Apparently, there is a delicate balance between AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell damage and that this balance hinges on the degrees of inhibition of ice recrystn. and of preferential growth of ice around the cells. Thus, under appropriate conditions, 1 of the proposed functions of AFPs in nature can be emulated, and perhaps have application, in cryopreservation of materials of biomedical interest.
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    Slowing the growth of ice with synthetic macromolecules: beyond antifreeze(glyco) proteins
    Gibson, Matthew I.
    Polymer Chemistry (2010), 1 (8), 1141-1152CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
    A review. Biol. antifreezes are a relatively large and diverse class of proteins (and very recently expanded to include lipopolysaccharides) which are capable of interacting with ice crystals in such a manner as to influence and, under the correct conditions, to prevent their growth. These properties allow for the survival of organisms which are either continuously or sporadically exposed to subzero temps. which would otherwise lead to cryo-injury/death. These proteins have been found in a range of organisms, including plants, bacteria, insects and fish, and the proteins themselves have a diverse range of chem. structures ranging from the highly conserved antifreeze glycoproteins (AFGPs) to the more diverse antifreeze proteins AFPs. Their unique abilities to non-colligatively decrease the f.p. of aq. solns., inhibit ice recrystn. and induce dynamic ice shaping suggest they will find many applications from cell/tissue/organ cryostorage, frozen food preservatives, texture enhancers or even as cryosurgery adjuvants. However, these applications have been limited by a lack of available material and also underlying questions regarding their mode of activity. The aim of this review article is to highlight the potential of polymeric materials to act as synthetic mimics of antifreeze(glyco) proteins, as well as to summarize the current general challenges in designing compds. capable of mimicking AF(G)Ps. This will cover the basic properties and modes of action of AF(G)Ps along with the methods commonly used to evaluate their activity. This section is essential to specifically define the 'antifreeze' terminol. in terms of these proteins' unique function and to distinguish them from conventional antifreezes. A detailed evaluation of the processes involved in AF(G)P activity is beyond the scope of this review, but the reader will be pointed towards relevant literature. This will then be placed in the context of modern polymer science, with a focus on the ability of synthetic polymers to display some type of specific antifreeze activity, which will be summarized. Finally, the potential applications of these materials will be highlighted and future avenues for their research and the challenges faced in achieving these goals suggested.
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    Graham, B.; Fayter, A. E. R.; Houston, J. E.; Evans, R. C.; Gibson, M. I. J. Am. Chem. Soc. 2018, 140 (17), 5682– 5685,  DOI: 10.1021/jacs.8b02066
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    Facially amphipathic glycopolymers inhibit ice recrystallization
    Graham, Ben; Fayter, Alice E. R.; Houston, Judith E.; Evans, Rachel C.; Gibson, Matthew I.
    Journal of the American Chemical Society (2018), 140 (17), 5682-5685CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Antifreeze glycoproteins (AFGPs) from polar fish are the most potent ice recrystn. (growth) inhibitors known, and synthetic mimics are required for low-temp. applications such as cell cryopreservation. Here we introduce facially amphipathic glycopolymers that mimic the three-dimensional structure of AFGPs. Glycopolymers featuring segregated hydrophilic and hydrophobic faces were prepd. by ring-opening metathesis polymn., and their rigid conformation was confirmed by small-angle neutron scattering. Ice recrystn. inhibition (IRI) activity was reduced when a hydrophilic oxo-ether was installed on the glycan-opposing face, but significant activity was restored by incorporating a hydrophobic dimethylfulvene residue. This biomimetic strategy demonstrates that segregated domains of distinct hydrophilicity/hydrophobicity are a crucial motif to introduce IRI activity, which increases our understanding of the complex ice crystal inhibition processes.
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  23. 23
    Deller, R. C.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Nat. Commun. 2014, 5, 3244,  DOI: 10.1038/ncomms4244
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    23
    Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing
    Deller Robert C; Vatish Manu; Mitchell Daniel A; Gibson Matthew I
    Nature communications (2014), 5 (), 3244 ISSN:.
    The cryopreservation of cells, tissue and organs is fundamental to modern biotechnology, transplantation medicine and chemical biology. The current state-of-the-art method of cryopreservation is the addition of large amounts of organic solvents such as glycerol or dimethyl sulfoxide, to promote vitrification and prevent ice formation. Here we employ a synthetic, biomimetic, polymer, which is capable of slowing the growth of ice crystals in a manner similar to antifreeze (glyco)proteins to enhance the cryopreservation of sheep and human red blood cells. We find that only 0.1 wt% of the polymer is required to attain significant cell recovery post freezing, compared with over 20 wt% required for solvent-based strategies. These results demonstrate that synthetic antifreeze (glyco)protein mimics could have a crucial role in modern regenerative medicine to improve the storage and distribution of biological material for transplantation.
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  24. 24
    Mitchell, D. E.; Lovett, J. R.; Armes, S. P.; Gibson, M. I. Angew. Chem., Int. Ed. 2016, 55 (8), 2801– 2804,  DOI: 10.1002/anie.201511454
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    24
    Combining Biomimetic Block Copolymer Worms with an Ice-Inhibiting Polymer for the Solvent-Free Cryopreservation of Red Blood Cells
    Mitchell, Daniel E.; Lovett, Joseph R.; Armes, Steven P.; Gibson, Matthew I.
    Angewandte Chemie, International Edition (2016), 55 (8), 2801-2804CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The first fully synthetic polymer-based approach for red-blood-cell cryopreservation without the need for any (toxic) org. solvents is reported. Highly hydroxylated block copolymer worms are shown to be a suitable replacement for hydroxyethyl starch as a extracellular matrix for red blood cells. When used alone, the worms are not a particularly effective preservative. However, when combined with poly(vinyl alc.), a known ice-recrystn. inhibitor, a remarkable additive cryopreservative effect is obsd. that matches the performance of hydroxyethyl starch. Moreover, these block copolymer worms enable post-thaw gelation by simply warming to 20 °C. This approach offers a new soln. for both the storage and transport of red blood cells and also a convenient matrix for subsequent 3D cell cultures.
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  25. 25
    Mitchell, D. E.; Cameron, N. R.; Gibson, M. I. Chem. Commun. 2015, 51 (65), 12977– 12980,  DOI: 10.1039/C5CC04647E
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    25
    Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation
    Mitchell, Daniel E.; Cameron, Neil R.; Gibson, Matthew I.
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (65), 12977-12980CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystn. inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystn. inhibiting polymer derived from a cheap commodity polymer, based on an ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromol. soln. to tissue storage.
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  26. 26
    Graham, B.; Bailey, T. L.; Healey, J. R. J.; Marcellini, M.; Deville, S.; Gibson, M. I. Angew. Chem., Int. Ed. 2017, 56, 15941– 15944,  DOI: 10.1002/anie.201706703
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    26
    Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers
    Graham, Ben; Bailey, Trisha L.; Healey, Joseph R. J.; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I.
    Angewandte Chemie, International Edition (2017), 56 (50), 15941-15944CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large vols. of solvent "antifreezes". Herein, the authors report polyproline as a min. (bio)synthetic mimic of antifreeze proteins that is accessible by soln., solid-phase, and recombinant methods. The authors demonstrate that polyproline has ice recrystn. inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromol. cryoprotectants.
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  27. 27
    Deller, R. C.; Pessin, J. E.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Biomater. Sci. 2016, 4, 1079,  DOI: 10.1039/C6BM00129G
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    27
    Enhanced non-vitreous cryopreservation of immortalized and primary cells by ice-growth inhibiting polymers
    Deller, Robert C.; Pessin, Jeffrey E.; Vatish, Manu; Mitchell, Daniel A.; Gibson, Matthew I.
    Biomaterials Science (2016), 4 (7), 1079-1084CODEN: BSICCH; ISSN:2047-4849. (Royal Society of Chemistry)
    Cell cryopreservation is an essential tool in modern biotechnol. and medicine. The ability to freeze, store and distribute materials underpins basic cell biol. and enables storage of donor cells needed for transplantation and regenerative medicine. However, many cell types do not survive freezing and the current state-of-the-art involves the addn. of significant amts. of org. solvents as cryoprotectants, which themselves can be cytotoxic, or simply interfere with assays. A key cause of cell death in cryopreservation is ice recrystn. (growth), which primarily occurs during thawing. Here it is demonstrated that the addn. of ice recrystalization inhibiting polymers to solns. contg. low (non vitrifying) concns. of DMSO enhance cell recovery rates by up to 75%. Cell functionality is also demonstrated using a placental cell line, and enhanced cryopreservation of primary rat hepatocytes is addnl. shown. The crucial role of the polymers architecture (chain length) is shown, with shorter polymers being more effective than longer ones.
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  28. 28
    Matsumura, K.; Bae, J. Y.; Kim, H. H.; Hyon, S. H. Cryobiology 2011, 63 (2), 76– 83,  DOI: 10.1016/j.cryobiol.2011.05.003
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    28
    Effective vitrification of human induced pluripotent stem cells using carboxylated ε-poly-L-lysine
    Matsumura, Kazuaki; Bae, Jung Yoon; Kim, Hak Hee; Hyon, Suong Hyu
    Cryobiology (2011), 63 (2), 76-83CODEN: CRYBAS; ISSN:0011-2240. (Elsevier B.V.)
    Derivation of human induced pluripotent stem (iPS) cells could enable their widespread application in future. Establishment of highly efficient and reliable methods for their preservation is a prerequisite for these applications. In this study, we developed a vitrification soln. comprising ethylene glycol (EG) and sucrose as well as carboxylated ε-poly-L-lysine (PLL); this soln. inhibited devitrification. Human iPS cells were vitrified in 200-μL vitrification solns. comprised 6.5 M EG, 0.75 M sucrose and 0 or 10% w/v carboxylated PLL with 65 mol% of the amino groups converted to carboxyl groups [PLL (0.65)] in a cryovial by directly immersing in liq. nitrogen. After warming, attached colony and recovery rates of human iPS cells vitrified by adding PLL (0.65) were significantly higher than those for cells without PLL (0.65) and vitrification soln. (DAP213: 2 M DMSO, 1 M acetamide and 3 M propylene glycol). Furthermore, even after warming at room temp., attached colony and recovery rates of iPS cells vitrified with PLL (0.65) were reduced to a lesser extent than those vitrified with either DAP213 or EG and sucrose without PLL (0.65). This could be attributed to inhibition of devitrification by PLL (0.65), as differential scanning calorimetry indicated less damage after vitrification with PLL (0.65). In addn., human iPS cells vitrified in the soln. with PLL (0.65) had normal karyotypes and maintained undifferentiated states and pluripotency as detd. by immunohistochem. and teratoma formation. Addn. of PLL (0.65) successfully vitrified human iPS cells with high efficiency. We believe that this method could aid future applications and increase utility of human iPS cells.
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  29. 29
    Congdon, T.; Notman, R.; Gibson, M. I. Biomacromolecules 2013, 14 (5), 1578– 1586,  DOI: 10.1021/bm400217j
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    29
    Antifreeze (Glyco)protein Mimetic Behavior of Poly(vinyl alcohol): Detailed Structure Ice Recrystallization Inhibition Activity Study
    Congdon, Thomas; Notman, Rebecca; Gibson, Matthew I.
    Biomacromolecules (2013), 14 (5), 1578-1586CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    This manuscript reports a detailed study on the ability of poly(vinyl alc.) to act as a biomimetic surrogate for AF(G)Ps ("antifreeze(glyco)proteins"), with a focus on the specific property of ice-recrystn. inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biol. antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alc.) (PVA) has been shown to display remarkable ice recrystn. inhibition activity despite its major structural differences to native antifreeze proteins. Here, controlled radical polymn. is used to synthesize well-defined PVA, which has enabled us to obtain the first quant. structure-activity relationships, to probe the role of mol. wt. and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight the idea that although hydrophobic domains are key components of IRI activity, the random inclusion of addnl. hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important.
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  30. 30
    Vail, N. S.; Stubbs, C.; Biggs, C. I.; Gibson, M. I. ACS Macro Lett. 2017, 6 (9), 1001– 1004,  DOI: 10.1021/acsmacrolett.7b00595
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    30
    Ultralow Dispersity Poly(vinyl alcohol) Reveals Significant Dispersity Effects on Ice Recrystallization Inhibition Activity
    Vail, Nicholas S.; Stubbs, Christopher; Biggs, Caroline I.; Gibson, Matthew I.
    ACS Macro Letters (2017), 6 (9), 1001-1004CODEN: AMLCCD; ISSN:2161-1653. (American Chemical Society)
    Polymer mimics of antifreeze proteins are emerging as an exciting class of macromol. cryoprotectants for the storage of donor cells and tissue. Poly(vinyl alc.), PVA, is the most potent polymeric ice growth inhibitor known, but its mode of action and the impact of valency (DP) are not fully understood. Herein, tandem RAFT polymn. and column chromatog. are used to isolate oligomers with dispersities <1.01 to enable the effect of mol. wt. distribution, as well as length, to be probed. It is found that polymers with equal no.-av. mol. wt., but lower dispersity, have significantly less activity, which can lead to false positives when identifying structure-property relationships. The min. chain length for PVA's unique activity, compared to other nonactive poly ols was identified. These results will guide the design of more active inhibitors, better cryopreservatives, and a deeper understanding of synthetic and biol. antifreeze macromols.
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  31. 31
    Inada, T.; Lu, S. S. Cryst. Growth Des. 2003, 3 (5), 747– 752,  DOI: 10.1021/cg0340300
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    31
    Inhibition of Recrystallization of Ice Grains by Adsorption of Poly(Vinyl Alcohol) onto Ice Surfaces
    Inada, Takaaki; Lu, Shu-Shen
    Crystal Growth & Design (2003), 3 (5), 747-752CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
    The effect of poly(vinyl alc.) (PVA) on recrystn. of ice was studied by comparison with the effect of antifreeze protein (AFP) type I. Polycryst. ice wafers consisting of numerous ice grains, whose initial size was <130 μm (i.e., less than the thickness of the ice wafer) were made from solns. contg. PVA or AFP type I at various concns. The ice wafers were annealed between -2.3 and -2.0° for 5 h, and then the size of the ice grains was measured using digital microscopy. Even at a PVA concn. as low as ∼5 × 10-7 mol/L, the size of the annealed ice grains made from the PVA soln. did not change significantly from the initial size, indicating that PVA is as effective as AFP type I in inhibiting ice recrystn. The effectiveness of PVA increased (i.e., the grain size decreased) with increasing molar concn., mol. wt., or degree of hydrolysis of PVA. The function of PVA mols. in the inhibition of recrystn. was analyzed by using the Langmuir adsorption equation.
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  32. 32
    Burkey, A. A.; Riley, C. L.; Wang, L. K.; Hatridge, T. A.; Lynd, N. A. Biomacromolecules 2018, 19 (1), 248– 255,  DOI: 10.1021/acs.biomac.7b01502
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    32
    Understanding Poly(vinyl alcohol)-Mediated Ice Recrystallization Inhibition through Ice Adsorption Measurement and pH Effects
    Burkey, Aaron A.; Riley, Christopher L.; Wang, Lyndsey K.; Hatridge, Taylor A.; Lynd, Nathaniel A.
    Biomacromolecules (2018), 19 (1), 248-255CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The development of improved cryopreservative materials is necessary to enable complete recovery of living cells and tissue after frozen storage. Remarkably, poly(vinyl alc.) (PVA) displays some of the same cryoprotective properties as many antifreeze proteins found in cold tolerant organisms. In particular, PVA is very effective at halting the Ostwald ripening of ice, a process that mech. damages cells and tissue. Despite the large practical importance of such a property, the mechanism by which PVA interacts with ice is poorly understood, hindering the development of improved cryoprotective materials. Herein, we quant. evaluated ice growth kinetics in the presence of PVA at different pH conditions and in the presence of a range of neutral salts. We demonstrated that pH, but not salt identity, alters the ability of PVA to halt ice grain coarsening. These observations are consistent with hydrogen-bonding playing a crucial role in PVA-mediated ice recrystn. inhibition. The evolution of the size distribution of ice crystals with annealing was consistent with incomplete surface coverage of ice with PVA. Binding assay measurements of dissolved fluorescently labeled PVA in an ice slurry showed that PVA interacts with ice through weak adsorption (<9%) to the ice crystal surface, which stands in contrast to fluorescently tagged type III antifreeze peptide, which binds strongly (ca. 64%) under the same conditions.
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    Inada, T.; Modak, P. R. Chem. Eng. Sci. 2006, 61 (10), 3149– 3158,  DOI: 10.1016/j.ces.2005.12.005
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    Growth control of ice crystals by poly(vinyl alcohol) and antifreeze protein in ice slurries
    Inada, Takaaki; Modak, Poly Rani
    Chemical Engineering Science (2006), 61 (10), 3149-3158CODEN: CESCAC; ISSN:0009-2509. (Elsevier Ltd.)
    Effect of poly(vinyl alc.) (PVA) in inhibiting an increase in ice crystal size in isothermal ice slurries was investigated, and then compared with the effect of an antifreeze protein (AFP), NaCl, and three other polymers, namely, poly(ethylene glycol), poly(vinyl pyrrolidone), and poly(acrylic acid). First, ice slurries, in which the initial size distribution of ice crystals was known, were isothermally preserved for given periods of time (typically 300 min) in the presence of PVA, AFP type I, NaCl, or the other three polymers. Then, the av. size of the ice crystals was measured using image processing. Both the PVA and AFP type I completely inhibited the increase in ice crystal size at such low concns. that the melting temp. of the soln. was -0.010°, whereas NaCl and the other three polymers clearly increased the ice crystal size due to Ostwald ripening. This inhibition effect of PVA and AFP type I was caused by thermal hysteresis, which is often taken as the primary manifestation of non-equil. antifreeze activity of these additives and defined as the difference between the melting temp. and non-equil. freezing temp. at which ice crystals start to grow in soln. The increase in ice crystal size was inhibited when the thermal hysteresis surpassed the driving potential for Ostwald ripening. Using PVA, which exhibits thermal hysteresis, is a novel technique to completely inhibit the increase in ice crystal size in isothermal ice slurries.
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    Budke, C.; Koop, T. ChemPhysChem 2006, 7 (12), 2601– 2606,  DOI: 10.1002/cphc.200600533
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    34
    Ice recrystallization inhibition and molecular recognition of ice faces by poly(vinyl alcohol)
    Budke, Carsten; Koop, Thomas
    ChemPhysChem (2006), 7 (12), 2601-2606CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The effects of poly(vinyl alc.) (PVA) on the Ostwald ripening of polycryst. ice samples are studied. At -6°, ice recrystn. in sucrose solns. is inhibited at PVA concns. down to 0.005 mg mL-1, with a recrystn. inhibition const. of 48.9 mL mg-1. Ice growth-habit expts. reveal mol. recognition of the arrangement of H2O mols. in the ice by PVA mols., and indicate that PVA mols. adsorb to the primary and secondary prism faces of hexagonal ice, 1h. Based on these observations, together with an anal. of the O-atom pattern in ice and the conformation of OH groups in PVA, an adsorption model is proposed. Probably PVA segments adsorb to the primary and secondary prism faces of ice parallel to the c axis with a linear misfit parameter of only 2.7%, most likely via multiple H bonds. The proposed adsorption mechanism is discussed in the light of recent thermal hysteresis and scanning tunneling microscopy expts.
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    Drori, R.; Li, C.; Hu, C.; Raiteri, P.; Rohl, A.; Ward, M. D.; Kahr, B. J. Am. Chem. Soc. 2016, 138 (40), 13396– 13401,  DOI: 10.1021/jacs.6b08267
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    35
    A Supramolecular Ice Growth Inhibitor
    Drori, Ran; Li, Chao; Hu, Chunhua; Raiteri, Paolo; Rohl, Andrew L.; Ward, Michael D.; Kahr, Bart
    Journal of the American Chemical Society (2016), 138 (40), 13396-13401CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Safranine O, a synthetic dye, was found to inhibit growth of ice at mM concns. with an activity comparable to that of highly evolved antifreeze glycoproteins. Safranine inhibits growth of ice crystals along the crystallog. a-axis, resulting in bipyramidal needles extended along the <0001> directions as well as and plane-specific thermal hysteresis (TH) activity. The interaction of safranine with ice is reversible, distinct from the previously reported behavior of antifreeze proteins. Spectroscopy and mol. dynamics indicate that safranine forms aggregates in aq. soln. at μM concns. Metadynamics simulations and aggregation theory suggested that as many as 30 safranine mols. were preorganized in stacks at the concns. where ice growth inhibition was obsd. The simulations and single-crystal x-ray structure of safranine revealed regularly spaced amino and Me substituents in the aggregates, akin to the ice-binding site of antifreeze proteins. Collectively, these observations suggest an unusual link between supramol. assemblies of small mols. and functional proteins.
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    Stubbs, C.; Lipecki, J.; Gibson, M. I. Biomacromolecules 2017, 18 (1), 295– 302,  DOI: 10.1021/acs.biomac.6b01691
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    36
    Regioregular Alternating Polyampholytes Have Enhanced Biomimetic Ice Recrystallization Activity Compared to Random Copolymers and the Role of Side Chain versus Main Chain Hydrophobicity
    Stubbs, Christopher; Lipecki, Julia; Gibson, Matthew I.
    Biomacromolecules (2017), 18 (1), 295-302CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Antifreeze proteins from polar fish species are potent ice recrystn. inhibitors (IRIs) effectively stopping all ice growth. Additives that have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regioregular polyampholytes are synthesized from maleic anhydride copolymers to enable stoichiometric installation of the charged groups, ensuring regioregularity, which is not possible using conventional random copolymn. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regioregular polymers was found to be superior to those derived from a std. random copolymn. with statistical incorporation of monomers, demonstrating that sequence compn. is crucial to the activity of IRI active polyampholytes.
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    Rajan, R.; Hayashi, F.; Nagashima, T.; Matsumura, K. Biomacromolecules 2016, 17 (5), 1882– 1893,  DOI: 10.1021/acs.biomac.6b00343
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    Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity
    Rajan, Robin; Hayashi, Fumiaki; Nagashima, Toshio; Matsumura, Kazuaki
    Biomacromolecules (2016), 17 (5), 1882-1893CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Cryopreservation enables long-term preservation of cells at ultralow temps. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA) (poly(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepd. two zwitterionic polymers, poly(sulfobetaine) (SPB) and poly(carboxymethyl betaine) (CMB), and compared their efficiency with poly(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the mol. mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Addnl., cryoprotective polymers, esp. their hydrophobic derivs., inhibit the recrystn. of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency.
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    Leclère, M.; Kwok, B. K.; Wu, L. K.; Allan, D. S.; Ben, R. N. Bioconjugate Chem. 2011, 22 (9), 1804– 1810,  DOI: 10.1021/bc2001837
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    C-Linked Antifreeze Glycoprotein (C-AFGP) Analogues as Novel Cryoprotectants
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    Significant cell damage occurs during cryopreservation resulting in a decreased no. of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystn. during freeze-thawing is one cause of decreased viability after cryopreservation. The authors have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystn. to increase cell viability after cryopreservation. The authors' results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) soln. of C-AFGP 1 is an excellent alternative to a 2.5% DMSO soln. for the cryopreservation of human embryonic liver cells.
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Cited By

This article is cited by 12 publications.

  1. Zhanhui Wang, Bin Yang, Zhuo Chen, Dan Liu, Lihong Jing, Chong Gao, Jian Li, Zhiyuan He, Jianjun Wang. Bioinspired Cryoprotectants of Glucose-Based Carbon Dots. ACS Applied Bio Materials 2020, 3 (6) , 3785-3791. https://doi.org/10.1021/acsabm.0c00376
  2. Pavithra M. Naullage, Valeria Molinero. Slow Propagation of Ice Binding Limits the Ice-Recrystallization Inhibition Efficiency of PVA and Other Flexible Polymers. Journal of the American Chemical Society 2020, 142 (9) , 4356-4366. https://doi.org/10.1021/jacs.9b12943
  3. Christopher Stubbs, Trisha L. Bailey, Kathryn Murray, Matthew I. Gibson. Polyampholytes as Emerging Macromolecular Cryoprotectants. Biomacromolecules 2020, 21 (1) , 7-17. https://doi.org/10.1021/acs.biomac.9b01053
  4. Bin Xue, Lishan Zhao, Xuehua Qin, Meng Qin, Jiancheng Lai, Wenmao Huang, Hai Lei, Jianjun Wang, Wei Wang, Ying Li, Yi Cao. Bioinspired Ice Growth Inhibitors Based on Self-Assembling Peptides. ACS Macro Letters 2019, 8 (10) , 1383-1390. https://doi.org/10.1021/acsmacrolett.9b00610
  5. Toru Ishibe, Thomas Congdon, Christopher Stubbs, Muhammad Hasan, Gabriele C. Sosso, Matthew I. Gibson. Enhancement of Macromolecular Ice Recrystallization Inhibition Activity by Exploiting Depletion Forces. ACS Macro Letters 2019, 8 (8) , 1063-1067. https://doi.org/10.1021/acsmacrolett.9b00386
  6. Trisha L. Bailey, Christopher Stubbs, Kathryn Murray, Ruben M. F. Tomás, Lucienne Otten, Matthew I. Gibson. Synthetically Scalable Poly(ampholyte) Which Dramatically Enhances Cellular Cryopreservation. Biomacromolecules 2019, 20 (8) , 3104-3114. https://doi.org/10.1021/acs.biomac.9b00681
  7. Bruno M. Guerreiro, Filomena Freitas, João C. Lima, Jorge C. Silva, Madalena Dionísio, Maria A.M. Reis. Demonstration of the cryoprotective properties of the fucose-containing polysaccharide FucoPol. Carbohydrate Polymers 2020, 245 , 116500. https://doi.org/10.1016/j.carbpol.2020.116500
  8. Panagiotis G. Georgiou, Ioanna Kontopoulou, Thomas R. Congdon, Matthew I. Gibson. Ice recrystallisation inhibiting polymer nano-objects via saline-tolerant polymerisation-induced self-assembly. Materials Horizons 2020, 7 (7) , 1883-1887. https://doi.org/10.1039/D0MH00354A
  9. Hong Xiang, Xiaohu Yang, Lei Ke, Yong Hu. The properties, biotechnologies, and applications of antifreeze proteins. International Journal of Biological Macromolecules 2020, 153 , 661-675. https://doi.org/10.1016/j.ijbiomac.2020.03.040
  10. Bo Liu, Qifa Zhang, Yunhui Zhao, Lixia Ren, Xiaoyan Yuan. Trehalose-functional glycopeptide enhances glycerol-free cryopreservation of red blood cells. Journal of Materials Chemistry B 2019, 7 (37) , 5695-5703. https://doi.org/10.1039/C9TB01089K
  11. Daniel E. Mitchell, Alice E. R. Fayter, Robert C. Deller, Muhammad Hasan, Jose Gutierrez-Marcos, Matthew I. Gibson. Ice-recrystallization inhibiting polymers protect proteins against freeze-stress and enable glycerol-free cryostorage. Materials Horizons 2019, 6 (2) , 364-368. https://doi.org/10.1039/C8MH00727F
  12. Christopher Stubbs, Thomas R Congdon, Matthew I. Gibson. Photo-polymerisation and study of the ice recrystallisation inhibition of hydrophobically modified poly(vinyl pyrrolidone) co-polymers. European Polymer Journal 2019, 110 , 330-336. https://doi.org/10.1016/j.eurpolymj.2018.11.047

  • Abstract

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    Figure 1

    Figure 1. Cryoprotectants used and IRI activity at concentrations relevant for this work. (A) Chemical structures. Cryomicrographs of ice wafers grown in the presence of (B) 100 mg mL–1 4 kDa PEG + 1 mg mL–1 10 kDa PVA, (C) 1 mg mL–1 10 kDa PVA, (D) 100 mg mL–1 4 kDa PEG, (E) 1 mg mL–1 AFPIII, (F) 50 mg mL–1 poly(ampholyte), and (G) PBS control. Scale bar = 100 μm.

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    Figure 2

    Figure 2. (A) Recovered colonies of E. coli after seven freeze (−196 °C)–thaw (20 °C) cycles. (B) Recovered colonies of E. coli after overnight incubation with cryoprotectants. Concentrations of cryoprotectants: [glycerol] = 25 wt %; [AFPIII] = 1 mg mL–1; [PVA] = 1 mg mL–1; [PEG/AFPIII] = 100 + 0.01 mg mL–1; [PEG/PVA] = 100 + 1 mg mL–1; [poly(ampholyte)] = 50 mg mL–1). Control is LB media alone.

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    Figure 3

    Figure 3. (A) Effect of varying PEG concentration on number of recovered E. coli colonies after seven freeze (−196 °C)–thaw (20 °C) cycles. (B) Live/dead viability testing on E. coli immediately after freeze–thaw cycle, with percentage of green (intact membrane) bacteria determined by confocal microscopy. [PEG/PVA] = 100 + 1 mg mL–1. Error bars represent SD from six repeats.

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    Figure 4

    Figure 4. Normalized cell recovery for three different bacteria upon addition of different cryoprotectants after seven freeze (−196 °C)–thaw (20 °C) cycles (red = control, blue = glycerol, black = PEG/PVA). Values obtained are normalized to themselves.

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    Figure 5

    Figure 5. E. coli growth profiles after seven freeze (−196 °C)–thaw (20 °C) cycles and then inoculation into LB media.

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      Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-mol. metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine - choline, trimethylamine N-oxide (TMAO) and betaine - were identified and then shown to predict risk for CVD in an independent large clin. cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a crit. role for dietary choline and gut flora in TMAO prodn., augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymic source of TMAO, segregated with atherosclerosis in hyperlipidemic mice. Discovery of a relationship between gut-flora-dependent metab. of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.
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      Sharon, G.; Sampson, T. R.; Geschwind, D. H.; Mazmanian, S. K. Cell 2016, 167 (4), 915– 932,  DOI: 10.1016/j.cell.2016.10.027
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      The Central Nervous System and the Gut Microbiome
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      A review. Neurodevelopment is a complex process governed by both intrinsic and extrinsic signals. While historically studied by researching the brain, inputs from the periphery impact many neurol. conditions. Indeed, emerging data suggest communication between the gut and the brain in anxiety, depression, cognition, and autism spectrum disorder (ASD). The development of a healthy, functional brain depends on key pre- and post-natal events that integrate environmental cues, such as mol. signals from the gut. These cues largely originate from the microbiome, the consortium of symbiotic bacteria that reside within all animals. Research over the past few years reveals that the gut microbiome plays a role in basic neurogenerative processes such as the formation of the blood-brain barrier, myelination, neurogenesis, and microglia maturation and also modulates many aspects of animal behavior. Herein, we discuss the biol. intersection of neurodevelopment and the microbiome and explore the hypothesis that gut bacteria are integral contributors to development and function of the nervous system and to the balance between mental health and disease.
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      Rea, K.; Dinan, T. G.; Cryan, J. F. Neurobiol. Stress 2016, 4, 23– 33,  DOI: 10.1016/j.ynstr.2016.03.001
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      The microbiome: A key regulator of stress and neuroinflammation
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      Neurobiology of stress (2016), 4 (), 23-33 ISSN:2352-2895.
      There is a growing emphasis on the relationship between the complexity and diversity of the microorganisms that inhabit our gut (human gastrointestinal microbiota) and health/disease, including brain health and disorders of the central nervous system. The microbiota-gut-brain axis is a dynamic matrix of tissues and organs including the brain, glands, gut, immune cells and gastrointestinal microbiota that communicate in a complex multidirectional manner to maintain homeostasis. Changes in this environment can lead to a broad spectrum of physiological and behavioural effects including hypothalamic-pituitary-adrenal (HPA) axis activation, and altered activity of neurotransmitter systems and immune function. While an appropriate, co-ordinated physiological response, such as an immune or stress response are necessary for survival, a dysfunctional response can be detrimental to the host contributing to the development of a number of CNS disorders. In this review, the involvement of the gastrointestinal microbiota in stress-mediated and immune-mediated modulation of neuroendocrine, immune and neurotransmitter systems and the consequential behaviour is considered. We also focus on the mechanisms by which commensal gut microbiota can regulate neuroinflammation and further aim to exploit our understanding of their role in stress-related disorders as a consequence of neuroinflammatory processes.
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      Navaneetharaja, N.; Griffiths, V.; Wileman, T.; Carding, S. J. Clin. Med. 2016, 5 (6), 55,  DOI: 10.3390/jcm5060055
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      A role for the intestinal microbiota and virome in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)?
      Navaneetharaja, Navena; Griffiths, Verity; Wileman, Tom; Carding, Simon R.
      Journal of Clinical Medicine (2016), 5 (6), 55/1-55/22CODEN: JCMOHK; ISSN:2077-0383. (MDPI AG)
      Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous disorder of significant societal impact that is proposed to involve both host and environmentally derived etiologies that may be autoimmune in nature. Immune-related symptoms of at least moderate severity persisting for prolonged periods of time are common in ME/CFS patients and B cell depletion therapy is of significant therapeutic benefit. The origin of these symptoms and whether it is infectious or inflammatory in nature is not clear, with seeking evidence of acute or chronic virus infections contributing to the induction of autoimmune processes in ME/CFS being an area of recent interest. This article provides a comprehensive review of the current evidence supporting an infectious etiology for ME/CFS leading us to propose the novel concept that the intestinal microbiota and in particular members of the virome are a source of the "infectious" trigger of the disease. Such an approach has the potential to identify disease biomarkers and influence therapeutics, providing much-needed approaches in preventing and managing a disease desperately in need of confronting.
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      Reshaping the gut microbiome with bacterial transplantation and antibiotic intake
      Manichanh, Chaysavanh; Reeder, Jens; Gibert, Prudence; Varela, Encarna; Llopis, Marta; Antolin, Maria; Guigo, Roderic; Knight, Rob; Guarner, Francisco
      Genome Research (2010), 20 (10), 1411-1419CODEN: GEREFS; ISSN:1088-9051. (Cold Spring Harbor Laboratory Press)
      The intestinal microbiota consists of over 1000 species, which play key roles in gut physiol. and homeostasis. Imbalances in the compn. of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were obsd. after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial compn. is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.
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      A review with 117 refs. Plants and ectothermic animals use a variety of substances and mechanisms to survive exposure to subfreezing temps. Proteinaceous ice nucleators trigger freezing at high subzero temps., either to provide cold protection from released heat of fusion or to establish a protective extracellular freezing in freeze-tolerant species. Freeze-avoiding species increase their supercooling potential by removing ice nucleators and accumulating polyols. Terrestrial invertebrates and polar marine fish stabilize their supercooled state by means of noncolligatively acting antifreeze proteins. Some organisms also depress their body fluid m.p. to ambient temp. by evapn. and/or solute accumulation. (c) 2000 Academic Press.
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      Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion
      Shu, Z.; Heimfeld, S.; Gao, D.
      Bone Marrow Transplantation (2014), 49 (4), 469-476CODEN: BMTRE9; ISSN:0268-3369. (Nature Publishing Group)
      A review. Transplantation of hematopoietic stem cells (HSCs) has been successfully developed as a part of treatment protocols for a large no. of clin. indications, and cryopreservation of both autologous and allogeneic sources of HSC grafts is increasingly being used to facilitate logistical challenges in coordinating the collection, processing, prepn., quality control testing and release of the final HSC product with delivery to the patient. Direct infusion of cryopreserved cell products into patients has been assocd. with the development of adverse reactions, ranging from relatively mild symptoms to much more serious, life-threatening complications, including allergic/gastrointestinal/cardiovascular/neurol. complications, renal/hepatic dysfunctions, and so on. In many cases, the cryoprotective agent (CPA) used-which is typically DMSO (DMSO)-is believed to be the main causal agent of these adverse reactions and thus many studies recommend depletion of DMSO before cell infusion. In this paper, we will briefly review the history of HSC cryopreservation, the side effects reported after transplantation, along with advances in strategies for reducing the adverse reactions, including methods and devices for removal of DMSO. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the DMSO concn. or using alternative CPAs for cryopreservation and removing DMSO before infusion. For DMSO removal, besides the traditional and widely applied method of centrifugation, new approaches have been explored in the past decade, such as filtration by spinning membrane, stepwise diln.-centrifugation using rotating syringe, diffusion-based DMSO extn. in microfluidic channels, dialysis and diln.-filtration through hollow-fiber dialyzers and some instruments (CytoMate, Sepax S-100, Cobe 2991, microfluidic channels, diln.-filtration system, etc.) as well. However, challenges still remain: development of the optimal (fast, safe, simple, automated, controllable, effective and low cost) methods and devices for CPA removal with min. cell loss and damage remains an unfilled need.
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      Capicciotti, C. J.; Kurach, J. D. R.; Turner, T. R.; Mancini, R. S.; Acker, J. P.; Ben, R. N. Sci. Rep. 2015, 5, 9692,  DOI: 10.1038/srep09692
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      Small Molecule Ice Recrystallization Inhibitors Enable Freezing of Human Red Blood Cells with Reduced Glycerol Concentrations
      Capicciotti, Chantelle J.; Kurach, Jayme D. R.; Turner, Tracey R.; Mancini, Ross S.; Acker, Jason P.; Ben, Robert N.
      Scientific Reports (2015), 5 (), 9692/1-9692/10CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      In North America, red blood cells (RBCs) are cryopreserved in a clin. setting using high glycerol concns. (40% w/v) with slow cooling rates (∼1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concns. with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small mol. ice recrystn. inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystn., a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addn. of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol soln. increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small mol. IRIs for the preservation of cells.
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      Microbial extremophiles at the limits of life
      Pikuta, Elena V.; Hoover, Richard B.; Tang, Jane
      Critical Reviews in Microbiology (2007), 33 (3), 183-209CODEN: CRVMAC; ISSN:1040-841X. (Informa Healthcare)
      A review. Prokaryotic extremophiles were the first representatives of life on Earth and they are responsible for the genesis of geol. structures during the evolution and creation of all currently known ecosystems. Flexibility of the genome probably allowed life to adapt to a wide spectrum of extreme environments. As a result, modern prokaryotic diversity formed in a framework of physico-chem. factors, and it is composed of: thermophilic, psychrophilic, acidophilic, alkalophilic, halophilic, barophilic, and radioresistant species. This artificial systematics cannot reflect the multiple actions of different environmental factors since one organism could unite characteristics of several extreme-groups. In this review we show the current status of studies in all fields of extremophiles and summarize the limits of life for different species of microbial extremophiles. We also discuss the finding of extremophiles from unusual places such as soils, and briefly review recent studies of microfossils in meteorites in the context of the significance of microbial extremophiles to Astrobiol.
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      Sidebottom, Chris; Buckley, Sarah; Pudney, Paul; Twigg, Sarah; Jarman, Carl; Holt, Chris; Telford, Julia; McArthur, Andrew; Worrall, Dawn; Hubbard, Rod; Lillford, Peter
      Nature (London) (2000), 406 (6793), 256CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      There is no expanded citation for this reference.
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      Biggs, C. I.; Bailey, T. L.; Ben Graham; Stubbs, C.; Fayter, A.; Gibson, M. I. Nat. Commun. 2017, 8 (1), 1546,  DOI: 10.1038/s41467-017-01421-7
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      Polymer mimics of biomacromolecular antifreezes
      Biggs Caroline I; Bailey Trisha L; Ben Graham; Stubbs Christopher; Fayter Alice; Gibson Matthew I; Gibson Matthew I
      Nature communications (2017), 8 (1), 1546 ISSN:.
      Antifreeze proteins from polar fish species are remarkable biomacromolecules which prevent the growth of ice crystals. Ice crystal growth is a major problem in cell/tissue cryopreservation for transplantation, transfusion and basic biomedical research, as well as technological applications such as icing of aircraft wings. This review will introduce the rapidly emerging field of synthetic macromolecular (polymer) mimics of antifreeze proteins. Particular focus is placed on designing polymers which have no structural similarities to antifreeze proteins but reproduce the same macroscopic properties, potentially by different molecular-level mechanisms. The application of these polymers to the cryopreservation of donor cells is also introduced.
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      Journal of Experimental Biology (1996), 199 (9), 2071-2076CODEN: JEBIAM; ISSN:0022-0949. (Company of Biologists)
      Antifreeze protein (AFP) types I, II and III were tested for their ability to protect red blood cells from lysis during warming, after cryopreservation in hydroxyethyl starch. All 3 types reduced hemolysis to 25% of control values at similar micromolar concns. but enhanced lysis as the AFP concn. approached millimolar levels. Site-directed mutants of type III AFP with different thermal hysteresis activities were tested for their ability to protect the cryopreserved cells from lysis. Their relative efficacy in protecting the cells correlated closely with their thermal hysteresis activity. Cryomicroscopy indicated that the protection of red cells by type III AFP and the mutant forms was due to inhibition of ice recrystn.
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      Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth
      Carpenter, John F.; Hansen, Thomas N.
      Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (19), 8953-7CODEN: PNASA6; ISSN:0027-8424.
      The influence of antifreeze proteins (AFPs) was detd. on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during warming. Relatively low concns. (e.g., 5-150 μg/mL) of winter flounder (Pseudopleuronectes americanus) AFP enhance survival of human red blood cells cryopreserved in hydroxyethyl starch solns. This effect is most apparent in samples warmed at suboptimal rates, i.e., where ice recrystn. would be exaggerated. Cryomicroscopy demonstrates that AFP inhibits ice recrystn. in the extracellular regions during the latter stages of the warming cycle. AFP concns. that enhance survival of red cells confer partial inhibition of recrystn. Relatively high concns. of AFP (e.g., 1.54 mg/mL) are much more effective at inhibiting extracellular recrystn. However, extensive growth of ice around the cell, and concomitant cell damage, is noted. The mechanism for this AFP-induced ice growth is unknown. Apparently, there is a delicate balance between AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell damage and that this balance hinges on the degrees of inhibition of ice recrystn. and of preferential growth of ice around the cells. Thus, under appropriate conditions, 1 of the proposed functions of AFPs in nature can be emulated, and perhaps have application, in cryopreservation of materials of biomedical interest.
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      Slowing the growth of ice with synthetic macromolecules: beyond antifreeze(glyco) proteins
      Gibson, Matthew I.
      Polymer Chemistry (2010), 1 (8), 1141-1152CODEN: PCOHC2; ISSN:1759-9962. (Royal Society of Chemistry)
      A review. Biol. antifreezes are a relatively large and diverse class of proteins (and very recently expanded to include lipopolysaccharides) which are capable of interacting with ice crystals in such a manner as to influence and, under the correct conditions, to prevent their growth. These properties allow for the survival of organisms which are either continuously or sporadically exposed to subzero temps. which would otherwise lead to cryo-injury/death. These proteins have been found in a range of organisms, including plants, bacteria, insects and fish, and the proteins themselves have a diverse range of chem. structures ranging from the highly conserved antifreeze glycoproteins (AFGPs) to the more diverse antifreeze proteins AFPs. Their unique abilities to non-colligatively decrease the f.p. of aq. solns., inhibit ice recrystn. and induce dynamic ice shaping suggest they will find many applications from cell/tissue/organ cryostorage, frozen food preservatives, texture enhancers or even as cryosurgery adjuvants. However, these applications have been limited by a lack of available material and also underlying questions regarding their mode of activity. The aim of this review article is to highlight the potential of polymeric materials to act as synthetic mimics of antifreeze(glyco) proteins, as well as to summarize the current general challenges in designing compds. capable of mimicking AF(G)Ps. This will cover the basic properties and modes of action of AF(G)Ps along with the methods commonly used to evaluate their activity. This section is essential to specifically define the 'antifreeze' terminol. in terms of these proteins' unique function and to distinguish them from conventional antifreezes. A detailed evaluation of the processes involved in AF(G)P activity is beyond the scope of this review, but the reader will be pointed towards relevant literature. This will then be placed in the context of modern polymer science, with a focus on the ability of synthetic polymers to display some type of specific antifreeze activity, which will be summarized. Finally, the potential applications of these materials will be highlighted and future avenues for their research and the challenges faced in achieving these goals suggested.
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      Graham, B.; Fayter, A. E. R.; Houston, J. E.; Evans, R. C.; Gibson, M. I. J. Am. Chem. Soc. 2018, 140 (17), 5682– 5685,  DOI: 10.1021/jacs.8b02066
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      Facially amphipathic glycopolymers inhibit ice recrystallization
      Graham, Ben; Fayter, Alice E. R.; Houston, Judith E.; Evans, Rachel C.; Gibson, Matthew I.
      Journal of the American Chemical Society (2018), 140 (17), 5682-5685CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Antifreeze glycoproteins (AFGPs) from polar fish are the most potent ice recrystn. (growth) inhibitors known, and synthetic mimics are required for low-temp. applications such as cell cryopreservation. Here we introduce facially amphipathic glycopolymers that mimic the three-dimensional structure of AFGPs. Glycopolymers featuring segregated hydrophilic and hydrophobic faces were prepd. by ring-opening metathesis polymn., and their rigid conformation was confirmed by small-angle neutron scattering. Ice recrystn. inhibition (IRI) activity was reduced when a hydrophilic oxo-ether was installed on the glycan-opposing face, but significant activity was restored by incorporating a hydrophobic dimethylfulvene residue. This biomimetic strategy demonstrates that segregated domains of distinct hydrophilicity/hydrophobicity are a crucial motif to introduce IRI activity, which increases our understanding of the complex ice crystal inhibition processes.
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      Deller, R. C.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Nat. Commun. 2014, 5, 3244,  DOI: 10.1038/ncomms4244
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      Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing
      Deller Robert C; Vatish Manu; Mitchell Daniel A; Gibson Matthew I
      Nature communications (2014), 5 (), 3244 ISSN:.
      The cryopreservation of cells, tissue and organs is fundamental to modern biotechnology, transplantation medicine and chemical biology. The current state-of-the-art method of cryopreservation is the addition of large amounts of organic solvents such as glycerol or dimethyl sulfoxide, to promote vitrification and prevent ice formation. Here we employ a synthetic, biomimetic, polymer, which is capable of slowing the growth of ice crystals in a manner similar to antifreeze (glyco)proteins to enhance the cryopreservation of sheep and human red blood cells. We find that only 0.1 wt% of the polymer is required to attain significant cell recovery post freezing, compared with over 20 wt% required for solvent-based strategies. These results demonstrate that synthetic antifreeze (glyco)protein mimics could have a crucial role in modern regenerative medicine to improve the storage and distribution of biological material for transplantation.
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      Mitchell, D. E.; Lovett, J. R.; Armes, S. P.; Gibson, M. I. Angew. Chem., Int. Ed. 2016, 55 (8), 2801– 2804,  DOI: 10.1002/anie.201511454
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      24
      Combining Biomimetic Block Copolymer Worms with an Ice-Inhibiting Polymer for the Solvent-Free Cryopreservation of Red Blood Cells
      Mitchell, Daniel E.; Lovett, Joseph R.; Armes, Steven P.; Gibson, Matthew I.
      Angewandte Chemie, International Edition (2016), 55 (8), 2801-2804CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The first fully synthetic polymer-based approach for red-blood-cell cryopreservation without the need for any (toxic) org. solvents is reported. Highly hydroxylated block copolymer worms are shown to be a suitable replacement for hydroxyethyl starch as a extracellular matrix for red blood cells. When used alone, the worms are not a particularly effective preservative. However, when combined with poly(vinyl alc.), a known ice-recrystn. inhibitor, a remarkable additive cryopreservative effect is obsd. that matches the performance of hydroxyethyl starch. Moreover, these block copolymer worms enable post-thaw gelation by simply warming to 20 °C. This approach offers a new soln. for both the storage and transport of red blood cells and also a convenient matrix for subsequent 3D cell cultures.
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    25. 25
      Mitchell, D. E.; Cameron, N. R.; Gibson, M. I. Chem. Commun. 2015, 51 (65), 12977– 12980,  DOI: 10.1039/C5CC04647E
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      25
      Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation
      Mitchell, Daniel E.; Cameron, Neil R.; Gibson, Matthew I.
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (65), 12977-12980CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Antifreeze (glyco) proteins AF(G)Ps are potent ice recrystn. inhibitors, which is a desirable property to enhance cryopreservation of donor tissue/cells. Here we present the rational synthesis of a new, biomimetic, ice-recrystn. inhibiting polymer derived from a cheap commodity polymer, based on an ampholyte structure. The polymer is used to enhance the cryopreservation of red blood cells, demonstrating a macromol. soln. to tissue storage.
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    26. 26
      Graham, B.; Bailey, T. L.; Healey, J. R. J.; Marcellini, M.; Deville, S.; Gibson, M. I. Angew. Chem., Int. Ed. 2017, 56, 15941– 15944,  DOI: 10.1002/anie.201706703
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      26
      Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers
      Graham, Ben; Bailey, Trisha L.; Healey, Joseph R. J.; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I.
      Angewandte Chemie, International Edition (2017), 56 (50), 15941-15944CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large vols. of solvent "antifreezes". Herein, the authors report polyproline as a min. (bio)synthetic mimic of antifreeze proteins that is accessible by soln., solid-phase, and recombinant methods. The authors demonstrate that polyproline has ice recrystn. inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromol. cryoprotectants.
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    27. 27
      Deller, R. C.; Pessin, J. E.; Vatish, M.; Mitchell, D. A.; Gibson, M. I. Biomater. Sci. 2016, 4, 1079,  DOI: 10.1039/C6BM00129G
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      27
      Enhanced non-vitreous cryopreservation of immortalized and primary cells by ice-growth inhibiting polymers
      Deller, Robert C.; Pessin, Jeffrey E.; Vatish, Manu; Mitchell, Daniel A.; Gibson, Matthew I.
      Biomaterials Science (2016), 4 (7), 1079-1084CODEN: BSICCH; ISSN:2047-4849. (Royal Society of Chemistry)
      Cell cryopreservation is an essential tool in modern biotechnol. and medicine. The ability to freeze, store and distribute materials underpins basic cell biol. and enables storage of donor cells needed for transplantation and regenerative medicine. However, many cell types do not survive freezing and the current state-of-the-art involves the addn. of significant amts. of org. solvents as cryoprotectants, which themselves can be cytotoxic, or simply interfere with assays. A key cause of cell death in cryopreservation is ice recrystn. (growth), which primarily occurs during thawing. Here it is demonstrated that the addn. of ice recrystalization inhibiting polymers to solns. contg. low (non vitrifying) concns. of DMSO enhance cell recovery rates by up to 75%. Cell functionality is also demonstrated using a placental cell line, and enhanced cryopreservation of primary rat hepatocytes is addnl. shown. The crucial role of the polymers architecture (chain length) is shown, with shorter polymers being more effective than longer ones.
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    28. 28
      Matsumura, K.; Bae, J. Y.; Kim, H. H.; Hyon, S. H. Cryobiology 2011, 63 (2), 76– 83,  DOI: 10.1016/j.cryobiol.2011.05.003
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      28
      Effective vitrification of human induced pluripotent stem cells using carboxylated ε-poly-L-lysine
      Matsumura, Kazuaki; Bae, Jung Yoon; Kim, Hak Hee; Hyon, Suong Hyu
      Cryobiology (2011), 63 (2), 76-83CODEN: CRYBAS; ISSN:0011-2240. (Elsevier B.V.)
      Derivation of human induced pluripotent stem (iPS) cells could enable their widespread application in future. Establishment of highly efficient and reliable methods for their preservation is a prerequisite for these applications. In this study, we developed a vitrification soln. comprising ethylene glycol (EG) and sucrose as well as carboxylated ε-poly-L-lysine (PLL); this soln. inhibited devitrification. Human iPS cells were vitrified in 200-μL vitrification solns. comprised 6.5 M EG, 0.75 M sucrose and 0 or 10% w/v carboxylated PLL with 65 mol% of the amino groups converted to carboxyl groups [PLL (0.65)] in a cryovial by directly immersing in liq. nitrogen. After warming, attached colony and recovery rates of human iPS cells vitrified by adding PLL (0.65) were significantly higher than those for cells without PLL (0.65) and vitrification soln. (DAP213: 2 M DMSO, 1 M acetamide and 3 M propylene glycol). Furthermore, even after warming at room temp., attached colony and recovery rates of iPS cells vitrified with PLL (0.65) were reduced to a lesser extent than those vitrified with either DAP213 or EG and sucrose without PLL (0.65). This could be attributed to inhibition of devitrification by PLL (0.65), as differential scanning calorimetry indicated less damage after vitrification with PLL (0.65). In addn., human iPS cells vitrified in the soln. with PLL (0.65) had normal karyotypes and maintained undifferentiated states and pluripotency as detd. by immunohistochem. and teratoma formation. Addn. of PLL (0.65) successfully vitrified human iPS cells with high efficiency. We believe that this method could aid future applications and increase utility of human iPS cells.
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    29. 29
      Congdon, T.; Notman, R.; Gibson, M. I. Biomacromolecules 2013, 14 (5), 1578– 1586,  DOI: 10.1021/bm400217j
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      29
      Antifreeze (Glyco)protein Mimetic Behavior of Poly(vinyl alcohol): Detailed Structure Ice Recrystallization Inhibition Activity Study
      Congdon, Thomas; Notman, Rebecca; Gibson, Matthew I.
      Biomacromolecules (2013), 14 (5), 1578-1586CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      This manuscript reports a detailed study on the ability of poly(vinyl alc.) to act as a biomimetic surrogate for AF(G)Ps ("antifreeze(glyco)proteins"), with a focus on the specific property of ice-recrystn. inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biol. antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alc.) (PVA) has been shown to display remarkable ice recrystn. inhibition activity despite its major structural differences to native antifreeze proteins. Here, controlled radical polymn. is used to synthesize well-defined PVA, which has enabled us to obtain the first quant. structure-activity relationships, to probe the role of mol. wt. and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight the idea that although hydrophobic domains are key components of IRI activity, the random inclusion of addnl. hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important.
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    30. 30
      Vail, N. S.; Stubbs, C.; Biggs, C. I.; Gibson, M. I. ACS Macro Lett. 2017, 6 (9), 1001– 1004,  DOI: 10.1021/acsmacrolett.7b00595
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      30
      Ultralow Dispersity Poly(vinyl alcohol) Reveals Significant Dispersity Effects on Ice Recrystallization Inhibition Activity
      Vail, Nicholas S.; Stubbs, Christopher; Biggs, Caroline I.; Gibson, Matthew I.
      ACS Macro Letters (2017), 6 (9), 1001-1004CODEN: AMLCCD; ISSN:2161-1653. (American Chemical Society)
      Polymer mimics of antifreeze proteins are emerging as an exciting class of macromol. cryoprotectants for the storage of donor cells and tissue. Poly(vinyl alc.), PVA, is the most potent polymeric ice growth inhibitor known, but its mode of action and the impact of valency (DP) are not fully understood. Herein, tandem RAFT polymn. and column chromatog. are used to isolate oligomers with dispersities <1.01 to enable the effect of mol. wt. distribution, as well as length, to be probed. It is found that polymers with equal no.-av. mol. wt., but lower dispersity, have significantly less activity, which can lead to false positives when identifying structure-property relationships. The min. chain length for PVA's unique activity, compared to other nonactive poly ols was identified. These results will guide the design of more active inhibitors, better cryopreservatives, and a deeper understanding of synthetic and biol. antifreeze macromols.
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    31. 31
      Inada, T.; Lu, S. S. Cryst. Growth Des. 2003, 3 (5), 747– 752,  DOI: 10.1021/cg0340300
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      31
      Inhibition of Recrystallization of Ice Grains by Adsorption of Poly(Vinyl Alcohol) onto Ice Surfaces
      Inada, Takaaki; Lu, Shu-Shen
      Crystal Growth & Design (2003), 3 (5), 747-752CODEN: CGDEFU; ISSN:1528-7483. (American Chemical Society)
      The effect of poly(vinyl alc.) (PVA) on recrystn. of ice was studied by comparison with the effect of antifreeze protein (AFP) type I. Polycryst. ice wafers consisting of numerous ice grains, whose initial size was <130 μm (i.e., less than the thickness of the ice wafer) were made from solns. contg. PVA or AFP type I at various concns. The ice wafers were annealed between -2.3 and -2.0° for 5 h, and then the size of the ice grains was measured using digital microscopy. Even at a PVA concn. as low as ∼5 × 10-7 mol/L, the size of the annealed ice grains made from the PVA soln. did not change significantly from the initial size, indicating that PVA is as effective as AFP type I in inhibiting ice recrystn. The effectiveness of PVA increased (i.e., the grain size decreased) with increasing molar concn., mol. wt., or degree of hydrolysis of PVA. The function of PVA mols. in the inhibition of recrystn. was analyzed by using the Langmuir adsorption equation.
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    32. 32
      Burkey, A. A.; Riley, C. L.; Wang, L. K.; Hatridge, T. A.; Lynd, N. A. Biomacromolecules 2018, 19 (1), 248– 255,  DOI: 10.1021/acs.biomac.7b01502
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      32
      Understanding Poly(vinyl alcohol)-Mediated Ice Recrystallization Inhibition through Ice Adsorption Measurement and pH Effects
      Burkey, Aaron A.; Riley, Christopher L.; Wang, Lyndsey K.; Hatridge, Taylor A.; Lynd, Nathaniel A.
      Biomacromolecules (2018), 19 (1), 248-255CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The development of improved cryopreservative materials is necessary to enable complete recovery of living cells and tissue after frozen storage. Remarkably, poly(vinyl alc.) (PVA) displays some of the same cryoprotective properties as many antifreeze proteins found in cold tolerant organisms. In particular, PVA is very effective at halting the Ostwald ripening of ice, a process that mech. damages cells and tissue. Despite the large practical importance of such a property, the mechanism by which PVA interacts with ice is poorly understood, hindering the development of improved cryoprotective materials. Herein, we quant. evaluated ice growth kinetics in the presence of PVA at different pH conditions and in the presence of a range of neutral salts. We demonstrated that pH, but not salt identity, alters the ability of PVA to halt ice grain coarsening. These observations are consistent with hydrogen-bonding playing a crucial role in PVA-mediated ice recrystn. inhibition. The evolution of the size distribution of ice crystals with annealing was consistent with incomplete surface coverage of ice with PVA. Binding assay measurements of dissolved fluorescently labeled PVA in an ice slurry showed that PVA interacts with ice through weak adsorption (<9%) to the ice crystal surface, which stands in contrast to fluorescently tagged type III antifreeze peptide, which binds strongly (ca. 64%) under the same conditions.
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    33. 33
      Inada, T.; Modak, P. R. Chem. Eng. Sci. 2006, 61 (10), 3149– 3158,  DOI: 10.1016/j.ces.2005.12.005
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      33
      Growth control of ice crystals by poly(vinyl alcohol) and antifreeze protein in ice slurries
      Inada, Takaaki; Modak, Poly Rani
      Chemical Engineering Science (2006), 61 (10), 3149-3158CODEN: CESCAC; ISSN:0009-2509. (Elsevier Ltd.)
      Effect of poly(vinyl alc.) (PVA) in inhibiting an increase in ice crystal size in isothermal ice slurries was investigated, and then compared with the effect of an antifreeze protein (AFP), NaCl, and three other polymers, namely, poly(ethylene glycol), poly(vinyl pyrrolidone), and poly(acrylic acid). First, ice slurries, in which the initial size distribution of ice crystals was known, were isothermally preserved for given periods of time (typically 300 min) in the presence of PVA, AFP type I, NaCl, or the other three polymers. Then, the av. size of the ice crystals was measured using image processing. Both the PVA and AFP type I completely inhibited the increase in ice crystal size at such low concns. that the melting temp. of the soln. was -0.010°, whereas NaCl and the other three polymers clearly increased the ice crystal size due to Ostwald ripening. This inhibition effect of PVA and AFP type I was caused by thermal hysteresis, which is often taken as the primary manifestation of non-equil. antifreeze activity of these additives and defined as the difference between the melting temp. and non-equil. freezing temp. at which ice crystals start to grow in soln. The increase in ice crystal size was inhibited when the thermal hysteresis surpassed the driving potential for Ostwald ripening. Using PVA, which exhibits thermal hysteresis, is a novel technique to completely inhibit the increase in ice crystal size in isothermal ice slurries.
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    34. 34
      Budke, C.; Koop, T. ChemPhysChem 2006, 7 (12), 2601– 2606,  DOI: 10.1002/cphc.200600533
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      34
      Ice recrystallization inhibition and molecular recognition of ice faces by poly(vinyl alcohol)
      Budke, Carsten; Koop, Thomas
      ChemPhysChem (2006), 7 (12), 2601-2606CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The effects of poly(vinyl alc.) (PVA) on the Ostwald ripening of polycryst. ice samples are studied. At -6°, ice recrystn. in sucrose solns. is inhibited at PVA concns. down to 0.005 mg mL-1, with a recrystn. inhibition const. of 48.9 mL mg-1. Ice growth-habit expts. reveal mol. recognition of the arrangement of H2O mols. in the ice by PVA mols., and indicate that PVA mols. adsorb to the primary and secondary prism faces of hexagonal ice, 1h. Based on these observations, together with an anal. of the O-atom pattern in ice and the conformation of OH groups in PVA, an adsorption model is proposed. Probably PVA segments adsorb to the primary and secondary prism faces of ice parallel to the c axis with a linear misfit parameter of only 2.7%, most likely via multiple H bonds. The proposed adsorption mechanism is discussed in the light of recent thermal hysteresis and scanning tunneling microscopy expts.
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      Drori, R.; Li, C.; Hu, C.; Raiteri, P.; Rohl, A.; Ward, M. D.; Kahr, B. J. Am. Chem. Soc. 2016, 138 (40), 13396– 13401,  DOI: 10.1021/jacs.6b08267
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      35
      A Supramolecular Ice Growth Inhibitor
      Drori, Ran; Li, Chao; Hu, Chunhua; Raiteri, Paolo; Rohl, Andrew L.; Ward, Michael D.; Kahr, Bart
      Journal of the American Chemical Society (2016), 138 (40), 13396-13401CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Safranine O, a synthetic dye, was found to inhibit growth of ice at mM concns. with an activity comparable to that of highly evolved antifreeze glycoproteins. Safranine inhibits growth of ice crystals along the crystallog. a-axis, resulting in bipyramidal needles extended along the <0001> directions as well as and plane-specific thermal hysteresis (TH) activity. The interaction of safranine with ice is reversible, distinct from the previously reported behavior of antifreeze proteins. Spectroscopy and mol. dynamics indicate that safranine forms aggregates in aq. soln. at μM concns. Metadynamics simulations and aggregation theory suggested that as many as 30 safranine mols. were preorganized in stacks at the concns. where ice growth inhibition was obsd. The simulations and single-crystal x-ray structure of safranine revealed regularly spaced amino and Me substituents in the aggregates, akin to the ice-binding site of antifreeze proteins. Collectively, these observations suggest an unusual link between supramol. assemblies of small mols. and functional proteins.
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    36. 36
      Stubbs, C.; Lipecki, J.; Gibson, M. I. Biomacromolecules 2017, 18 (1), 295– 302,  DOI: 10.1021/acs.biomac.6b01691
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      36
      Regioregular Alternating Polyampholytes Have Enhanced Biomimetic Ice Recrystallization Activity Compared to Random Copolymers and the Role of Side Chain versus Main Chain Hydrophobicity
      Stubbs, Christopher; Lipecki, Julia; Gibson, Matthew I.
      Biomacromolecules (2017), 18 (1), 295-302CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Antifreeze proteins from polar fish species are potent ice recrystn. inhibitors (IRIs) effectively stopping all ice growth. Additives that have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regioregular polyampholytes are synthesized from maleic anhydride copolymers to enable stoichiometric installation of the charged groups, ensuring regioregularity, which is not possible using conventional random copolymn. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regioregular polymers was found to be superior to those derived from a std. random copolymn. with statistical incorporation of monomers, demonstrating that sequence compn. is crucial to the activity of IRI active polyampholytes.
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      Rajan, R.; Hayashi, F.; Nagashima, T.; Matsumura, K. Biomacromolecules 2016, 17 (5), 1882– 1893,  DOI: 10.1021/acs.biomac.6b00343
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      Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity
      Rajan, Robin; Hayashi, Fumiaki; Nagashima, Toshio; Matsumura, Kazuaki
      Biomacromolecules (2016), 17 (5), 1882-1893CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Cryopreservation enables long-term preservation of cells at ultralow temps. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA) (poly(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepd. two zwitterionic polymers, poly(sulfobetaine) (SPB) and poly(carboxymethyl betaine) (CMB), and compared their efficiency with poly(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the mol. mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Addnl., cryoprotective polymers, esp. their hydrophobic derivs., inhibit the recrystn. of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency.
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      Leclère, M.; Kwok, B. K.; Wu, L. K.; Allan, D. S.; Ben, R. N. Bioconjugate Chem. 2011, 22 (9), 1804– 1810,  DOI: 10.1021/bc2001837
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      38
      C-Linked Antifreeze Glycoprotein (C-AFGP) Analogues as Novel Cryoprotectants
      Leclere, Mathieu; Kwok, Bonnie K.; Wu, Luke K.; Allan, David S.; Ben, Robert N.
      Bioconjugate Chemistry (2011), 22 (9), 1804-1810CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Significant cell damage occurs during cryopreservation resulting in a decreased no. of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystn. during freeze-thawing is one cause of decreased viability after cryopreservation. The authors have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystn. to increase cell viability after cryopreservation. The authors' results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) soln. of C-AFGP 1 is an excellent alternative to a 2.5% DMSO soln. for the cryopreservation of human embryonic liver cells.
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    39. 39
      Mangiagalli, M.; Bar-Dolev, M.; Tedesco, P.; Natalello, A.; Kaleda, A.; Brocca, S.; de Pascale, D.; Pucciarelli, S.; Miceli, C.; Braslavsky, I.; Lotti, M. FEBS J. 2017, 284 (1), 163– 177,  DOI: 10.1111/febs.13965
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      39
      Cryo-protective effect of an ice-binding protein derived from Antarctic bacteria
      Mangiagalli, Marco; Bar-Dolev, Maya; Tedesco, Pietro; Natalello, Antonino; Kaleda, Aleksei; Brocca, Stefania; de Pascale, Donatella; Pucciarelli, Sandra; Miceli, Cristina; Bravslavsky, Ido; Lotti, Marina
      FEBS Journal (2017), 284 (1), 163-177CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslSrtbw%253D&md5=0c9d8fa2706ef40fd6adcbe322340bf7
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht1Citb3L&md5=f58aebd1abda979f92bd638015bf69ad
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXjtl2mug%253D%253D&md5=29eb136cce512ae445aca91119c032a1
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      A review on the oral toxicity of polyvinyl alc. (PVA). Polyvinyl alcs. (PVA) (CAS no. 9002-89-5) are synthetic polymers used in a wide range of industrial, com., medical and food applications. The purpose of this review, this crit. evaluation of the available information on PVA, is to support the safety of PVA as a coating agent for pharmaceutical and dietary supplement products. All the available information on PVA gleaned from a comprehensive search of the scientific literature were critically evaluated. Orally administered PVA is relatively harmless. The safety of PVA is based on the following: (1) the acute oral toxicity of PVA is very low, with LD50s in the range of 15-20 g/kg; (2) orally administered PVA is very poorly absorbed from the gastrointestinal tract; (3) PVA does not accumulate in the body when administered orally; (4) PVA is not mutagenic or clastogenic; and (5) NOAELs of orally administered PVA in male and female rats were 5000 mg/kg body wt./day in the 90-day dietary study and 5000 mg/kg body wt./day in the two-generation reprodn. study, which was the highest dose tested. A crit. evaluation of the existing information on PVA supports its safety for use as a coating agent for pharmaceutical and dietary supplement products.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xps12mu78%253D&md5=5669ccac90038aaf698b4481051aad13
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      Polyethylene glycol (PEG) is a polymer of choice in drug delivery systems. This USFDA-approved polymer is popular due to its tunable properties and well-established safety profile: prime requisites considered during the selection of any excipient in formulation development. The unique properties and applications of PEG have been discussed at length in the existing literature. However, a proper guidance on selection of PEG grade to cater to one's purpose is lacking. This article provides preliminary guidelines to formulators on selection of appropriate PEG grade, typically based on its physico-chem. properties and role-based functional application in pharmaceuticals. It should be noted that the aim article is not to deep dive in each application area. Guidance on PEG application and grade of choice is lacking in the available literature. The authors have discussed and provided guidance to formulators on the appropriate PEG grade selection for particular application based on the available in vitro and in vivo literature data. In this review a State-of-the-art use of PEG in therapeutic applications, its clin. status and com. use is also summarized. Nevertheless, toxicities related to different PEG grades and related impurities are discussed in this review.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xot1ymtLo%253D&md5=c30ce55d835e6c1b1154544a36f74a07
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      DeMerlis, C. C.; Schoneker, D. R. Food Chem. Toxicol. 2003, 41, 319– 326,  DOI: 10.1016/S0278-6915(02)00258-2
      [Crossref], [PubMed], [CAS], Google Scholar
      46
      Review of the oral toxicity of polyvinyl alcohol (PVA)
      DeMerlis, C. C.; Schoneker, D. R.
      Food and Chemical Toxicology (2003), 41 (3), 319-326CODEN: FCTOD7; ISSN:0278-6915. (Elsevier Science Ltd.)
      A review on the oral toxicity of polyvinyl alc. (PVA). Polyvinyl alcs. (PVA) (CAS no. 9002-89-5) are synthetic polymers used in a wide range of industrial, com., medical and food applications. The purpose of this review, this crit. evaluation of the available information on PVA, is to support the safety of PVA as a coating agent for pharmaceutical and dietary supplement products. All the available information on PVA gleaned from a comprehensive search of the scientific literature were critically evaluated. Orally administered PVA is relatively harmless. The safety of PVA is based on the following: (1) the acute oral toxicity of PVA is very low, with LD50s in the range of 15-20 g/kg; (2) orally administered PVA is very poorly absorbed from the gastrointestinal tract; (3) PVA does not accumulate in the body when administered orally; (4) PVA is not mutagenic or clastogenic; and (5) NOAELs of orally administered PVA in male and female rats were 5000 mg/kg body wt./day in the 90-day dietary study and 5000 mg/kg body wt./day in the two-generation reprodn. study, which was the highest dose tested. A crit. evaluation of the existing information on PVA supports its safety for use as a coating agent for pharmaceutical and dietary supplement products.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xps12mu78%253D&md5=5669ccac90038aaf698b4481051aad13

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