The new ice age

Cryo-storage is a multibillion-dollar industry, but surprisingly we are not very good at it. Here, Alice Fayter and Matthew Gibson talk extremophiles, ice and a new generation of biological cold storage

Cryonics and cryogenics are words with a sci-fi sort of feel – think of Han Solo, or Fry from Futurama. They sound like wizardry with the supposed aim to preserve life or provide an extended timeline to find cures to currently incurable diseases.

These concepts may not even be possible, but cryopreservation itself- the sub-zero storage of biological material is very much science fact, and underpins huge areas of clinical, fundamental and applied science, from food to fertility.

Cold-storage, particularly cryo-storage, of biological materials is a multibillion dollar industry; at least 20% of clinical trials today require cold chain logistics (8ºC to -80ºC) and between 14 and 35% of vaccine shipments require sub-zero temperatures at some stage.^1,2 These biologics can be cell-based; there is growing research into the microbiome and its involvement in a range of medical disorders thus there is a significant need for storage of bacteria; they can also be proteins, viruses and antibodies; such as those employed in vaccines.

Cell-based processes and various biotechnologies are underpinned by the recovery of intact and viable cells after storage. These cells need to survive multiple stages such as preparation, growth, storage, transport, recovery, formulation and transferal to patients, many of which require cold temperatures. However there are issues with limited storage lifetime and degradation. This process needs to be considered when producing a potential therapeutic, as an effective and efficient chain leads to a better quality product.

Research in our group at the University of Warwick focuses on biomaterials science and its potential to impact on global healthcare challenges, one in particular is cryopreservation, which is commonly used in academic, medical and industrial settings. Our team is multidisciplinary, including chemists, biologists and medical school staff, aiming to collaborate and exploit their skills ranging from microbiology, polymer chemistry and biophysics, to innovate solutions for these challenges. One hope is to utilize our specialities and knowledge to develop new biomaterials for long term, successful cold storage of biologics, particularly to reduce or replace traditional ‘solvent’ based antifreeze agents.

An icy issue It might surprise you to know that water, and its solid form, ice, are two of the most studied materials on Earth, and yet there is still an incomplete understanding of their properties. Ice crystal formation and growth is a serious problem, particularly in relation to cryopreservation and storage and transport of biological materials, as well as in technological applications such as aeronautical engineering and agriculture. Compounds that exhibit effects on the ice structure (antifreezes, cryoprotectants etc.) are quite interesting and their mechanisms of action are not fully understood. By employing physical, biological and chemical principles, our work explores the effect of different compounds on ice morphology and growth, with the aim of elucidating their mechanisms of action, furthering our understanding of ice and improving cryo-storage.

Nature has evolved a great range of mechanisms to allow its survival in extreme conditions; at sub-zero temperatures there are insects, fish and plants that survive; these include various Antarctic species including ocean pout, eel pout and Alaskan plaice as well as snowfleas, certain beetles and overwintering grasses.^3–6 One of the key methods used by extremophiles to survive is the production of antifreeze proteins and antifreeze glycoproteins (AF(G)Ps). These are of great interest in our group and are an exciting source of inspiration for our synthetic chemists.⁷

These AF(G)Ps can lower the freezing point of water, thus preventing freezing, without significantly altering the melting point (thermal hysteresis (TH)), but can also inhibit ice crystal growth (ice recrystallization inhibition (IRI)), so even if the water is cold enough to freeze, the ice crystals are inhibited from growing. Yet, when considering their use in storage of biological materials such as red blood cells or bacteria (potential use in microbiome research), there are immunogenicity and toxicity issues, as well as the problem of ice shaping – these proteins can cause the crystals to be smaller but also can shape the ice crystals into needles, which may burst or damage frozen cells.

Protein mimicry This unique ability to inhibit ice recrystallization (IRI) is of great interest in our lab, and, alongside trying to overcome toxicity challenges, has inspired us to produce IRI-active polymers and other mimics that reproduce the same macroscopic properties and to test whether they protect cells in a similar way to AF(G)Ps.⁸

Ice crystal growth during freezing and thawing are major contributors to failure of cryopreservation techniques. Biological materials are stored at sub-zero temperatures using cryoprotectants, which traditionally are organic solvents such as dimethyl sulfoxide or glycerol to mitigate ice damage as well as to prevent osmotic stress and membrane rupturing, which otherwise would lead to cell death. However, these have some toxicity issues and impact downstream applications and assays. We are working on solvent-free storage techniques, focussing on cryopreservation exploiting protein mimics. We use low cost polymers, which are simple to produce and may be synthesised in a range of molecular weights and structures; making them versatile and usable for a range of biological materials. The idea is to use non-toxic, low concentration, IRI-active compounds to protect biological materials at sub-zero temperatures. We may be able to help improve longevity of donated blood stocks as well as assist with consistency of the cold chain process – by reducing the possibility of ice crystal growth during unavoidable temperature fluctuations.

We have utilised a range of methods including solid-state nuclear magnetic resonance spectroscopy (ssNMR), X-ray diffraction, differential scanning calorimetry and microscopy to aid characterisation and analysis by monitoring structural changes in ice upon addition of cryoprotectant as well as studying the viability of cell lines post cryo-storage. It is of great importance to further our understanding of ice and the mechanism of ice crystal growth, especially when considering possible cryoprotectants as this will lead to the improvement of techniques for the prevention of ice formation and increased cell/tissue survival during cryopreservation.

So far we have successfully managed to cryopreserve a range of cell lines as well as different proteins, showing that IRI-active polymers can be used successfully as an alternative cryopreservation method to the traditional solvent-based method. We observed an increase in viable cell recovery post thaw for bacterial cells compared to that of the gold standard, glycerol, when using lower concentrations of our cryoprotectants.⁹

Our findings motivate us to continue studying antifreeze-protein mimics, using a range of biophysical and chemical techniques with the aim of ultimately reducing the huge disconnect between donor cells and tissue (or even organs) and the recipients in transplantation medicine as well as improving cell-based therapies. Organs cannot be stored and must be transported rapidly to the recipient, with failure rates increasing with the time since removal. The ability to cryo-store and bank tissue and organs would address an important unmet medical need. We have started a cryopreservation trial, allowing us to share our unique, Warwick-developed technology with the aim to provide real benefit to other research groups’ biochemical and structural biology research.

Despite being extremely far from a world of cryonics and cryogenics, we are getting closer to improved biologic cryo-storage and we are excited for the future.

References

1. PAREXEL. Cold Chain Logistics. (2018). Available at: https://www.parexel.com/solutions/clinical-research/clinical-trial-supplies-and-logistics/cold-chain-logistics.
2. Kartoglu, Ü., Özgüler, N. K., Wolfson, L. J. & Kurzatkowski, W. Validation of the shake test for detecting freeze damage to adsorbed vaccines. Bull. World Health Organ. 88, 624–631 (2010).`
3. Davies, P. L. Ice-binding proteins: A remarkable diversity of structures for stopping and starting ice growth. Trends in Biochemical Sciences 39, 548–555 (2014).
4. Marshall, C. B., Fletcher, G. L. & Davies, P. L. Hyperactive antifreeze protein in a fish. Nature 429, 153 (2004).
5. Sidebottom, C. et al. Heat-stable antifreeze protein from grass. Nature 406, 256 (2000).
6. Ben, R. N. Antifreeze Glycoproteins-Preventing the Growth of Ice. ChemBioChem 2, 161–166 (2001).
7. Biggs, C. I. et al. Polymer mimics of biomacromolecular antifreezes. Nat. Commun. 8, 1546 (2017). https://www.nature.com/articles/s41467-017-01421-7
8. Gibson, M. I. Slowing the growth of ice with synthetic macromolecules: beyond antifreeze(glyco) proteins. Polym. Chem. 1, 1141 (2010).
9. Hasan, M., Fayter, A. E. R. & Gibson, M. I. Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Micro-organisms. Biomacromolecules 19, 3371–3376 (2018). https://pubs.acs.org/doi/10.1021/acs.biomac.8b00660

Authors: Alice E.R Fayter joined the Gibson group in 2016 and began a PhD studying antifreeze proteins and synthetic compounds with IRI activity

Professor Matthew I. Gibson leads a diverse research group using materials to address healthcare challenges; cryopreservation, diagnostics, glycobiology and polymer chemistry