Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - CRYOMAT (Antifreeze GlycoProtein Mimetic Polymers)

Teaser

The storage and transplant of donor cells is a major societal and industrial challenge – once any material is taken from a body it rapidly degrades and hence must either be quickly transfused, transplanted or placed into longer term storage. In analogy to the food industry...

Summary

The storage and transplant of donor cells is a major societal and industrial challenge – once any material is taken from a body it rapidly degrades and hence must either be quickly transfused, transplanted or placed into longer term storage. In analogy to the food industry, freezing is the most convenient and desirable storage method. However, cells do not tolerate freezing and cryoprotectants - ‘antifreezes’ must be added to ensure the cells are still viable after thawing. The overall aim of this ambitious ERC funded project is to develop new synthetic materials which mimic one of Nature’s most active cryoprotectants; Antifreeze Glycoproteins (AFGPs). AFGPs are specialised proteins found in polar fish species which help them survive, with a particular property of slowing the rate of ice crystal growth (not formation). By using synthetic mimics we aim to be able to both understand how these fascinating proteins work (they can ‘see the difference between ice and water’ which is already very complex!), scale up their synthesis and translate it to aid our ability to freeze donor cells and tissue.

So far we have developed 3 unique classes of materials based on synthetic polymers (‘plastics’) which are all very potent at stopping ice crystal growth. We have been able to link their structure to their function and then use them to store cells. In particular, we have shown new ways of storing red blood cells – the largest volume of cells transfused globally and used in nearly every major surgery. Using our polymers we have successfully frozen red blood cells without the need for any traditional antifreezes, which are based on organic solvents such as glycerol.
In addition to blood, we have begun to freeze nucleated cells too – these are more complex than blood cells and include examples of cells which are needed medically, such as bone marrow (stem) cells, islet cells and hepatocytes. We showed a new method to freeze cells whilst they are still attached to the surface they grow on – this is a major breakthrough as this makes it easier to process the cells and more useful for biotechnology applications.
Our work has also been highlighted in many scientific and general audience media outlets during this period.

Work performed

Significant progress has been made towards the aims of the project and more than 10 publications have emerged from this work including a major review article introducing this new field which was published in Nature Communications.


We have made particular progress in Workpackages A and B which involved the design, synthesis and biophysical testing of antifreeze glycoprotein mimetic materials. In particular we have made huge strides into understanding our model polymer – poly(vinyl alcohol) (PVA) and its interaction with ice. We have developed new architectures of PVA including block copolymers and star-shaped polymers and demonstrated how the ice growth activity is not affected by these changes, with all activity being retained. We have developed new synthetic methods to allow us to access degradable polymers based on PVA, which being based entirely on a carbon backbone is normally stable in solution.
We have also investigated a new class of ice growth inhibitors based on poly(ampholytes) – polymers with positive and negative charged groups. Using a synthetic strategy to give alternating polymers, we were able to identify the key structural motifs needed for activity, including the role of hydrophobic groups.
In collaboration with partners at Warwick, we have also developed a new class of ice growth inhibitors based on self-assembled optically pure metallo-helicies and used these to test theories about the importance of ice binding, verses amphiphilicity in inhibiting ice growth. Finally, we introduced polyproline as a moderately active ice growth inhibiting polymer and linked its secondary structure to the ice growth properties. A patent has been filed relating to some of the above results and we are in discussions with partners about exploitation of this.

The other aim of this work (Workpackage C) is improved cryopreservation methodolgoies. In this we have made good progress and by using various cell-based models we have shown how we can increase the number of recovery cells post-thaw by modulating ice growth with our polymers. We have shown preliminary results in this area using red blood cells, but also cultured monolayers. In the next period we will investigate this area a lot more, using the panel of new polymers which we have synthesised.

In summary we have published more than 10 papers and given > 10 presentations on this work to a range of audiences in the UK, EU and beyond. We have also undertaken public engagement though both press releases and media reports as well as outreach activities. A patent has also been filed based on this work and we are seeking commercial partners.

Final results

Before this work, nearly all cryoprotectants were ‘small molecule’ solvents. We have now progressed the state of the art such that the potential of ‘macromolecular cryoprotectants’ is validated and is an emerging theme of research. As a marker of this, several other research groups around the world are now engaging and forming their own research programs based on our work, which we anticipate will help the field advance rapidly.

One important advance was our demonstration that hydroxyl groups (alcohols) are not essential motifs for an ice-inhibiting compound. It had been speculated that these were essential to hydrogen bond with a growing ice crystal plane. However, we have introduced two diverse sets of materials which have no hydroxyl groups at all, and in fact no obvious ice-binding face. However, these were still very active. Self-assembled metallo-helicies were shown to be remarkably potent and their ‘patchy’ surface was linked to their activity. Secondly, polyproline was shown to have moderate ice growth activity but to be very potent in enhancing the cryopreservation of nucleated cell monolayers. These two key results demonstrate the un-tapped chemical space for new ice growth inhibitors and their exciting potential to advance biomedicine through improved cell storage.

By the end of the project we aim to have demonstrated another new macromolecular ice growth inhibitor, and have strong data to support this, and to have developed some guidelines for how to predict if something will be active. By the end of the project we will also have extended the cryopreservation technology to a far wider range of cells.