Liver hepatocytes are the front-line cells for screening new compounds for toxicity. However, hepatocytes are stored frozen in vials, not in monolayers meaning they need substantial processing to be ‘usable’ especially for high throughput screening. To solve this the Gibson and Dallman groups have collaborated, so show that immortalised and primary hepatocytes can be cryopreserved whilst attached to 96 well microplates. These can then be taken from the freezer, and simple thawed and are ready to use in under 24 hours. Post-thaw the cells show equal performance to fresh. This was achieved by careful consideration of the molecular mechanism damage during freezing, with the team using patent-pending controlled ice nucleation technology, rather than a traditional re-formulation of cryoprotectants approach. This work was in collaboration with Cryologyx, a University of Warwick Spin out, which has commercialised aspects of this technology.  
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Trafficking of proteins, lipids, and other molecules between cellular compartments is carried out by vesicular carriers. Material destined for transfer is packaged into a small trafficking vesicle at the donor compartment; the vesicle must then travel to its destination, before fusing with the target compartment to deliver the material.

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Our 2023 Summer Graduation took place on Thursday 20 July, celebrating the fantastic achievements of our very first cohort of undergraduate BSc Health & Medical Sciences students, our MB ChB, Master's and PhD students.

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Dr John James' lab have used a cellular reconstitution of preTCR function to investigate the trafficking dynamics of this developmentally important immune receptor, work which has just been published in the Journal of Cell Biology.

Expression of the pre-T cell receptor (preTCR) is an important checkpoint during the development of T cells, an essential cell type of our adaptive immune system. The preTCR complex is only transiently expressed and rapidly internalized in developing T cells and is thought to signal in a ligand-independent manner. However, identifying a mechanistic basis for these unique features of the preTCR compared with the final TCR complex has been confounded by the concomitant signaling that is normally present. Thus, we have reconstituted preTCR expression in non-immune cells to uncouple receptor trafficking dynamics from its associated signaling. We find that all the defining features of the preTCR are intrinsic properties of the receptor itself, driven by exposure of an extracellular hydrophobic region, and are not the consequence of receptor activation. Finally, we show that transitory preTCR cell surface expression can sustain tonic signaling in the absence of ligand binding, suggesting how the preTCR can nonetheless drive αβTCR lineage commitment.

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Advanced cell-based therapies are often delivered to the patient frozen. Hence, any technology which increases the number of recovered, viable, cells post-thaw may improve clinical outcomes or allow more treatments per donation. The GibsonGroup have previously shown strategies to protect cells during freezing. In this latest work, in collaboration with Cytiva, the team show that incubating a model t-cell line with proline before cryopreservation leads to increased post-thaw cell yields. All proline is removed before cryopreservation so the actual freezing and thawing processes are unchanged. It was shown that proline limits cell proliferation, which might be contributing to its mode of action similar to ‘metabolic pre-conditioning’ which has been shown before.

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Glycans (sugars) dictate cell-cell communication, are sites for pathogen invasion and are a key part of our immune systems. Current synthetic platforms to display glycans to investigate their biology almost always are imperfect, with heterogeneity in terms of number of glycans and the synthesis is not reproducible batch to batch. The Gibson and Ward (chemistry) groups have collaborated on a Leverhulme-Trust funded project to create ‘programmable’ glyco-clusters - before entering the lab, this method enables a research to know exactly how many glycans and their 3-D location are present on a material, and gives zero heterogeneity. This is achieved using metal co-ordination cages - 3D structures formed by spontaneous self-assembly. The team used these with model glycan-binding proteins to identify key interactions which would not be possible with traditional materials. The team are now using this to interrogate a range of targets including toxins, for diagnostics.

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Spheroids (and organoids) can reproduce key aspects of human biological responses, and since the FDA simplification act it is possible (in some cases) to bypass animal testing in the development of new drugs where quality tissue models exist. However, these are not accessible ‘off the shelf’ so are not widely used, with monolayer culture then animal studies common. The GibsonGroup working with the WhaleGroup have recently shown how controlled nucleation (making ice form) can actually improve cryopreservation outcomes by reducing intracellular ice formation. In this latest work they combine nucleation with proline-pre conditioning which ‘prepares’ cells for cryopreservation. This shows how joined-up thinking of cryopreservation as a biochemical and biophysical problem can make a major impact on cell-storage platform technologies.

Read the paper here.