| dc.description.abstract | Tissue and stem cell culture methods have been dominated by glass and plastic substrates such as Tissue culture plastic. These solid substrates, although widely used, are associated with poor scalability for adherent stem cell expansion in systems such as 3D bioreactors and the design of parallel culture systems. Therefore, investigating strategies to bypass these obstacles in stem cell expansion is essential to enable the wider translation of stem cell technologies. An alternative strategy recently proposed consists in using a liquid surface instead, such as an oil, and associated oil droplets. Indeed, emulsions can be formed using protein nanosheets to stabilise oil/water interfaces to promote the adhesion of stem cells and enable their proliferation. These nanosheets exhibit enhanced interfacial mechanics and allow the introduction of bioactive components via recombinant protein expression to promote bioactivity. Beyond the application of resulting bioemulsions for the expansion of Mesenchymal stem cells, the impact of these bioactive interfaces on the differentiation of iPSCs and the development of cerebral organoids will be presented. The Bovine serum albumin protein was recombinantly modified to attach an N-terminal Avi-Tag, this was expressed and purified from the yeast P. pastoris expression system. The Avi-tag was then biotinylated in vitro by recombinantly expressed BirA. Emulsions of a specific size were formed using the newly biotinylated Bt-BSA protein and functionalized with a cascade of components to mimic cell-cell ligands, this resulted in bioemulsions with a bioactive surface that can interact with surrounding cells. These functionalised droplets were integrated into developing cerebral organoids and their impact on phenotype was studied. The droplets were found not to deform sufficiently to allow mechanical forces to be measured, yet the many of these droplets were retained within the organoids which led to an interesting phenotype within the organoids. The developing rosettes were found to develop enlarged lumens shown by an increase in area, this phenotype did not impact the differentiation into the cerebral lineage depicted by immunohistochemistry of hallmark marker of neuronal differentiation within organoids retaining droplets. The interfacial mechanics of fibrinogen nanosheets treated with varying concentrations of thrombin was studied using interfacial shear rheology. The effect of thrombin significantly altered the interfacial mechanics with the lower concentration of thrombin significantly increasing the toughness multiple folds and decreasing the elasticity of the nanosheets. Additionally, the nanostructure of nanosheets was studied using SEM and TEM and traditional fibrin fibres were found to not form at these interfaces, but local rearrangements and retractions in the thrombin treated nanosheets were observed. Finally, these enhanced mechanical properties promoted the proliferation and expansion of Mesenchymal stem cells on quasi-2D and 3D interfaces. | en_US |
