Biology subjects

Chrysanthou, A.

Publications and source records attributed to Chrysanthou, A..

5 recordsLinked to original sources

bioRxiv2023

Engineered Protein Nanosheets for the Scale up of Mesenchymal Stem Cell Culture on Bioemulsions

The rapid progress in cell therapies and stem cell technologies requires the development of novel bioprocessing and biomanufacturing pipelines able to cope with the scale up of cell manufacturing. In this respect, microdroplet technologies have already revolutionised the field of biotechnologies, but remain ill-suited to the culture of adherent cells. In this report, we describe the engineering of albumins with cell adhesive peptides for the stabilisation of microdroplets enabling the scale up of mesenchymal stem cell (MSC) expansion. We characterise the modified albumins prior to study their self-assembly at liquid-liquid interfaces via interfacial shear rheology, and mechanical strengthening through the formation of crosslinked nanosheets. The biofunctionalisation of these protein nanosheets is then characterised by fluorescence microscopy. In turn, the ability of the resulting bioactive microdroplets to promote rapid cell adhesion and expansion is examined and the extensive deposition of matrix associated with such cultures is characterised. The culture of MSCs is then scaled up 100 fold, first at the surface of fluorinated oil emulsions, then on plant-based emulsions stabilised by engineered protein nanosheets and the phenotype of resulting cells is characterised. The microdroplet culture system presented displays attractive advantages over existing technologies, in terms of simplicity of processing, compatibility with regulatory expectations and costs of production, and offers exciting opportunities for translation to cell manufacturing, for cell therapies and cultivated meat applications.

bioengineering↗

bioRxiv2023

Matrix Nanoscale Mechanics Regulates Exosome Production by Mesenchymal Stem Cells

Complex biotherapeutics such exosomes offer attractive opportunities for cell-free treatment of disease and conditions difficult to address with single, defined compounds. However their production remains challenging as adherent cells proposed to secrete therapeutic extra cellular vesicles require scalable platforms. In addition, the role of biomaterials design parameters on processes regulating vesicular secretory phenotypes is unclear. Here we propose the use of bioactive microdroplets, or bioemulsions, as microcarriers for the culture of mesenchymal stem cells and production of exosomes. We demonstrate 100% increase in the output of extracellular vesicles on bioemulsions. The impact of matrix mechanics on this process is then investigated, and in particular interfacial shear mechanical properties of corresponding liquid-liquid interfaces forming microdroplets. We find that such local nanoscale mechanics regulates not only cell adhesion, but also exosome output. We find that exosomes generated by cells cultured on bioemulsion microdroplets retain a high content of protein and RNA cargos. Finally, we demonstrate that the cold-shock protein YBox 1, previously associated with RNA packaging, is modulated by matrix mechanics and regulates exosome production. Together, these results demonstrate the impact of local interfacial mechanics on the adhesion and secretory machinery and provide a proof of concept for the application of bioemulsions for the production of complex biotherapeutics.

bioengineering↗

bioRxiv2023

Strong Elastic Protein Nanosheets Enable the Culture and Differentiation of Induced Pluripotent Stem Cells on Microdroplets

Advances in stem cell technologies, revolutionising regenerative therapies and advanced in vitro testing, require novel cell manufacturing pipelines able to cope with scale up and parallelisation. Microdroplet technologies, which have transformed single cell sequencing and other cell-based assays, are attractive in this context, but the inherent soft mechanics of liquid-liquid interfaces is typically thought to be incompatible with the expansion of induced pluripotent stem cells (iPSCs), and their differentiation. In this work, we report the design of protein nanosheets stabilising liquid-liquid interfaces and enabling the adhesion, expansion and retention of stemness by iPSCs. We use microdroplet microfluidic chips to control the formulation of droplets with defined dimensions and size distributions and demonstrate that these sustain high expansion rates, with excellent retention of stem cell marker expression. We further demonstrate that iPSCs cultured in such conditions retain the capacity to differentiate into cardiomyocytes and demonstrate such process on droplets. This work provides clear evidence that local nanoscale mechanics, associated with interfacial viscoelasticity, provides strong cues able to regulate and maintain pluripotency, as well as to support commitment in defined differentiation conditions. Microdroplet technologies appear as attractive candidates to transform cell manufacturing pipelines, bypassing significant hurdles paused by solid substrates and microcarriers.

bioengineering↗

bioRxiv2022

Engineering of Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilisation of Bioemulsions Enabling Adherent Cell Expansion

Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion to liquid substrates have been based on co-assemblies of protein and reactive co-surfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and {beta}-lactoglobulin (BLG), biofunctionalised with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without co-surfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalisation mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of co-surfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without co-surfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

bioengineering↗

bioRxiv2022

Supercharged Protein Nanosheets for Cell Expansion on Bioemulsions

Cell culture at liquid-liquid interfaces, for example at the surface of oil microdroplets, is an attractive strategy to scale up adherent cell manufacturing whilst replacing the use of microplastics. Such process requires the adhesion of cells at interfaces stabilized and reinforced by protein nanosheets displaying high elasticity, but also presenting cell adhesive ligands able to bind integrin receptors. In this report, supercharged albumins are found to form strong elastic protein nanosheets and mediate extracellular matrix (ECM) protein adsorption and cell adhesion. The interfacial mechanical properties and elasticity of supercharged nanosheets is characterized by interfacial rheology and behaviors are compared to those of native bovine serum albumin, human serum albumin and -lactalbumin. ECM protein adsorption to resulting supercharged nanosheets is then quantified via surface plasmon resonance and fluorescence microscopy, demonstrating the dual role supercharged albumins are proposed to play, as scaffold proteins structuring liquid-liquid interfaces and substrates for the capture of ECM molecules. Finally, the adhesion and proliferation of primary human epidermal stem cells is investigated, at pinned droplets, as well as on bioemulsions stabilized by corresponding supercharged nanosheets. This study demonstrates the potential of supercharged proteins for the engineering of biointerfaces for stem cell manufacturing, and draws structure-property relationships that will guide further engineering of associated systems.

bioengineering↗