Biology subjects

Gautrot, J.

Publications and source records attributed to Gautrot, J..

8 recordsLinked to original sources

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

Biomimetic Artificial Bone Marrow Niches for the Scale Up of Hematopoietic Stem Cells

Hematopoietic stem cell (HSC) transplantation to treat haematological disorders is greatly restricted by poor cell availability. Ex vivo expansion of HSCs as a strategy to overcome this limitation has shown limited success so far, due to the loss of stem cell properties in culture. Therefore, engineering of culture platforms that mimic the physiological properties of the bone marrow (BM) in a scalable format is an important target to enable the translation of HSC therapies. Here, we report the design of biomimetic BM niches that enable the culture of HSCs in a scalable 3D platform. Beyond cellular and biochemical components (e.g., matrix and growth factors), an important element of the BM microenvironment is its architecture, dense in adipocytes, with relatively limited matrix and anisotropic mechanical properties. To capture this context, we propose the use of bioemulsions1,2 in which oil microdroplets and associated mechanical anisotropy recreate important architectural features of the hematopoietic niche. Mesenchymal stem cells (MSCs) grown at the surface of such bioemulsion remodelled this environment, assembling an interstitial matrix mimicking that of the BM microenvironment composition. In addition, MSCs secreted important factors underpinning the crosstalk between stromal cells and HSCs in the native environment. HSCs cultured in the resulting artificial BM niches maintained stemness whilst expanding significantly (> 33-fold compared to HSCs cultured in suspension) and enabling scale up of expansion in conical flask bioreactors, to produce 2M cells in a single batch. This platform therefore harnesses engineered BM microenvironments and the processability of bioemulsions and microdroplet technologies to produce HSCs in a scalable format, for application in cell-based therapies.

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↗

bioRxiv2023

Vascularised Cardiac Spheroids-on-a-Chip for Testing the Toxicity of Therapeutics

Microfabricated organ-on-a-chip tissue models are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but recreating microvascularised tissue models remains difficult in many cases. This is particularly relevant to mimic the systemic delivery of therapeutics, to capture the complex multi-step processes associated with trans-endothelial migration, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac tissue spheroids embedded in microfluidic chips. The embedding of spheroids within vascularised multi-compartment microfluidic chips was investigated to identify the importance of the spheroid processing, and co-culture with pericytes on the integration of the spheroid within the microvascular networks formed. The architecture of the resulting models, the expression of cardiac and endothelial markers and the perfusion of the system was then investigated. The ability to retain beating over prolonged periods of time was quantified, over a period of 25 days, demonstrating not only perfusability but also functional performance of the tissue model. Finally, as a proof-of-concept of therapeutic testing, the toxicity of one therapeutic associated with cardiac disfunction was evaluated, identifying differences between direct in vitro testing on suspended spheroids and vascularised models.

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↗

bioRxiv2022

Impact of Pericytes on the Stabilisation of Microvascular Networks in Microfluidic Systems in Response to Nanotoxicity

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in vitro models and organ-on-chip design. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. This report presents a microfluidic model exploring interactions between endothelial cells and pericytes. We identify basal conditions required to form stable and reproducible endothelial networks. We then investigate interactions between endothelial cells and pericytes via direct co-culture. In our system, pericytes inhibited vessel hyperplasia and maintained vessel length in prolonged culture (>10 days). In addition, these vessels displayed barrier function and expression of junction markers associated with vessel maturation, including VE-cadherin, {beta}-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and inhibited vessel regression, in contrast to the striking dissociation of networks in endothelial monocultures. This response was also observed when endothelial/pericyte co-cultures were exposed to high concentrations of moderately toxic cationic nanoparticles used for gene delivery. This study highlights the importance of pericytes in protecting vascular networks from stress and external agents and their importance to the design of advanced in vitro models, including for the testing of nanotoxicity, to better recapitulate physiological response and avoid false positives.

bioengineering↗

bioRxiv2022

Design of an Integrated Microvascularised Human Skin-on-a-Chip Tissue Equivalent Model

Tissue engineered skin constructs have been under development since the 1980s as a replacement for human skin tissues and animal models for therapeutics and cosmetic testing. These have evolved from simple single cell-assays to increasingly complex models with integrated dermal equivalents and multiple cell types including a dermis, epidermis and vasculature. The development of micro-engineered platforms and biomaterials has enabled scientists to better recreate and capture the tissue microenvironment in vitro, including the vascularization of tissue models and their integration into microfluidic chips. However, to date, microvascularised human skin equivalents in a microfluidic context have not been reported. Here we present the design of a novel skin-on-a-chip model integrating human derived primary and immortalized cells in a full thickness skin equivalent. The model is housed in a microfluidic device, in which a microvasculature was previously established. We characterize the impact of our chip design on the quality of the microvascular networks formed and evidence that this enables the formation of more homogenous networks. We developed a methodology to harvest tissues from embedded chips, after 14 days of culture, and characterize the impact of culture conditions and vascularization (including with pericyte co-cultures) on the stratification of the epidermis in the resulting skin equivalents. Our results indicate that vascularization enhances stratification and differentiation (thickness, architecture and expression of terminal differentiation markers such as involucrin and transglutaminase 1), allowing formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, thanks to their realistic microvasculature, may find application for the testing efficacy and safety of therapeutics delivered systemically, in a human context.

bioengineering↗