Biology subjects

Bosch-Fortea, M.

Publications and source records attributed to Bosch-Fortea, M..

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

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↗

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↗

bioRxiv2021

Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

biophysics↗

bioRxiv2019

The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains

The primary cilium is a specialized plasma membrane protrusion that harbors receptors involved in important signaling pathways. Despite its central role in regulating cellular behavior, the biogenesis of the primary cilium is not fully understood. In fact, the source of the ciliary membrane remains a mystery in cell types that assemble their primary cilium entirely at the cell surface, such as polarized renal epithelial cells. After cytokinesis, the remnant of the midbody of these cells moves to the center of the apical surface, where it licenses the centrosome for ciliogenesis through an unidentified mechanism. Here, to investigate the origin of the ciliary membrane and the role of the midbody remnant, we analyzed membrane compaction and lipid dynamics at the microscale and nanoscale in living renal epithelial MDCK cells. We found that a specialized patch made of condensed membranes with restricted lipid lateral mobility surrounds the midbody remnant. This patch accompanies the remnant on its journey towards the centrosome and, once the two structures have met, the remnant delivers part of membranes of the patch to build the ciliary membrane. In this way, we have determined the origin of the ciliary membrane and the contribution of the midbody remnant to primary cilium formation in cells whose primary cilium is assembled at the plasma membrane.

cell biology↗