BMP-Binding Polysulfonate Brushes to Control Growth Factor Presentation and Regulate Matrix Remodelling.

Bone morphogenetic proteins (BMPs) are important targets to incorporate in biomaterial scaffolds to orchestrate tissue repair. Glycosaminoglycans (GAGs) such as heparin allow the capture of BMPs and their retention at the surface of biomaterials at safe concentrations. Although heparin has strong affinities for BMP2 and BMP4, two important types of growth factors regulating bone and tissue repair, it remains difficult to embed stably at the surface of a broad range of biomaterials and degrades rapidly in vitro and in vivo. In this report, biomimetic poly(sulfopropyl methacrylate) (PSPMA) brushes are proposed as sulfated GAG mimetic interfaces for the stable capture of BMPs. The growth of PSPMA brushes via a surface-initiated activator regenerated by electron transfer polymerization is investigated via ellipsometry, prior to characterization of swelling and surface chemistry via X-ray photoelectron spectroscopy and Fourier transform infrared. The capacity of PSPMA brushes to bind BMP2 and BMP4 is then characterized via surface plasmon resonance. BMP2 is found to anchor particularly stably and at high density at the surface of PSPMA brushes, and a strong impact of the brush architecture on binding capacity is observed. These results are further confirmed using a quartz crystal microbalance with dissipation monitoring, providing some insights into the mode of adsorption of BMPs at the surface of PSPMA brushes. Primary adsorption of BMP2, with relatively little infiltration, is observed on thick dense brushes, implying that this growth factor should be accessible for further binding of corresponding cell membrane receptors. Finally, to demonstrate the impact of PSPMA brushes for BMP2 capture, dermal fibroblasts were then cultured at the surface of functionalized PSPMA brushes. The presence of BMP2 and the architecture of the brush are found to have a significant impact on matrix deposition at the corresponding interfaces. Therefore, PSPMA brushes emerge as attractive coatings for scaffold engineering and stable capture of BMP2 for regenerative medicine applications.

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

Advances in stem cell technologies, revolutionizing regenerative therapies and advanced in vitro testing, require novel cell manufacturing pipelines able to cope with scale up and parallelization. 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, the design of protein nanosheets stabilizing liquid-liquid interfaces and enabling the adhesion, expansion and retention of stemness by iPSCs is reported. Microdroplet microfluidic chips are used to control the formulation of droplets with defined dimensions and size distributions. The resulting emulsions sustain high expansion rates, with excellent retention of stem cell marker expression. iPSCs cultured in such conditions retain the capacity to differentiate into cardiomyocytes. 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.

Interfacial mechanics of ß-casein and albumin mixed protein assemblies at liquid-liquid interfaces.

Protein emulsifiers play an important role in formulation science, from food product development to emerging applications in biotechnologies. The impact of mixed protein assemblies on surface composition and interfacial shear mechanics remains broadly unexplored, in comparison to the impact that formulation has on dilatational mechanics and surface tension or pressure. In this report, we use interfacial shear rheology to quantify the evolution of interfacial shear moduli as a function of composition in bovine serum albumin (BSA)/ß-casein mixed assemblies. We present the pronounced difference in mechanics of these two protein, at oil interfaces, and observe the dominance of ß-casein in regulating interfacial shear mechanics. This observation correlates well with the strong asymmetry of adsorption of these two proteins, characterised by fluorescence microscopy. Using neutron reflectometry and fluorescence recovery after photobleaching, we examine the architecture of corresponding protein assemblies and their surface diffusion, providing evidence for distinct morphologies, but surprisingly comparable diffusion profiles. Finally, we explore the impact of crosslinking and sequential protein adsorption on the interfacial shear mechanics of corresponding assemblies. Overall, this work indicates that, despite comparable surface densities, BSA and ß-casein assemblies at liquid-liquid interfaces display almost 2 orders of magnitude difference in interfacial shear storage modulus and markedly different viscoelastic profiles. In addition, co-adsorption and sequential adsorption processes are found to further modulate interfacial shear mechanics. Beyond formulation science, the understanding of complex mixed protein assemblies and mechanics may have implications for the stability of emulsions and may underpin changes in the mechanical strength of corresponding interfaces, for example in tissue culture or in physiological conditions.

Vascularised cardiac spheroids-on-a-chip for testing the toxicity of therapeutics.

Microfabricated organ-on-a-chips 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 transport or diffusion, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac spheroids embedded in microfluidic chips. Different protocols used for embedding spheroids within vascularised multi-compartment microfluidic chips were investigated first 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. This confirmed the excellent stability of the vascular networks formed, as well as the persistent expression of cardiomyocyte markers such as cTNT and the assembly of striated F-actin, myosin and α-actinin cytoskeletal networks typically associated with contractility and beating. The ability to retain beating over prolonged periods of time was quantified, over 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.

Thiyl radical induced cis/trans isomerism in double bond containing elastomers.

This report presents an evaluation of thiyl radical-induced cis/trans isomerism in double bond-containing elastomers, such as natural, polychloroprene, and polybutadiene rubbers. The study aims to extensively investigate structural changes in polymers after functionalisation using thiol-ene chemistry, a useful click reaction for modifying polymers and developing materials with new functionalities. The paper reports on the use of different thiols, and cis/trans isomerism was detected through 1H NMR analysis, even at very low alkene/thiol mole ratios. The study finds that the configurational arrangements between non-functionalised elastomer units and thiolated units followed a trans-functionalised-cis units arrangement up to an alkene/thiol mole feed ratio of 0.3, while from 0.4 onward, a combination of trans-functionalised-cis and cis-functionalised-trans configurations are found. Additionally, it is observed that by increasing the level of functionalisation, the glass transition temperature of the resulting modified elastomer also increases. Overall, this study provides valuable insights into the effects of thiol-ene chemistry on the structure and properties of elastomers and could have important implications for the development of new materials with enhanced functionality.

A guide to functionalisation and bioconjugation strategies to surface-initiated polymer brushes.

Since the first introduction of their concept in the 1980s and 90s, polymer brushes have been the focus of intense research efforts to identify novel physico-chemical properties and responsiveness, and optimise the properties of associated interfaces for an ever growing range of applications. To a large extent, this effort has been enabled by progress in surface initiated controlled polymerisation techniques, allowing a huge diversity of monomers and macromolecular architectures to be harnessed and achieved. However, polymer functionalisation through chemical coupling of various moieties and molecular structures has also played an important role in expanding the molecular design toolbox of the field of polymer brush science. This perspective article reviews recent progress in polymer brush functionalisation, discussing a broad range of strategies for the side chain and end chain chemical modification of these polymer coatings. The impact of the brush architecture on associated coupling is also examined. In turn, the role that such functionalisation approaches play in the patterning and structuring of brushes, as well as their conjugation with biomacromolecules for the design of biofunctional interfaces is then reviewed and discussed.

Programmed Self-Assembly of DNA Nanosheets with Discrete Single-Molecule Thickness and Interfacial Mechanics: Design, Simulation, and Characterization.

DNA is programmed to hierarchically self-assemble into superstructures spanning from nanometer to micrometer scales. Here, we demonstrate DNA nanosheets assembled out of a rationally designed flexible DNA unit (F-unit), whose shape resembles a Feynman diagram. F-units were designed to self-assemble in two dimensions and to display a high DNA density of hydrophobic moieties. oxDNA simulations confirmed the planarity of the F-unit. DNA nanosheets with a thickness of a single DNA duplex layer and with large coverage (at least 30 µm × 30 µm) were assembled from the liquid phase at the solid/liquid interface, as unambiguously evidenced by atomic force microscopy imaging. Interestingly, single-layer nanodiscs formed in solution at low DNA concentrations. DNA nanosheet superstructures were further assembled at liquid/liquid interfaces, as demonstrated by the fluorescence of a double-stranded DNA intercalator. Moreover, the interfacial mechanical properties of the nanosheet superstructures were measured as a response to temperature changes, demonstrating the control of interfacial shear mechanics based on DNA nanostructure engineering. The rational design of the F-unit, along with the presented results, provide an avenue toward the controlled assembly of reconfigurable/responsive nanosheets and membranes at liquid/liquid interfaces, to be potentially used in the characterization of biomechanical processes and materials transport.

The impact of pericytes on the stability of microvascular networks in response to nanoparticles.

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in-vitro models and organ-on-chip designs. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. The use of such co-culture for the testing of therapeutics and nanoparticle safety is increasingly considered for the validation of therapeutic strategies. This report presents the use of a microfluidic model for such applications. Interactions between endothelial cells and pericytes are first explored. 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 prevented 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, ß-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and prevented 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.

Mechanical Evaluation of Hydrogel-Elastomer Interfaces Generated through Thiol-Ene Coupling.

The formation of hybrid hydrogel-elastomer scaffolds is an attractive strategy for the formation of tissue engineering constructs and microfabricated platforms for advanced in vitro models. The emergence of thiol-ene coupling, in particular radical-based, for the engineering of cell-instructive hydrogels and the design of elastomers raises the possibility of mechanically integrating these structures without relying on the introduction of additional chemical moieties. However, the bonding of hydrogels (thiol-ene radical or more classic acrylate/methacrylate radical-based) to thiol-ene elastomers and alkene-functional elastomers has not been characterized in detail. In this study, we quantify the tensile mechanical properties of hybrid hydrogel samples formed of two elastomers bonded to a hydrogel material. We examine the impact of radical thiol-ene coupling on the crosslinking of both elastomers (silicone or polyesters) and hydrogels (based on thiol-ene crosslinking or diacrylate chemistry) and on the mechanics and failure behavior of the resulting hybrids. This study demonstrates the strong bonding of thiol-ene hydrogels to alkene-presenting elastomers with a range of chemistries, including silicones and polyesters. Overall, thiol-ene coupling appears as an attractive tool for the generation of strong, mechanically integrated, hybrid structures for a broad range of applications.

Growth of mesenchymal stem cells at the surface of silicone, mineral and plant-based oils.

Bioemulsions are attractive platforms for the expansion of adherent cells in bioreactors. Their design relies on the self-assembly of protein nanosheets at liquid-liquid interfaces, displaying strong interfacial mechanical properties and promoting integrin-mediated cell adhesion. However, most systems developed to date have focused on fluorinated oils, which are unlikely to be accepted for direct implantation of resulting cell products for regenerative medicine, and protein nanosheets self-assembly at other interfaces has not been investigated. In this report, the composition of aliphatic pro-surfactants palmitoyl chloride and sebacoyl chloride, on the assembly kinetics of poly(L-lysine) at silicone oil interfaces and characterisation of ultimate interfacial shear mechanics and viscoelasticity is presented. The impact of the resulting nanosheets on the adhesion of mesenchymal stem cells (MSCs) is investigated via immunostaining and fluorescence microscopy, demonstrating the engagement of the classic focal adhesion-actin cytoskeleton machinery. The ability of MSCs to proliferate at the corresponding interfaces is quantified. In addition, expansion of MSCs at other non-fluorinated oil interfaces, based on mineral and plant-based oils is investigated. Finally, the proof-of-concept of such non-fluorinated oil systems for the formulation of bioemulsions supporting stem cell adhesion and expansion is demonstrated.

Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization 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 at liquid substrates have been based on coassemblies of protein and reactive cosurfactants, 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 ß-lactoglobulin (BLG), biofunctionalized 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 cosurfactant 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. Biofunctionalization 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 cosurfactant, 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 cosurfactants 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.

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 while replacing the use of microplastics. Such a process requires the adhesion of cells at interfaces stabilized and reinforced by protein nanosheets displaying not only 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 when co-assembling with the co-surfactant pentafluorobenzoyl chloride (PFBC) and mediate extracellular matrix (ECM) protein adsorption and cell adhesion. The interfacial mechanical properties and elasticity of supercharged nanosheets are characterized by interfacial rheology, and behaviors are compared to those of native bovine serum albumin, human serum albumin, and α-lactalbumin. The impact of PFBC on such assembly is investigated. ECM protein adsorption to resulting supercharged nanosheets is then quantified via surface plasmon resonance and fluorescence microscopy, demonstrating that the dual role supercharged albumins are proposed to play as scaffold protein structuring liquid-liquid interfaces and substrates for the capture of ECM molecules. Finally, the adhesion and proliferation of primary human epidermal stem cells are 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.

Mesenchymal Stem Cells Sense the Toughness of Nanomaterials and Interfaces.

Stem cells are known to sense and respond to the mechanical properties of biomaterials. In turn, cells exert forces on their environment that can lead to striking changes in shape, size and contraction of associated tissues, and may result in mechanical disruption and functional failure. However, no study has so far correlated stem cell phenotype and biomaterials toughness. Indeed, disentangling toughness-mediated cell response from other mechanosensing processes has remained elusive as it is particularly challenging to uncouple Youngs' or shear moduli from toughness, within a range relevant to cell-generated forces. In this report, it is shown how the design of the macromolecular architecture of polymer nanosheets regulates interfacial toughness, independently of interfacial shear storage modulus, and how this controls the expansion of mesenchymal stem cells at liquid interfaces. The viscoelasticity and toughness of poly(l-lysine) nanosheets assembled at liquid-liquid interfaces is characterised via interfacial shear rheology. The local (microscale) mechanics of nanosheets are characterised via magnetic tweezer-assisted interfacial microrheology and the thickness of these assemblies is determined from in situ ellipsometry. Finally, the response of mesenchymal stem cells to adhesion and culture at corresponding interfaces is investigated via immunostaining and confocal microscopy.

Design of an Integrated Microvascularized 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, microvascularized 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 the formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, because of their realistic microvasculature, may find applications for testing efficacy and safety of therapeutics delivered systemically, in a human context.

Multi-Scale Analysis of the Composition, Structure, and Function of Decellularized Extracellular Matrix for Human Skin and Wound Healing Models.

The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.

Protein Ligand Nanopattern Size Selects for Cellular Adhesion via Hemidesmosomes over Focal Adhesions.

Hemidesmosomes (HDs) are multiprotein complexes that firmly anchor epidermal cells to the basement membrane of skin through the interconnection of the cytoplasmic intermediate filaments with extracellular laminin 332 (Ln332). Considerably less attention has been paid to HDs compared to focal complexes/focal adhesions (FC/FAs) in mechanistic single-cell structures due to the lack of suitable in vitro model systems. Here nanopatterns of Ln332 (100-1000 nm) are created to direct and study the formation of HD in adherent HaCaT cells. It is observed that HaCaT cells at Ln 332 nanopatterns adhere via hemidesmosomes, in stark contrast to cells at homogeneous Ln332 surfaces that adhere via FC/FAs. Clustering of α6 integrin is observed at nanopatterned Ln332 of 300 nm patches and larger. Cells at 500 nm diameter patterns show strong colocalization of α6 integrin with ColXVII or pan-cytokeratin compared to 300 nm/1000 nm indicating a threshold for HD initiation >100 nm but a pattern size selection for maturation of HDs. It is demonstrated that the pattern of Ln332 can determine the cellular selection of adhesion types with a size-dependent initiation and maturation of HDs. The protein nanopatterning approach that is presented provides a new in vitro route to study the role of HDs in cell signaling and function.

Impact of the multiscale viscoelasticity of quasi-2D self-assembled protein networks on stem cell expansion at liquid interfaces.

Although not typically thought to sustain cell adhesion and expansion, liquid substrates have recently been shown to support such phenotypes, providing protein nanosheets could be assembled at corresponding liquid-liquid interfaces. However, the precise mechanical properties required from such quasi-2D nanoassemblies and how these correlate with molecular structure and nanoscale architecture has remained unclear. In this report, we screen a broad range of surfactants, proteins, oils and cell types and correlate interfacial mechanical properties with stem cell expansion. Correlations suggest an impact of interfacial viscoelasticity on the regulation of such behaviour. We combine interfacial rheology and magnetic tweezer-based interfacial microrheology to characterise the viscoelastic profile of protein nanosheets assembled at liquid-liquid interfaces. Based on neutron reflectometry and transmission electron microscopy data, we propose that the amorphous nanoarchitecture of quasi-2D protein nanosheets controls their multi-scale viscoelasticity which, in turn, correlates with cell expansion. This understanding paves the way for the rational design of protein nanosheets for microdroplet and bioemulsion-based stem cell manufacturing and screening platforms.

Highly Stretchable Conductive Covalent Coacervate Gels for Electronic Skin.

Highly stretchable electrically conductive hydrogels have been extensively researched in recent years, especially for applications in strain and pressure sensing, electronic skin, and implantable bioelectronic devices. Herein, we present a new cross-linked complex coacervate approach to prepare conductive hydrogels that are both highly stretchable and compressive. The gels involve a complex coacervate between carboxylated nanogels and branched poly(ethylene imine), whereby the latter is covalently cross-linked by poly(ethylene glycol) diglycidyl ether (PEGDGE). Inclusion of graphene nanoplatelets (Gnp) provides electrical conductivity as well as tensile and compressive strain-sensing capability to the hydrogels. We demonstrate that judicious selection of the molecular weight of the PEGDGE cross-linker enables the mechanical properties of these hydrogels to be tuned. Indeed, the gels prepared with a PEGDGE molecular weight of 6000 g/mol defy the general rule that toughness decreases as strength increases. The conductive hydrogels achieve a compressive strength of 25 MPa and a stretchability of up to 1500%. These new gels are both adhesive and conformal. They provide a self-healable electronic circuit, respond rapidly to human motion, and can act as strain-dependent sensors while exhibiting low cytotoxicity. Our new approach to conductive gel preparation is efficient, involves only preformed components, and is scalable.

Long term expansion profile of mesenchymal stromal cells at protein nanosheet-stabilised bioemulsions for next generation cell culture microcarriers.

Tremendous progress in the identification, isolation and expansion of stem cells has allowed their application in regenerative medicine and tissue engineering, and their use as advanced in vitro models. As a result, stem cell manufacturing increasingly requires scale up, parallelisation and automation. However, solid substrates currently used for the culture of adherent cells are poorly adapted for such applications, owing to their difficult processing from cell products, relatively high costs and their typical reliance on difficult to recycle plastics and microplastics. In this work, we show that bioemulsions formed of microdroplets stabilised by protein nanosheets displaying strong interfacial mechanics are well-suited for the scale up of adherent stem cells such as mesenchymal stromal cells (MSCs). We demonstrate that, over multiple passages (up to passage 10), MSCs retain comparable phenotypes when cultured on such bioemulsions, solid microcarriers (Synthemax II) and classic 2D tissue culture polystyrene. Phenotyping (cell proliferation, morphometry, flow cytometry and differentiation assays) of MSCs cultured for multiple passages on these systems indicate that, although stemness is lost at late passages when cultured on these different substrates, stem cell phenotypes remained comparable between different culture conditions, at any given passage. Hence our study validates the use of bioemulsions for the long term expansion of adherent stem cells and paves the way to the design of novel 3D bioreactors based on microdroplet microcarriers.

Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors.

In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.