Objective: This project aims to study the behavior of nanoparticles and formation of nanoparticle corona (NPC) in biomimetic liquid, lung lining fluid (LLF), and on a biomimetic surface, an artificial cilia surface to mimic the ciliated lung epithelium. The interactions of NPs with cells in these environments will be investiated towards nanotoxicology. Significance: Nanoparticle corona is the dynamical corona of associated biomolecules on a nanoparticle, that determines its biological identity. Current knowledge and efforts on nanoparticle corona studies are limited to use of serum as the biological media, however, lung fluid and lung surfaces should be focused on for understanding how the cell will see the nanoparticle, for the resulting cellular and toxicological response. Specific Aims: Different sized silica (SiO2) and cerium oxide (CeO2) nanoparticles will be investigated in terms of their aggregation and stability behavior with the formed corona composed of biomolecules that is composed of the real physiological constituents of LLF, in the two different biomimetic millieu: 1) lung fluid as found in the alveolar regions covering the alveolar epithelium, 2) the lung fluid covering the ciliated epithelial tissues of the airway surfaces. The first liquid will be mimicked by proteins and phospholipids purified from porcine lung. The second liquid and the ciliated surface will be mimicked by preparation of magnetically actuatable cilia surface and growing in-situ and ex-situ protein secreting cells on this surface. Nanoparticle corona formations will be studied in both systems, using light scattering, differential sedimentation centrifuge, field flow fractionation, gel electrophoresis to dechiper the corona structure. Also, the behavior of the nanoparticles will be investigated in presence of the artificial cilia beating on the biomimietic cilia surface, in presence of the sol-gel composed of mucus and the periciliary liquid, utilising optical microscopy. Interactions with different types of lung epithelial cells will be investigated towards better characterization of NPC for their toxicological response. Impact: The project will have a significant impact for both nanotoxicology and nanomedicine, as the fate of the nanoparticles that are deposited in the lungs is strongly dependent on the behavior of NPCs and the interactions in different environments. The project will result in better characterization standards for nanotoxicology studies, as well as aiding in better development of engineered nanoparticles for nanomedicine.

In the 70's, the unicellular internode of acorticated characeae helped proving plant growth is pressure-driven and that growth and mechanical anisotropies are equal. The internode of the related corticated characeae is constituted by one giant cylindrical cell surounded by a layer of smaller elongated cells, the cortications; our project introduces this minimal multicellular model organism to quantitatify the interplay between turgor pressure, mechanical anisotropy and adhesion for spiral growth. A set-up to measure the elongation and rotation while controling the pressure inside the central cell will be mounted. “Constitutive laws”, estimated on the central cell, will be used to numerically extrapolate the elongation-rotation relation of corticated characeae and compare them to direct experiments thus precising the extent of the mechanical role of pressure on cell-wall growth. The respective roles of anisotropy and adhesion on rotation will be quantified using biochemical treatment.

One of the most striking features of embryonic development is that differentiation is happening in a spatially ordered fashion: tissue self-organize to form well-defined patterns that pre-figure the body plan. During gastrulation, the cells of the embryo are allocated into three germ layers: ectoderm, mesoderm and endoderm. During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. Studying the spatio-temporal dynamics of pattern formation is difficult in live embryos, because of their inherent lack of observability (especially in organisms that develop in utero) but also because it is not possible in an embryo to control in a quantitative manner the parameters that are likely to be relevant for the establishment of the multi cellular pattern such as the size and shape of the tissue and its physical and chemical environment. Recently, we took the first step toward in vitro recapitulation of early embryonic patterning by showing that human Embryonic Stem Cells (ESCs) confined to circular disks comparable in size to mammalian embryos using micro-patterning technology and treated with the gastrulation inducing signal BMP4 differentiate to an outer trophectoderm-like ring followed by the three embryonic germ layers in an ordered, reproducible radial pattern. Quite notably, at the level of the Brachyury positive ring of mesodermal cells, hESC colonies on micro-patterned glass substrates show the molecular (up regulation of Snail) and morphological the (Epithelial-Mesenchymal Transition) signature of gastrulating cells. These results demonstrate that geometric confinement is sufficient to trigger self-organized patterning in ESCs and that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries. ESC culture on micro-patterned substrates thus provides a quantitative assay to study early development. The observed pattern doesn’t however recapitulate all the features of the patterning observed in embryos, suggesting that the imposed boundary conditions are too stringent or incorrectly defined. By taking advantage the micro-fabrication and the micro-fluidics toolbox, we propose here to study how the physical (stiffness of the substrate, size and shape of the tissue) and chemical (spatio-temporal profile of differentiating signals) properties of the cells micro-environment is affecting the final pattern, in order to define what is the minimal set of boundary conditions allowing for proper gastrulation. With this series of experiments, we expect to better understand the physical rules governing the self-organization of differentiating tissues. This knowledge is necessary if we want to be able one day to grow organs in a dish.

In the peripheral nervous system, Schwann cells (SCs) govern the neuron myelination, constituting a key player in many neuropathies where current treatments usually lead to poor functional recovery. Peripheral neuropathies affect about 5% of the general population after the age of 50 and are caused by a wide set of factors among whom diabetes, genetic causes, toxic (such as chemotherapies, pollutants) and life habits. The oxidative stress on neurons and Schwann Cells plays a central role in them and the current treatments. Extracellular vesicles (EV) seem to play an important role in regulating this stress and more generally in the myelination / demyelination / remyelination mechanisms. They constitute a potential avenue for new biotherapeutics, especially for the peripheral neuropathies. In OXIMOCHI, we will develop a neurofluidic myelinating model of the peripheral nervous system. Taking advantage of compartmentalized architectures and soft styrenic block copolymer for microfluidics, we will monitor the structure and function of myelinating axons as well as the demyelination / remyelination induced by oxidative stress in time. Our model of the peripheral nervous system (PNS) is based on sensory neurons cultured from embryonic dorsal root ganglia (DRG). It allows a better control on neuron and SCs differentiation starting at precursor state until myelination. The OXIMOCHI chips will be compatible with high content screening for neuropharmacological assays. We will quantitatively assess the myelin regeneration potency of 3 standard of care: lithium chloride, benzatropine and methylcobalamin. This physiologically relevant and augmented model will allow to recapitulate the myelin for healthy and diseased configuration both for physiological studies of the myelin as well as neuropharmacological assays. We will then develop an analytical workflow around the vesicles secreted in vitro by SCs (SC-EVs) and neuron to follow up their signature function of myelin health. Thanks to the current analytical techniques for EVs, including field flow fractionation hyphenated analysis AF4-MALS-DLS, we will analyze the SCs secretome to perform the first screening of the myelinated neuron signature and generate a database function of the status of the myelin. The use of proteomics and secretomics will allow to relate these different signatures to known myelin factors and obtain a database clarifying the role of EVs in the myelin state of our model and its role in the oxidative stress regulation. This project will provide a new microfluidic technology and an analytical methodology for the fundamental understanding of myelin in the peripheral nervous system and the role of EVs in its physiology as well as for facilitating assessing therapeutic potency of treatments in the context of myelin diseases, in particular those related to oxidative stress. The database provided will contribute to identify potential therapeutical targets and contribute to the emergence of EV-based biotherapeutics for myelin neuropathies related to oxidative stress.

Fistulas are a major neglected health burden related to Crohn's disease or secondary to surgery, cancer therapy or trauma. Post-surgical digestive fistulas are challenging conditions associated with low remission rates and high refractoriness. There is an urgent need of novel therapeutic approaches for this disease. FisTher investigates an alternative to cell therapy approach, by proposing a minimally-invasive cell-free local therapy based on the regenerative effect of extracellular vesicles (EVs) from mesenchymal stem/stromal cells (MSCs). We consider that MSC EVs represent an eligible alternative for fistula therapy, as they recapitulate the regenerative effect of their mother cells while mitigating risks of uncontrolled replication, differentiation and vascular occlusion, offering “off-the-shelf”, storage and shelf-life gains. The main challenges for rendering EV-based regenerative medicine clinically feasible are large-scale high-yield standardized EV production and EV optimized administration. Concerning EV manufacturing, stringent requirements must be considered such as up-scaled and high-yield production fulfilling uniformity, consistency, purity and reproducibility criteria based on standardized and reliable quality control. The way EVs are administered also represents a main concern considering that systemic administration results in rapid EV clearance and localization in off-target organs. Building on the PI previous work, our strong preliminary results, our intellectual property and complimentary collaborators, FisTher has the ambition to render viable the implementation of EV-based therapy by tackling EV production and administration technical barriers. FisTher proposes large-scale high-yield EV production based on our patented concept of turbulence-vesiculation complying with a standardized production in GMP bioreactors in line with regulatory issues. FisTher set-up relies on the generation of a controlled turbulent flow in which turbulence microvortices will elicit a shear stress on cells triggering EV release. This turbulence-based strategy is (i) time-saving enabling massive EV release in some hours, (ii) integrated as it is based on tuning the own GMP bioreactor stirring system, (iii) straightforward as no further processing is required to eliminate the trigger (turbulence disappearing when stirring is turned off) and (iv) scalable based on turbulence flow parameters. FisTher also proposes a thermo-actuated EV delivery in the fistula tract for eliciting an enhanced therapeutic effect in situ. FisTher strategy is expected to avoid systemic administration clearance and overcome difficulties related to local delivery, such as fistula secretions (washing-out the therapeutic agent) and fistula tract inaccessibility (sometimes irregular large defects of several centimeters). FisTher relies on dual biomaterial/EV component for fistula therapy. The thermoresponsive hydrogel biomaterial component is expected to cope with fistula local delivery difficulties promoting an occlusive effect, retaining EVs in the fistula tract and preventing EV wash-out by fistula secretions, while enabling the filling of the entire fistula tract despite its size and irregular morphology. Biomaterial choice was based on material physical and therapeutic properties and considered a clinical translation perspective. Building on strong preliminary results, we intend to investigate the combination of turbulence EVs with a poloxamer 407 hydrogel. FisTher proposes the off-label use of this hydrogel, which is a vessel occlusive medical device authorized in Europe, as an innovative fistula occlusive EV vehicle. FisTher fully considers key regulatory and manufacturing issues in the project choices to set the basis for implementing the first future clinical trial on MSC EVs for the therapy of post-surgical digestive fistulas.