Organotypic culture of human brain explants as a preclinical model for AI-driven antiviral studies.

Viral neuroinfections represent a major health burden for which the development of antivirals is needed. Antiviral compounds that target the consequences of a brain infection (symptomatic treatment) rather than the cause (direct-acting antivirals) constitute a promising mitigation strategy that requires to be investigated in relevant models. However, physiological surrogates mimicking an adult human cortex are lacking, limiting our understanding of the mechanisms associated with viro-induced neurological disorders. Here, we optimized the Organotypic culture of Post-mortem Adult human cortical Brain explants (OPAB) as a preclinical platform for Artificial Intelligence (AI)-driven antiviral studies. OPAB shows robust viability over weeks, well-preserved 3D cytoarchitecture, viral permissiveness, and spontaneous local field potential (LFP). Using LFP as a surrogate for neurohealth, we developed a machine learning framework to predict with high confidence the infection status of OPAB. As a proof-of-concept, we showed that antiviral-treated OPAB could partially restore LFP-based electrical activity of infected OPAB in a donor-dependent manner. Together, we propose OPAB as a physiologically relevant and versatile model to study neuroinfections and beyond, providing a platform for preclinical drug discovery.

Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.

Protruding cantilever microelectrode array to monitor the inner electrical activity of cerebral organoids.

Stem cell-derived cerebral organoids are artificially grown miniature organ-like structures mimicking embryonic brain architecture. They are composed of multiple neural cell types with 3D cell layer organization exhibiting local field potential. Measuring the extracellular electrical activity by means of conventional planar microelectrode arrays is particularly challenging due to the 3D architecture of organoids. In order to monitor the intra-organoid electrical activity of thick spheroid-shaped samples, we developed long protruding microelectrode arrays able to penetrate the inner regions of cerebral organoids to measure the local potential of neurons within the organoids. A new microfabrication process has been developed which, thanks to the relaxation of internal stresses of a stack of materials deposited over a sacrificial layer, allows one to build a protruding cantilever microelectrode array placed at the apex of beams which rise vertically, over two hundred microns. These slender beams inserted deeply into the organoids give access to the recording of local field potential from neurons buried inside the organoid. This novel device shall provide valuable tools to study neural functions in greater detail.

Viro-fluidics: Real-time analysis of virus production kinetics at the single-cell level.

Real-time visualization and quantification of viruses released by a cell are crucial to further decipher infection processes. Kinetics studies at the single-cell level will circumvent the limitations of bulk assays with asynchronous virus replication. We have implemented a "viro-fluidic" method, which combines microfluidics and virology at single-cell and single-virus resolutions. As an experimental model, we used standard cell lines producing fluorescent HIV-like particles (VLPs). First, to scale the strategy to the single-cell level, we validated a sensitive flow virometry system to detect VLPs in low concentration samples (≥104 VLPs/mL). Then, this system was coupled to a single-cell trapping device to monitor in real-time the VLPs released, one at a time, from single cells under cell culture conditions. Our results revealed an average production rate of 50 VLPs/h/cell similar to the rate estimated for the same cells grown in population. Thus, the virus-producing capacities of the trapped cells were preserved and its real-time monitoring was accurate. Moreover, single-cell analysis revealed a release of VLPs with stochastic bursts with typical time intervals of few minutes, revealing the existence of limiting step(s) in the virus biogenesis process. Our tools can be applied to other pathogens or to extracellular vesicles to elucidate the dissemination mechanisms of these biological nanoparticles.

Human neuromuscular junction on micro-structured microfluidic devices implemented with a custom micro electrode array (MEA).

In the neuromuscular system, signal transmission from motor neurons (MNs) to innervated muscle fibers is crucial for their synaptic function, viability, and maintenance. In order to better understand human neuromuscular junction (hNMJ) functionality, it is important to develop on-a-chip devices with human cells. To investigate this cell network, microfluidic platforms are useful to grow different cell types in isolated compartments. Such devices have already been developed to study in vitro neuronal circuitry. Here, we combined microfluidics with two techniques: soft lithography and custom microelectrodes array (MEA). Our goal was to create hNMJs on a specific pattern of electrodes to stimulate pre-synaptic axons and record post-synaptic muscle activity. Micromachining was used to create structurations to guide muscle growth above electrodes, without impairing axon propagation, therefore optimizing the effectiveness of activity recording. Electrodes were also arranged to be aligned with the microfluidic chambers in order to specifically stimulate axons that were growing between the two compartments. Isolation of the two cell types allows for the selective treatment of neurons or muscle fibers to assess NMJ functionality hallmarks. Altogether, this microfluidic/microstructured/MEA platform allowed mature and functional in vitro hNMJ modelling. We demonstrate that electrical activation of MNs can trigger recordable extracellular muscle action potentials. This study provides evidence for a physiologically relevant model to mimic a hNMJ that will in the future be a powerful tool, more sensitive than calcium imaging, to better understand and characterize NMJs and their disruption in neurodegenerative diseases.

High-Resolution Photonic Force Microscopy Based on Sharp Nanofabricated Tips.

Although near-field imaging techniques reach sub-nanometer resolution on rigid samples, it remains extremely challenging to image soft interfaces, such as biological membranes, due to the deformations induced by the probe. In photonic force microscopy, optical tweezers are used to manipulate and measure the scanning probe, allowing imaging of soft materials without force-induced artifacts. However, the size of the optically trapped probe still limits the maximum resolution. Here, we show a novel and simple nanofabrication protocol to massively produce optically trappable quartz particles which mimic the sharp tips of atomic force microscopy. Imaging rigid nanostructures with our tips, we resolve features smaller than 80 nm. Scanning the membrane of living malaria-infected red blood cells reveals, with no visible artifacts, submicron features termed knobs, related to the parasite activity. The use of nanoengineered particles in photonic force microscopy opens the way to imaging soft samples at high resolution.

Micro/Nanostructure Engineering of Epitaxial Piezoelectric α-Quartz Thin Films on Silicon.

The monolithic integration of sub-micron quartz structures on silicon substrates is a key issue for the future development of piezoelectric devices as prospective sensors with applications based on the operation in the high-frequency range. However, to date, it has not been possible to make existing quartz manufacturing methods compatible with integration on silicon and structuration by top-down lithographic techniques. Here, we report an unprecedented large-scale fabrication of ordered arrays of piezoelectric epitaxial quartz nanostructures on silicon substrates by the combination of soft-chemistry and three lithographic techniques: (i) laser interference lithography, (ii) soft nanoimprint lithography on Sr-doped SiO2 sol-gel thin films, and (iii) self-assembled SrCO3 nanoparticle reactive nanomasks. Epitaxial α-quartz nanopillars with different diameters (from 1 µm down to 50 nm) and heights (up to 2 µm) were obtained. This work demonstrates the complementarity of soft-chemistry and top-down lithographic techniques for the patterning of epitaxial quartz thin films on silicon while preserving its epitaxial crystallinity and piezoelectric properties. These results open up the opportunity to develop a cost-effective on-chip integration of nanostructured piezoelectric α-quartz MEMS with enhanced sensing properties of relevance in different fields of application.

Microfluidic blood vasculature replicas using backside lithography.

Blood vessels in living tissues are an organized and hierarchical network of arteries, arterioles, capillaries, veinules and veins. Their sizes, lengths, shapes and connectivity are set up for an optimum perfusion of the tissues in which they deploy. In order to study the hemodynamics and hemophysics of blood flows and also to investigate artificial vasculature for organs on a chip, it is essential to reproduce most of these geometric features. Common microfluidic techniques produce channels with a uniform height and a rectangular cross section that do not capture the size hierarchy observed in vivo. This paper presents a new single-mask photolithography process using an optical diffuser to produce a backside exposure leading to microchannels with both a rounded cross section and a direct proportionality between local height and local width, allowing a one-step design of intrinsically hierarchical networks.

Tailoring the crystal growth of quartz on silicon for patterning epitaxial piezoelectric films.

Epitaxial films of piezoelectric α-quartz could enable the fabrication of sensors with unprecedented sensitivity for prospective applications in electronics, biology and medicine. However, the prerequisites are harnessing the crystallization of epitaxial α-quartz and tailoring suitable film microstructures for nanostructuration. Here, we bring new insights into the crystallization of epitaxial α-quartz films on silicon (100) from the devitrification of porous silica and the control of the film microstructures: we show that by increasing the quantity of devitrifying agent (Sr) it is possible to switch from an α-quartz microstructure consisting of a porous flat film to one dominated by larger, fully dense α-quartz crystals. We also found that the film thickness, relative humidity and the nature of the surfactant play an important role in the control of the microstructure and homogeneity of the films. Via a multi-layer deposition method, we have extended the maximum thickness of the α-quartz films from a few hundreds of nm to the µm range. Moreover, we found a convenient method to combine this multilayer approach with soft lithography to pattern silica films while preserving epitaxial crystallization. This improved control over crystallization and the possibility of preparing patterned films of epitaxial α-quartz on Si substrates pave the path to future developments in applications based on electromechanics, optics and optomechanics.

An integrated microfluidic/microelectrode array for the study of activity-dependent intracellular dynamics in neuronal networks.

In the central nervous system, neurons are organized in specific neural networks with distinct electrical patterns, input integration capacities, and intracellular dynamics. In order to better understand how neurons process information, it is crucial to keep the complex organization of brain circuits. However, performing subcellular investigations with high spatial and temporal resolution in vivo is technically challenging, especially in fine structures, such as axonal projections. Here, we present an on-a-chip system that combines a microfluidic platform with a dedicated matrix of electrodes to study activity-dependent dynamics in the physiological context of brain circuits. Because this system is compatible with high-resolution video-microscopy, it is possible to simultaneously record intracellular dynamics and electrical activity in presynaptic axonal projections and in their postsynaptic neuronal targets. Similarly, specific patterns of electrical activity can be applied to both compartments in order to investigate how intrinsic and network activities influence intracellular dynamics. The fluidic isolation of each compartment further allows the selective application of drugs at identified sites to study activity-dependent synaptic transmission. This integrated microfluidic/microelectrode array (microMEA) platform is a valuable tool for studying various intracellular and synaptic dynamics in response to neuronal activity in a physiologically relevant context that resembles in vivo brain circuits.

Neuronal network maturation differently affects secretory vesicles and mitochondria transport in axons.

Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons in vivo is technically challenging. In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility.

Unexpected Hard Protein Behavior of BSA on Gold Nanoparticle Caused by Resveratrol.

The understanding of the interactions between nanomaterials, biomolecules, and polyphenols is fundamental in food chemistry, toxicology, and new emerging fields, such as nanomedicine. Here, we investigated the effect of the resveratrol, a principal actor in drug-delivery application on the interaction between bovine serum albumin (BSA), employed as a vector for the delivery of polyphenol drugs, and gold nanoparticle (gNP), the most promising tool in theranostic applications. Through a combination of experimental techniques, which includes an initial evaluation by dynamic light scattering and surface plasmon resonance spectroscopy, we were able to evaluate the evolution of the gold nanoparticle aggregation with increasing ionic strength and the consequences of the BSA and resveratrol addition. To investigate the mechanisms of the interactions, we pursued at the single-molecule level using solid-state nanopore and fluorescence correlation spectroscopy. Our results show that without resveratrol, the BSA is adsorbed on the gNP in water or saline solution. In the presence of resveratrol, the BSA is normally absorbed on gNP in water, but the salt addition leads to its desorption. The resveratrol clearly plays a fundamental role, changing the protein behavior and making the BSA adsorption a reversible process in the presence of salt.

Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington's Disease.

Huntington's disease (HD), a devastating neurodegenerative disorder, strongly affects the corticostriatal network, but the contribution of pre- and postsynaptic neurons in the first phases of disease is unclear due to difficulties performing early subcellular investigations in vivo. Here, we have developed an on-a-chip approach to reconstitute an HD corticostriatal network in vitro, using microfluidic devices compatible with subcellular resolution. We observed major defects in the different compartments of the corticostriatal circuit, from presynaptic dynamics to synaptic structure and transmission and to postsynaptic traffic and signaling, that correlate with altered global synchrony of the network. Importantly, the genetic status of the presynaptic compartment was necessary and sufficient to alter or restore the circuit. This highlights an important weight for the presynaptic compartment in HD that has to be considered for future therapies. This disease-on-a-chip microfluidic platform is thus a physiologically relevant in vitro system for investigating pathogenic mechanisms and for identifying drugs.

Millisecond infrared laser pulses depolarize and elicit action potentials on in-vitro dorsal root ganglion neurons.

This work focuses on the optical stimulation of dorsal root ganglion (DRG) neurons through infrared laser light stimulation. We show that a few millisecond laser pulse at 1875 nm induces a membrane depolarization, which was observed by the patch-clamp technique. This stimulation led to action potentials firing on a minority of neurons beyond an energy threshold. A depolarization without action potential was observed for the majority of DRG neurons, even beyond the action potential energy threshold. The use of ruthenium red, a thermal channel blocker, stops the action potential generation, but has no effects on membrane depolarization. Local temperature measurements reveal that the depolarization amplitude is sensitive to the amplitude of the temperature rise as well as to the time rate of change of temperature, but in a way which may not fully follow a photothermal capacitive mechanism, suggesting that more complex mechanisms are involved.

Role of red cells and plasma composition on blood sessile droplet evaporation.

The morphology of dried blood droplets derives from the deposition of red cells, the main components of their solute phase. Up to now, evaporation-induced convective flows were supposed to be at the base of red cell distribution in blood samples. Here, we present a direct visualization by videomicroscopy of the internal dynamics in desiccating blood droplets, focusing on the role of cell concentration and plasma composition. We show that in diluted suspensions, the convection is promoted by the rich molecular composition of plasma, whereas it is replaced by an outward red blood cell displacement front at higher hematocrits. We also evaluate by ultrasounds the effect of red cell deposition on the temporal evolution of sample rigidity and adhesiveness.

Influence of Adsorption on Proteins and Amyloid Detection by Silicon Nitride Nanopore.

For the past 2 decades, emerging single-nanopore technologies have opened the route to multiple sensing applications. Besides DNA sensing, the identification of proteins and amyloids is a promising field for early diagnosis. However, the influence of the interactions between the nanopore surface and proteins should be taken into account. In this work, we have selected three proteins (avidin, lysozyme, and IgG) that exhibit different affinities with the SiNx surface, and we have also examined lysozyme amyloid. Our results show that the piranha treatment of SiNx significantly decreases protein adsorption. Moreover, we have successfully detected all proteins (pore diameter 17 nm) and shown the possibility of discriminating between denatured lysozyme and its amyloid. For all proteins, the capture rates are lower than expected, and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that proteins interacting only with the nanopore surface wall stay long enough to be detected. For lysozyme amyloid, we show that the use of the nanopore is suitable for determining the number of monomer units even if only the proteins interacting with the nanopore are detected.

PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation.

The treatment of anterior cruciate ligament (ACL) failures remains a current clinical challenge. The present study aims at providing suitable degradable scaffolds for ligament tissue engineering. First, we focus on the design and the evaluation of poly(lactide)/poloxamer or poly(lactide)/poloxamine multiblock copolymers selected and developed to have suitable degradation and mechanical properties to match ACL repair. In the second part, it is shown that the copolymers can be processed in the form of microfibers and scaffolds consisting of a combination of twisted/braided fibers to further modulate the mechanical properties and prepare scaffold prototypes suitable for ligament application. Finally, after assessment of their cytocompatibility, the polymer scaffolds are associated with mesenchymal stem cells (MSCs). MSC differentiation toward a ligament fibroblast phenotype is promoted by a dual stimulation including an inductive culture medium and cyclic mechanical loads. RT-qPCR analyses confirm the potential of our scaffolds and MSCs for ACL regeneration with upregulation of some differentiation markers including Scleraxis, Tenascin-C and Tenomodulin.

Neurite growth acceleration of adult Dorsal Root Ganglion neurons illuminated by low-level Light Emitting Diode light at 645 nm.

The effect of a 645 nm Light Emitting Diode (LED) light irradiation on the neurite growth velocity of adult Dorsal Root Ganglion (DRG) neurons with peripheral axon injury 4-10 days before plating and without previous injury was investigated. The real amount of light reaching the neurons was calculated by taking into account the optical characteristics of the light source and of media in the light path. The knowledge of these parameters is essential to be able to compare results of the literature and a way to reduce inconsistencies. We found that 4 min irradiation of a mean irradiance of 11.3 mW/cm(2) (corresponding to an actual irradiance reaching the neurons of 83 mW/cm(2)) induced a 1.6-fold neurite growth acceleration on non-injured neurons and on axotomized neurons. Although the axotomized neurons were naturally already in a rapid regeneration process, an enhancement was found to occur while irradiating with the LED light, which may be promising for therapy applications. Dorsal Root Ganglion neurons (A) without previous injury and (B) subjected to a conditioning injury.

Electronics manufacturing infrastructures for education and commercialization.

Many medical and biological applications require custom integrated hardware. Infrastructures for the design of CMOS and MEMS hardware allow the education and commercialization at a low cost and in a short time. This paper is reviewing the most recent developments at CMP for the manufacturing of ICs and MEMS. These custom hardware vehicles allow to target many medical and biological applications. Examples are provided through the paper. Such infrastructures may help the Medical and Biological community the same way they helped the Microelectronics community at the time of the VLSI revolution.