RESUMEN
Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and no automation. Here, we introduce the sliding insert micropipette aspiration method, which permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.
RESUMEN
Conventional culture conditions are oftentimes insufficient to study tissues, organisms, or 3D multicellular assemblies. They lack both dynamic chemical and mechanical control over the microenvironment. While specific microfluidic devices have been developed to address chemical control, they often do not allow the control of compressive forces emerging when cells proliferate in a confined environment. Here, we present a generic microfluidic device to control both chemical and mechanical compressive forces. This device relies on the use of sliding elements consisting of microfabricated rods that can be inserted inside a microfluidic device. Sliding elements enable the creation of reconfigurable closed culture chambers for the study of whole organisms or model micro-tissues. By confining the micro-tissues, we studied the biophysical impact of growth-induced pressure and showed that this mechanical stress is associated with an increase in macromolecular crowding, shedding light on this understudied type of mechanical stress. Our mechano-chemostat allows the long-term culture of biological samples and can be used to study both the impact of specific conditions as well as the consequences of mechanical compression.
Asunto(s)
Microfluídica , Estrés Mecánico , PresiónRESUMEN
An efficient superhydrophobic concentrator is developed using a hierarchical superhydrophobic surface on which the evaporation of a sessile droplet (6 µL) drives the nonvolatile elements it contains on a predefined micrometric analytical surface (pedestal of 80 µm diameter). This hierarchical silicon surface exhibits a surface texture made of etched nanopillars and consists of micropillars and guiding lines, arranged in radial symmetry around the central pedestal. The guiding lines ensure the overall convergence of the sessile droplet toward the central pedestal during evaporation. The nanopillar texturing induced a delay in the Cassie-Baxter to Wenzel regime transition, until the edge of the droplet reaches the periphery of the pedestal. Experiments performed with polymer microparticles suspended in ultrapure water or with DNA molecules solubilized in ultrapure water at sub-fM concentrations demonstrated that the totality of the nonvolatile elements in the liquid microvolume is delivered on or close to the pedestal area, in a very reproducible manner. The very high concentration capacity of the device enabled the discrimination of the degree of purity of ultrapure water samples from different origins. The concentrator also turned out to be functional for raw water samples, opening possible applications to environmental analysis.
Asunto(s)
Silicio , Agua , Agua/química , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de Superficie , Silicio/química , Polímeros/químicaRESUMEN
Flexible intracerebral probes for neural recording and electrical stimulation have been the focus of many research works to achieve better compliance with the surrounding tissue while minimizing rejection. Strategies have been explored to find the best way to insert flexible probes into the brain while maintaining their flexibility once positioned. Here, we present a novel and versatile scalable batch fabrication approach to deliver ultrathin and flexible probes consisting of a silk-parylene bilayer. The biodegradable silk layer, whose degradation time is programmable, provides a temporary and programmable stiffener to allow the insertion of ultrathin parylene-based flexible devices. Our innovative and robust batch fabrication technology allows complete freedom over probe design in terms of materials, size, shape, and thickness. We demonstrate successful ex vivo insertion of the probe with acute high-fidelity recordings of epileptic seizures in field potentials as well as single-unit action potentials in mouse brain slices. Our novel technological solution for implanting ultraflexible devices in the brain while minimizing rejection risks shows high potential for use in both brain research and clinical therapies.
RESUMEN
Microdevices composed of microwell arrays integrating nanoelectrodes (OptoElecWell) are developed to achieve dual high-resolution optical and electrochemical detections on single Saccharomyces cerevisiae yeast cells. Each array consists of 1.6 × 105 microwells measuring 8 µm in diameter and 5 µm height, with a platinum nanoring electrode for in situ electrochemistry, all integrated on a transparent thin wafer for further high-resolution live-cell imaging. After optimizing the filling rate, 32% of cells are effectively trapped within microwells. This allows to analyse S. cerevisiae metabolism associated with basal respiration while simultaneously measuring optically other cellular parameters. In this study, the impact of glucose concentration on respiration and intracellular rheology is focused. It is found that while the oxygen uptake rate decreases with increasing glucose concentration, diffusion of tracer nanoparticles increases. The OptoElecWell-based respiration methodology provides similar results compared to the commercial gold-standard Seahorse XF analyzer, while using 20 times fewer biological samples, paving the way to achieve single cell metabolomics. In addition, it facilitates an optical route to monitor the contents within single cells. The proposed device, in combination with the dual detection analysis, opens up new avenues for measuring cellular metabolism, and relating it to cellular physiological indicators at single cell level.
Asunto(s)
Saccharomyces cerevisiae , Saccharomycetales , Electrodos , Oxígeno , ReologíaRESUMEN
Giant cell arteritis is a systemic vasculitis that affects arteries of medium and large caliber, mainly the aorta artery and its main branches. It is more frequent in women older than 50 years. The most common symptoms are fever, jaw claudication, headache, hyperesthesia of the scalp and loss of vision with anterior ischemic optic nerve disease. But, in a minority of cases, less frequent symptoms are observed that delay and make more difficult the diagnosis. Here, we present the case of a 76-year-old woman who came to our consultation having pain in the oral cavity and presenting tongue and neck edema for 48 hours. She had also suffered from headaches during the previous month. Because the physical examination showed clinical signs of lingual ischemia, a presumptive diagnosis of ischemic involvement due to giant cell arteritis was considered. She started a treatment with systemic corticosteroids and a temporal artery biopsy was performed. We conclude, that giant cell arteritis should be suspected in patients presenting lingual ischemia symptoms in order to start the specific treatment early enough to avoid irreversible complications.
La arteritis de células gigantes es una vasculitis sistémica que compromete arterias de mediano y gran calibre, principalmente la arteria aorta y sus ramas. Su prevalencia es mayor en mujeres a partir de los 50 años, típicamente se manifiesta con fiebre, claudicación mandibular, cefalea, hiperestesia del cuero cabelludo y pérdida de la visión con neuropatía óptica isquémica anterior, en una minoría de casos aparecen síntomas menos frecuentes que dificultan y retrasan el diagnóstico. Se presenta el caso de una mujer de 76 años que consultó por dolor en la cavidad bucal con edema lingual y en cuello de 48 horas de evolución asociado a cefalea el mes previo. En el examen físico presentaba signos clínicos de isquemia lingual, por lo que se consideró como diagnóstico presuntivo compromiso isquémico por arteritis de células gigantes, e inició tratamiento con corticoides sistémicos realizándose una biopsia de arteria temporal que evidenció infiltrado linfocitario panparietal con engrosamiento de la túnica íntima y hallazgos compatibles con panarteritis. La arteritis de células gigantes debe ser sospechada en pacientes con manifestaciones de isquemia lingual, iniciándose en forma precoz el tratamiento para evitar complicaciones irreversibles.
Asunto(s)
Arteritis de Células Gigantes/patología , Enfermedades de la Lengua/patología , Anciano , Diagnóstico Diferencial , Femenino , Arteritis de Células Gigantes/diagnóstico , Arteritis de Células Gigantes/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Humanos , Necrosis , Enfermedades de la Lengua/diagnóstico , Enfermedades de la Lengua/tratamiento farmacológicoRESUMEN
La arteritis de células gigantes es una vasculitis sistémica que compromete arterias de mediano y gran calibre, principalmente la arteria aorta y sus ramas. Su prevalencia es mayor en mujeres a partir de los 50 años, típicamente se manifiesta con fiebre, claudicación mandibular, cefalea, hiperestesia del cuero cabelludo y pérdida de la visión con neuropatía óptica isquémica anterior, en una minoría de casos aparecen síntomas menos frecuentes que dificultan y retrasan el diagnóstico. Se presenta el caso de una mujer de 76 años que consultó por dolor en la cavidad bucal con edema lingual y en cuello de 48 horas de evolución asociado a cefalea el mes previo. En el examen físico presentaba signos clínicos de isquemia lingual, por lo que se consideró como diagnóstico presuntivo compromiso isquémico por arteritis de células gigantes, e inició tratamiento con corticoides sistémicos realizándose una biopsia de arteria temporal que evidenció infiltrado linfocitario panparietal con engrosamiento de la túnica íntima y hallazgos compatibles con panarteritis. La arteritis de células gigantes debe ser sospechada en pacientes con manifestaciones de isquemia lingual, iniciándose en forma precoz el tratamiento para evitar complicaciones irreversibles.
Giant cell arteritis is a systemic vasculitis that affects arteries of medium and large caliber, mainly the aorta artery and its main branches. It is more frequent in women older than 50 years. The most common symptoms are fever, jaw claudication, headache, hyperesthesia of the scalp and loss of vision with anterior ischemic optic nerve disease. But, in a minority of cases, less frequent symptoms are observed that delay and make more difficult the diagnosis. Here, we present the case of a 76-year-old woman who came to our consultation having pain in the oral cavity and presenting tongue and neck edema for 48 hours. She had also suffered from headaches during the previous month. Because the physical examination showed clinical signs of lingual ischemia, a presumptive diagnosis of ischemic involvement due to giant cell arteritis was considered. She started a treatment with systemic corticosteroids and a temporal artery biopsy was performed. We conclude, that giant cell arteritis should be suspected in patients presenting lingual ischemia symptoms in order to start the specific treatment early enough to avoid irreversible complications.
Asunto(s)
Humanos , Femenino , Anciano , Arteritis de Células Gigantes/patología , Enfermedades de la Lengua/patología , Arteritis de Células Gigantes/diagnóstico , Arteritis de Células Gigantes/tratamiento farmacológico , Enfermedades de la Lengua/diagnóstico , Enfermedades de la Lengua/tratamiento farmacológico , Diagnóstico Diferencial , Glucocorticoides/uso terapéutico , NecrosisRESUMEN
In vivo, immune cells migrate through a wide variety of tissues, including confined and constricting environments. Deciphering how cells apply forces when infiltrating narrow areas is a critical issue that requires innovative experimental procedures. To reveal the distribution and dynamics of the forces of cells migrating in confined environments, we designed a device combining microchannels of controlled dimensions with integrated deformable micropillars serving as sensors of nanoscale subcellular forces. First, a specific process composed of two steps of photolithography and dry etching was tuned to obtain micrometric pillars of controlled stiffness and dimensions inside microchannels. Second, an image-analysis workflow was developed to automatically evaluate the amplitude and direction of the forces applied on the micropillars by migrating cells. Using this workflow, we show that this microdevice is a sensor of forces with a limit of detection down to 64 pN. Third, by recording pillar movements during the migration of macrophages inside the confining microchannels, we reveal that macrophages bent the pillars with typical forces of 0.3 nN and applied higher forces at the cell edges than around their nuclei. When the degree of confinement was increased, we found that forces were redirected from inward to outward. By providing a microdevice that allows the analysis of force direction and force magnitude developed by confined cells, our work paves the way for investigating the mechanical behavior of cells migrating though 3D constricted environments.
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Técnicas de Cultivo de Célula , Núcleo Celular/química , Dispositivos Laboratorio en un Chip , Macrófagos/química , Técnicas Biosensibles/métodos , Adhesión Celular/genética , Movimiento Celular/genética , Núcleo Celular/genética , Microambiente Celular/genética , Voluntarios Sanos , Humanos , Fenómenos Mecánicos , Monocitos/químicaRESUMEN
We report the design, fabrication and evaluation of an array of microdevices composed of high aspect ratio PDMS pillars, dedicated to the study of tumour spheroid mechanical properties. The principle of the microdevice is to confine a spheroid within a circle of micropillars acting as peripheral flexible force sensors. We present a technological process for fabricating high aspect ratio micropillars (300 µm high) with tunable feature dimensions (diameter and spacing) enabling production of flexible PDMS pillars with a height comparable to spheroid sizes. This represents an upscale of 10 along the vertical direction in comparison to more conventional PDMS pillar force sensors devoted to single cell studies, while maintaining their force sensitivity in the same order of magnitude. We present a method for keeping these very high aspect ratio PDMS pillars stable and straight in liquid solution. We demonstrate that microfabricated devices are biocompatible and adapted to long-term spheroid growth. Finally, we show that the spheroid interaction with the micropillars' surface is dependent on PDMS cellular adhesiveness. Time-lapse recordings of growth-induced micropillars' bending coupled with a software program to automatically detect and analyse micropillar displacements are presented. The use of these microdevices as force microsensors opens new prospects in the fields of tissue mechanics and pharmacological drug screening.