Tissue homeostasis depends critically on the exchange of interstitial fluid between blood and tissues, as well as lymphatic drainage. While mechanical forces, such as external compression during physical activity, are known to accelerate interstitial fluid clearance, arterial pulsatility is hypothesized to play a similar role, especially in regions lacking lymphatic vasculature, such as the central nervous system. Here, we investigate how vascular pulsations modulate solute transport using a novel multi-microvessel pulsatile interstitial flow (PIF) chip. This platform enables real-time visualization of deformation and solute dynamics within a tissue-mimetic collagen hydrogel. Our results reveal a clipped Gent-like porohyperelastic response in acellular lumens, which is further amplified in vascularized lumens exhibiting asymmetric expansion-retraction behavior. Notably, microvessels induce strain retention within the hydrogel bulk, which is abruptly released during each pressure cycle. This cyclic strain retention and release mechanism coincides with a 1.3-fold increase in solute velocity, accelerating tracer clearance under pulsatile actuation compared to constant pressure. These findings highlight a vasculature-driven transport mechanism, where deformation-induced flow enhances interstitial clearance. These results provide new insights into how vascular dynamics may contribute to tissue homeostasis and interstitial fluid clearance. ### Competing Interest Statement The authors have declared no competing interest. WINGS-QSTEP MEXT, JPMXP1223UT-0331

The poroelastic properties of tissues regulate molecular transport and mechanical signaling, yet their evolution during aging remains poorly understood. In particular, senescent fibroblasts accumulate in aged tissues, contributing to extracellular matrix (ECM) remodeling, but their impact on tissue mechanics and permeability is unclear. In this study, we develop a microfluidic-based in vitro model to assess the poroelastic properties of collagen gels embedded with senescent fibroblasts over time. Our approach integrates periodic pressure actuation with real-time pressure monitoring in a sealed air cavity, enabling the detection of fluid permeation and solid matrix deformations. We analyze our data using analytical and numerical models based on a porohyperelastic framework that combines the compressible neo-Hookean elasticity with the Kozeny-Carman permeability relationship. We demonstrate that senescent fibroblasts induce a progressive softening of the ECM without altering its permeability. Immunostaining reveals that this softening correlates with structural reorganization of the collagen network characterized by increased branching and network remodeling. Our findings provide insights into the biomechanical effects of senescent fibroblasts on ECM homeostasis. We further argue that our platform offers a unique solution to investigate ECM remodeling not only in aging but also in fibrosis, cancer progression, or regenerative medicine strategies.

In response to mechanical cues, endothelial cells elicit highly sensitive cellular response pathways that contribute to the regulation of the physiology and disorders of the vascular system. However, it remains relatively unexplored how endothelial tissues process and integrate the intraluminal pressure, and in turn regulate the permeation flow across the vessel wall. Leveraging a tissue engineering approach to create microvessels (MVs), we measured real-time permeation flow induced by intraluminal pressures ranging from 0.1 to 2.0 kPa. Our findings reveal that mechanically stimulated MVs strengthen their barrier function within seconds of exposure to pressures below 1 kPa, with this enhanced barrier function persisting for 30 minutes. We demonstrate that this barrier reinforcement is linked to the closure of paracellular gaps. Additionally, we observe that it is associated with, and depends on, actin cytoskeleton reorganization, including the accumulation of stress fibers near intercellular junctions and the broadening of adherence junction protein localization. These findings provide insights into the ability of endothelial tissues to regulate interstitial fluid flow in response to sudden increases in blood pressure.

Epidermal growth factor receptor (EGFR) mutation detection is now commonly used in the management of cancer patients, particularly those diagnosed with non-small cell lung cancer. Molecular beacon-based sensing is direct and rapid, but its sensitivity is low. Conversely, high-sensitivity detection methodologies based on amplification are robust and sensitive but are limited by relatively require long turnaround times. In this study, we utilized a size-resolved, molecular beacon-based strategy for the rapid detection of EGFR genomic alterations, specifically exon 19 deletions and L858R point mutation. This technology combines a concentration and separation module, which allows us to successfully demonstrate the detection of deletions and point mutations of EGFR in five minutes with a mutant allele sensitivity of 10%. The use of a dual-color detection insures fast detection with a reduced risk of false positives. This work represents a first step toward the fast and specific detection of genetic mutations to improve the management of patients with hard-to-treat tumors.

The poroelastic properties of tissues regulate molecular transport and mechanical signaling, yet their evolution during aging remains poorly understood. In particular, senescent fibroblasts accumulate in aged tissues, contributing to extracellular matrix (ECM) remodeling, but their impact on tissue mechanics and permeability is unclear. In this study, we developed a microfluidic-based in vitro model to assess the poroelastic properties of collagen gels embedded with senescent fibroblasts over time. Our approach integrates periodic pressure actuation with real-time pressure monitoring in a sealed air cavity, enabling the detection of fluid permeation and solid matrix deformations. We analyze our data using analytical and numerical models based on a porohyperelastic framework. This framework combines compressible Neo-Hookean elasticity with the Kozeny–Carman permeability relationship. We demonstrate that senescent fibroblasts induce a progressive softening of the ECM without altering its permeability. Immunostaining reveals that this softening correlates with structural reorganization of the collagen network, characterized by increased branching and network remodeling. Our findings provide insights into the biomechanical effects of senescent fibroblasts on ECM homeostasis. We further argue that our platform offers a unique solution to investigate ECM remodeling not only in aging but also fibrosis, cancer progression, or regenerative medicine strategies. ### Competing Interest Statement The authors have declared no competing interest. JSPS postdoctoral felloship, , LIMMS-CNRS, , Grant-in-Aid for JSPS Fellows (20F20806), , AMED P-CREATE (JP18cm0106239h0001), , JSPS Core-to-Core Program (JPJSCCA-20190006)., ,

The μLAS technology enables in-line DNA concentration and separation in a microchannel. Here, we describe its operation to analyze the size profile of cell-free DNA (cfDNA) extracted from blood plasma. Operated on commercial systems for capillary electrophoresis, we provide the size distribution of healthy individuals or patients using an input of 10 μL.

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ’s extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

Organ -on -a -chip technologies enable the fabrication of endothelial tissues, socalled microvessels (MVs), which emulate the endothelial barrier function in healthy or disease conditions. In this protocol, we describe the fabrication of perfusable open -chamber style MVs embedded in collagen gels. We then report a simple technology to characterize the MV barrier properties in static or under pressure based on fluorescence confocal imaging. Finally, we provide quantification techniques that enable us to infer the structure of MV paracellular pores. For complete details on the use and execution of this protocol, please refer to Cacheux et al. 1

The endothelial layers of the microvasculature regulate the transport of solutes to the surrounding tissues. It remains unclear how this barrier function is affected by blood flow-induced intraluminal pressure. Using a 3D microvessel model, we compare the transport of macromolecules through endothelial tissues at mechanical rest or with intraluminal pressure, and correlate these data with electron microscopy of endothelial junctions. On application of an intraluminal pressure of 100 Pa, we demonstrate that the flow through the tissue increases by 2.35 times. This increase is associated with a 25% expansion of microvessel diameter, which leads to tissue remodeling and thinning of the paracellular junctions. We recapitulate these data with the deformable monopore model, in which the increase in paracellular transport is explained by the augmentation of the diffusion rate across thinned junctions under mechanical stress. We therefore suggest that the deformation of microvasculatures contributes to regulate their barrier function.

The mechanisms of solute transport in brain tissues are still under debate. The medical relevance of this topic has put the blood-brain barrier and the mechanisms of solute transport through the brain parenchyma in the spotlight, notably in the context of brain clearance. In the last decade, the classical view of pure diffusive flow across the brain parenchyma was tested against the recent proposal of an active, convectional fluid flow model known as the glymphatic model. Experimental studies of brain transport on living humans and animals have temporal and spatial limitations to validate any of these models. Therefore, detailed microscopic observations, mostly ex vivo tissue and simplified in vitro brain models with the support from computational models, are necessary to understand transport mechanisms in brain tissues. However, standardization is lacking between these experimental approaches, which tends to limit the generality of conclusions. In this review, we provide an overview of the output and limitations of modern brain solute transport studies to search for key parameters comparable across experimental setups. We emphasize that in vitro models relying on physiological material and reproducing the biophysical setting of the brain, as well as computational/mathematical models constitute powerful solutions to understand the solute transport phenomena inside of the brain tissue. Finally, we suggest the blood-brain barrier permeability and the apparent diffusion coefficient through the brain parenchyma to be robust biophysical parameters for the extraction of cross-model conclusion.

The Starling principle describes exchanges between blood and tissues based on the balance of hydrostatic and osmotic flows. However, the permeation properties of the main constituent of tissues, namely, collagen, in response to the stress exerted by blood pressure remain poorly characterized. Here, we develop an instrument to determine the elasticity and permeability of collagen gels under tensile and compressive stress based on measuring the temporal change in pressure in an air cavity sealed at the outlet of a collagen slab. Data analysis with an analytical model reveals a drop in the permeability and enhanced strain stiffening of native collagen gels under compression versus tension, both effects being essentially lost after chemical cross-linking. Furthermore, we report the control of the permeability of native collagen gels using sinusoidal fluid injection, an effect explained by the asymmetric response in tension and compression. We lastly suggest that blood-associated pulsations could contribute to exchanges within tissues.

DNA size fractionation is an essential tool in molecular biology and is used to isolate targets in a mixture characterized by a broad molecular-weight distribution. Microfluidics was thought to provide the opportunity to create devices capable of enhancing and speeding up the classical fractionation processes. However, this conjecture met limited success due to the low mass or volume throughput of these technologies. We describe the μLAF (μ-laboratory for DNA fractionation) technology for DNA size selection based on the stacking of molecules on films of ∼100 μm in thickness with 105 cm-2 pores ∼2 μm in diameter. Size selection is achieved by controlling the regime of electrohydrodynamic migration through the temporal modulation of an electric field. This technology allows the processing of milliliter-scale samples containing a DNA mass of several hundreds of ng within ∼10 min and the selection of DNA in virtually any size window spanning 200 to 1000 bp. We demonstrate that one operation suffices to fractionate sheared genomic DNA in up to six fractions with collection efficiencies of ∼20-40% and enrichment factors of ∼1.5-3-fold. These performances compare favorably in terms of speed and versatility to those of the current standards.

Speeding up and enhancing the performances of nucleic acid biosensing technologies have remained drivers for innovation. Here, we optimize a fluorimetry-based technology for DNA detection based on the concentration of linear targets paired with probes. The concentration module consists of a microfluidic channel with the shape of a funnel in which we monitor a viscoelastic flow and a counter-electrophoretic force. We report that the technology performs better with a target longer than 100 nucleotides (nt) and a probe shorter than 30 nt. We also prove that the control of the funnel geometry in 2.5D using grayscale lithography enhances sensitivity by 100-fold in comparison to chips obtained by conventional photolithography. With these optimized settings, we demonstrate a limit of detection of 4 fM in 30 s and a detection range of more than five decades. This technology hence provides an excellent balance between sensitivity and time to result.

We present a fluorimetry-based technology for micro-RNA-21 (miR-21) sensing based on the concentration of miR-molecular beacon (MB) complexes and flushing of unbound MB. This concentration module consists of a microfluidic channel with the shape of a funnel operated with electrohydrodynamic actuation. We report a limit of detection of 2 pM in less than 1 min for miR-21 alone, and then demonstrate that miR-21 levels, measured in fine needle biopsy samples, from patients with pancreatic cancer correlate with the reference technique of reverse-transcription polymerase chain reaction (RT-PCR). Altogether, this technology has promising clinical performances for the follow-up of patients with cancer.

Electrohydrodynamic migration, which is based on hydrodynamic actuation with an opposing electrophoretic force, enables the separation of DNA molecules of 3-100 kbp in glass capillary within 1 h. Here, we wish to enhance these performances using microchip technologies. This study starts with the fabrication of microchips with uniform surfaces, as motivated by our observation that band splitting occurs in microchannels made out of heterogeneous materials such as glass and silicon. The resulting glass-adhesive-glass microchips feature the highest reported bonding strength of 11 MPa for such materials (115 kgf/cm2), a high lateral resolution of critical dimension 5 μm, and minimal auto-fluorescence. These devices enable us to report the separation of 13 DNA bands in the size range of 1-150 kbp in one experiment of 5 min, i.e. 13 times faster than with capillary. In turn, we observe that bands split during electrohydrodynamic migration in heterogeneous glass-silicon but not in homogeneous glass-adhesive-glass microchips. We suggest that this effect arises from differential Electro-Osmotic Flow (EOF) in between the upper and lower walls of heterogeneous channels, and provide evidence that this phenomenon of differential EOF causes band broadening in electrophoresis during microchip electrophoresis. We finally prove that our electrohydrodynamic separation compares very favorably to microchip technologies in terms of resolution length and features the broadest analytical range reported so far.

The combination of hydrodynamic actuation with an opposing electrophoretic force in viscoelastic liquids enables the separation, concentration, and purification of DNA. Obtaining good analytical performances despite the use of hydrodynamic flow fields, which dramatically enhance band broadening due to Taylor dispersion, constitutes a paradox that remains to be clarified. Here, we study the mechanism of band broadening in electrohydrodynamic migration with an automated microfluidic platform that allows us to track the migration of a 600 bp band in the pressure-electric field parameter space. We demonstrate that diffusion in the electrohydrodynamic regime is controlled predominantly by the electric field and marginally by the hydrodynamic flow velocity. We explain this response with an analytical model of diffusion based on Taylor dispersion arguments. Furthermore, we demonstrate that the electric field can be modulated over time to monitor and minimize the breadth of a DNA band, and suggest guidelines to enhance the resolution of DNA separation experiments. Altogether, our report is a leap towards to the development of high-performance analytical technologies based on electrohydrodynamic actuation.