Online monitoring of epithelial barrier kinetics and cell detachment during cisplatin-induced toxicity of renal proximal tubule cells.

Real-time and non-invasive assessment of tissue health is crucial for maximizing the potential of microphysiological systems (MPS) for drug-induced nephrotoxicity screening. Although impedance has been widely considered as a measure of the barrier function, it has not been incorporated to detect cell detachment in MPS with top and bottom microfluidic channels separated by a porous membrane. During cell delamination from the porous membrane, the resistance between both channels decreases, while capacitance increases, allowing the detection of such detachment. Previously reported concepts have solely attributed the decrease in the resistance to the distortion of the barrier function, ignoring the resistance and capacitance changes due to cell detachment. Here, we report a two-channel MPS with integrated indium tin oxide (ITO) electrodes capable of measuring impedance in real time. The trans-epithelial electrical resistance (TEER) and tissue reactance (capacitance) were extracted from the impedance profiles. We attributed the anomalous initial increase observed in TEER, upon cisplatin administration, to the distortion of tight junctions. Cell detachment was captured by sudden jumps in capacitance. TEER profiles illuminated the effects of cisplatin and cimetidine treatments in a dose-dependent and polarity-dependent manner. The correspondence between TEER and barrier function was validated for a continuous tissue using the capacitance profiles. These results demonstrate that capacitance can be used as a real-time and non-invasive indicator of confluence and will support the accuracy of the drug-induced cytotoxicity assessed by TEER profiles in the two-channel MPS for the barrier function of a cell monolayer.

Random Matrix Statistics in Propagating Correlation Fronts of Fermions.

We theoretically study propagating correlation fronts in noninteracting fermions on a one-dimensional lattice starting from an alternating state, where the fermions occupy every other site. We find that, in the long-time asymptotic regime, all the moments of dynamical fluctuations around the correlation fronts are described by the universal correlation functions of Gaussian orthogonal and symplectic random matrices at the soft edge. Our finding here sheds light on a hitherto unknown connection between random matrix theory and correlation propagation in quantum dynamics.

SARS-CoV-2-induced disruption of a vascular bed in a microphysiological system caused by type-I interferon from bronchial organoids.

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, we developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

Deciphering potential vascularization factors of on-chip co-cultured hiPSC-derived cerebral organoids.

The lack of functional vascular system in stem cell-derived cerebral organoids (COs) limits their utility in modeling developmental processes and disease pathologies. Unlike other organs, brain vascularization is poorly understood, which makes it particularly difficult to mimic in vitro. Although several attempts have been made to vascularize COs, complete vascularization leading to functional capillary network development has only been achieved via transplantation into a mouse brain. Understanding the cues governing neurovascular communication is therefore imperative for establishing an efficient in vitro system for vascularized cerebral organoids that can emulate human brain development. Here, we used a multidisciplinary approach combining microfluidics, organoids, and transcriptomics to identify molecular changes in angiogenic programs that impede the successful in vitro vascularization of human induced pluripotent stem cell (iPSC)-derived COs. First, we established a microfluidic cerebral organoid (CO)-vascular bed (VB) co-culture system and conducted transcriptome analysis on the outermost cell layer of COs cultured on the preformed VB. Results revealed coordinated regulation of multiple pro-angiogenic factors and their downstream targets. The VEGF-HIF1A-AKT network was identified as a central pathway involved in the angiogenic response of cerebral organoids to the preformed VB. Among the 324 regulated genes associated with angiogenesis, six transcripts represented significantly regulated growth factors with the capacity to influence angiogenic activity during co-culture. Subsequent on-chip experiments demonstrated the angiogenic and vasculogenic potential of cysteine-rich angiogenic inducer 61 (CYR61) and hepatoma-derived growth factor (HDGF) as potential enhancers of organoid vascularization. Our study provides the first global analysis of cerebral organoid response to three-dimensional microvasculature for in vitro vascularization.

Pericytes and shear stress each alter the shape of a self-assembled vascular network.

Blood vessel morphology is dictated by mechanical and biochemical cues. Flow-induced shear stress and pericytes both play important roles, and they have previously been studied using on-chip vascular networks to uncover their connection to angiogenic sprouting and network stabilization. However, it is unknown which shear stress values promote angiogenesis, how pericytes are directed to sprouts, and how shear stress and pericytes affect the overall vessel morphology. Here, we employed a microfluidic device to study these phenomena in three-dimensional (3D) self-assembled vasculature. Computational fluid dynamics solver (COMSOL) simulations indicated that sprouts form most frequently at locations of relatively low shear stresses (0.5-1.5 dyn cm-2). Experimental results show that pericytes limit vascular diameter. Interestingly, when treated with imatinib or crenolanib, which are chemotherapeutic drugs and inhibitors of platelet-derived growth factor receptor ß (PDGFRß), the pericyte coverage of vessels decreased significantly but vessel diameter remained unchanged. This furthers our understanding of the mechanisms underlying vascular development and demonstrates the value of this microfluidic device in future studies on drug development and vascular biology.

Long-term effect of sodium selenite on the integrity and permeability of on-chip microvasculature.

Development of the robust and functionally stable three-dimensional (3D) microvasculature remains challenging. One often-overlooked factor is the presence of potential anti-angiogenic agents in culture media. Sodium selenite, an antioxidant commonly used in serum-free media, demonstrates strong anti-angiogenic properties and has been proposed as an anticancer drug. However, its long-term effects on in vitro microvascular systems at the concentrations used in culture media have not been studied. In this study, we used a five-channel microfluidic device to investigate the concentration and temporal effects of sodium selenite on the morphology and functionality of on-chip preformed microvasculature. We found that high concentrations (∼3.0 µM) had adverse effects on microvasculature perfusion, permeability, and overall integrity within the first few days. Moreover, even at low concentrations (∼3.0 nM), a long-term culture effect was observed, resulting in an increase in vascular permeability without any noticeable changes in morphology. A further analysis suggested that vessel leakage may be due to vascular endothelial growth factor dysregulation, disruption of intracellular junctions, or both. This study provides important insight into the adverse effects caused by the routinely present sodium selenite on 3D microvasculature in long-term studies for its application in disease modeling and drug screening.

Durability of Aligned Microtubules Dependent on Persistence Length Determines Phase Transition and Pattern Formation in Collective Motion.

Collective motion is a ubiquitous phenomenon in nature. The collective motion of cytoskeleton filaments results mainly from dynamic collisions and alignments; however, the detailed mechanism of pattern formation still needs to be clarified. In particular, the influence of persistence length, which is a measure of filament flexibility, on collective motion is still unclear and lacks experimental verifications although it is likely to directly affect the orientational flexibility of filaments. Here, we investigated the collective motion of microtubules with different persistence lengths using a microtubule-kinesin motility system. We showed that local interactions between microtubules significantly vary depending on their persistence length. We demonstrated that the bundling of microtubules is enhanced by more durable alignment, rather than by greater likelihood of alignment. An agent-based computational model confirmed that the rigidity-dependent durability of microtubule alignment dominates their collective behavior.

Impact of Dissipation on Universal Fluctuation Dynamics in Open Quantum Systems.

Recent theoretical and experimental works have explored universal dynamics related to surface growth physics in isolated quantum systems. In this Letter, we theoretically elucidate that dissipation drastically alters universal particle-number-fluctuation dynamics associated with surface-roughness growth in one-dimensional free fermions and bosons. In a system under dephasing that causes loss of spatial coherence, we numerically find that a universality class of surface-roughness dynamics changes from the ballistic class to a class with the Edwards-Wilkinson scaling exponents and an unconventional scaling function. We provide the analytical derivation of the diffusion equation from the dephasing Lindblad equation via a renormalization-group technique and succeed in explaining the drastic change. Furthermore, we numerically find the same change of the universality class under a more nontrivial dissipation, i.e., symmetric incoherent hopping.

Linear-Zero Mode Waveguides for Single-Molecule Fluorescence Observation of Nucleotides in Kinesin-Microtubule Motility Assay.

Single-molecule fluorescence microscopy is a key tool to investigate the chemo-mechanical coupling of microtubule-associated motor proteins, such as kinesin. However, a major limitation of the implementation of single-molecule observation is the concentration of fluorescently labeled molecules. For example, in total internal reflection fluorescence microscopy, the available concentration is of the order of 10 nM. This concentration is much lower than the concentration of adenosine triphosphate (ATP) in vivo, hindering the single-molecule observation of fluorescently labeled ATP hydrolyzed by motor proteins under the physiologically relevant conditions. Here, we provide a method for the use of single-molecule fluorescence microscopy in the presence of ~500 nM of fluorescently labeled ATP. To achieve this, a device equipped with nano-slits is used to confine excitation light into its slits as an expansion of zero-mode waveguides (ZMWs). Conventional ZMWs equip apertures with a diameter smaller than the wavelength of light to suppress background noise from the labeled molecules diffusing outside of the apertures. While they are not compatible with filamentous objects, our linear-ZMWs enable the usage of filamentous objects, such as microtubules. An experiment using linear-ZMWs demonstrated the successful exploration of the interaction between kinesin and ATP using single-molecule fluorescence microscopy.

Three-dimensional tissue model in direct contact with an on-chip vascular bed enabled by removable membranes.

Three-dimensional (3D) tissue culture is a powerful tool for understanding physiological events. However, 3D tissues still have limitations in their size, culture period, and maturity, which are caused by the lack of nutrients and oxygen supply through the vasculature. Here, we propose a new method for culturing a 3D tissue-a spheroid-directly on an 'on-chip vascular bed'. The method can be applied to any 3D tissue because the vascular bed is preformed, so that angiogenic factors from the tissue are not necessary to induce vasculature. The essential component of the assay system is the removable membrane that initially separates the 3D tissue culture well and the microchannel in which a uniform vascular bed is formed, and then allows the tissue to be settled directly onto the vascular bed following its removal. This in vitro system offers a new technique for evaluating the effects of vasculature on 3D tissues.

Dynamical Scaling of Surface Roughness and Entanglement Entropy in Disordered Fermion Models.

Localization is one of the most fundamental interference phenomena caused by randomness, and its universal aspects have been extensively explored from the perspective of one-parameter scaling mainly for static properties. We numerically study dynamics of fermions on disordered one-dimensional potentials exhibiting localization and find dynamical one-parameter scaling for surface roughness, which represents particle-number fluctuations at a given length scale, and for entanglement entropy when the system is in delocalized phases. This dynamical scaling corresponds to the Family-Vicsek scaling originally developed in classical surface growth, and the associated scaling exponents depend on the type of disorder. Notably, we find that partially localized states in the delocalized phase of the random-dimer model lead to anomalous scaling, where destructive interference unique to quantum systems leads to exponents unknown for classical systems and clean systems.

Growth rate-dependent flexural rigidity of microtubules influences pattern formation in collective motion.

BACKGROUND: Microtubules (MTs) are highly dynamic tubular cytoskeleton filaments that are essential for cellular morphology and intracellular transport. In vivo, the flexural rigidity of MTs can be dynamically regulated depending on their intracellular function. In the in vitro reconstructed MT-motor system, flexural rigidity affects MT gliding behaviors and trajectories. Despite the importance of flexural rigidity for both biological functions and in vitro applications, there is no clear interpretation of the regulation of MT flexural rigidity, and the results of many studies are contradictory. These discrepancies impede our understanding of the regulation of MT flexural rigidity, thereby challenging its precise manipulation. RESULTS: Here, plausible explanations for these discrepancies are provided and a new method to evaluate the MT rigidity is developed. Moreover, a new relationship of the dynamic and mechanic of MTs is revealed that MT flexural rigidity decreases through three phases with the growth rate increases, which offers a method of designing MT flexural rigidity by regulating its growth rate. To test the validity of this method, the gliding performances of MTs with different flexural rigidities polymerized at different growth rates are examined. The growth rate-dependent flexural rigidity of MTs is experimentally found to influence the pattern formation in collective motion using gliding motility assay, which is further validated using machine learning. CONCLUSION: Our study establishes a robust quantitative method for measurement and design of MT flexural rigidity to study its influences on MT gliding assays, collective motion, and other biological activities in vitro. The new relationship about the growth rate and rigidity of MTs updates current concepts on the dynamics and mechanics of MTs and provides comparable data for investigating the regulation mechanism of MT rigidity in vivo in the future.

Magnetic Solitons in a Spin-1 Bose-Einstein Condensate.

Vector solitons are a type of solitary or nonspreading wave packet occurring in a nonlinear medium composed of multiple components. As such, a variety of synthetic systems can be constructed to explore their properties, from nonlinear optics to ultracold atoms, and even in metamaterials. Bose-Einstein condensates have a rich panoply of internal hyperfine levels, or spin components, which make them a unique platform for exploring these solitary waves. However, existing experimental work has focused largely on binary systems confined to the Manakov limit of the nonlinear equations governing the soliton behavior, where quantum magnetism plays no role. Here we observe, using a "magnetic shadowing" technique, a new type of soliton in a spinor Bose-Einstein condensate, one that exists only when the underlying interactions are antiferromagnetic and which is deeply embedded within a full spin-1 quantum system. Our approach opens up a vista for future studies of "solitonic matter" whereby multiple solitons interact with one another at deterministic locations.

Family-Vicsek Scaling of Roughness Growth in a Strongly Interacting Bose Gas.

Family-Vicsek scaling is one of the most essential scale-invariant laws emerging in surface-roughness growth of classical systems. In this Letter, we theoretically elucidate the emergence of the Family-Vicsek scaling even in a strongly interacting quantum bosonic system by introducing a surface-height operator. This operator is comprised of a summation of local particle-number operators at a simultaneous time, and thus the observation of the surface roughness in the quantum many-body system and its scaling behavior are accessible to current experiments of ultracold atoms.

Synthetic dissipation and cascade fluxes in a turbulent quantum gas.

Scale-invariant fluxes are the defining property of turbulent cascades, but their direct measurement is a challenging experimental problem. Here we perform such a measurement for a direct energy cascade in a turbulent quantum gas. Using a time-periodic force, we inject energy at a large length scale and generate a cascade in a uniformly trapped three-dimensional Bose gas. The adjustable trap depth provides a high-momentum cutoff k D, which realizes a synthetic dissipation scale. This gives us direct access to the particle flux across a momentum shell of radius k D, and the tunability of k D allows for a clear demonstration of the zeroth law of turbulence. Moreover, our time-resolved measurements give unique access to the pre-steady-state dynamics, when the cascade front propagates in momentum space.

Flemish Strings of Magnetic Solitons and a Nonthermal Fixed Point in a One-Dimensional Antiferromagnetic Spin-1 Bose Gas.

Thermalization in a quenched one-dimensional antiferromagnetic spin-1 Bose gas is shown to proceed via a nonthermal fixed point through annihilation of Flemish-string bound states of magnetic solitons. A possible experimental situation is discussed.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 µM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

Unconventional Universality Class of One-Dimensional Isolated Coarsening Dynamics in a Spinor Bose Gas.

By studying the coarsening dynamics of a one-dimensional spin-1 Bose-Hubbard model in a superfluid regime, we analytically find an unconventional universal dynamical scaling for the growth of the spin correlation length, which is characterized by the exponential integral unlike the conventional power law or simple logarithmic behavior, and numerically confirmed with the truncated Wigner approximation.

Dynamic formation of a microchannel array enabling kinesin-driven microtubule transport between separate compartments on a chip.

Microtubules driven by kinesin motors have been utilised as "molecular shuttles" in microfluidic environments with potential applications in autonomous nanoscale manipulations such as capturing, separating, and/or concentrating biomolecules. However, the conventional flow cell-based assay has difficulty in separating bound target molecules from free ones even with buffer flushing because molecular manipulations by molecular shuttles take place on a glass surface and molecular binding occurs stochastically; this makes it difficult to determine whether molecules are carried by molecular shuttles or by diffusion. To address this issue, we developed a microtubule-based transport system between two compartments connected by a single-micrometre-scale channel array that forms dynamically via pneumatic actuation of a polydimethylsiloxane membrane. The device comprises three layers-a control channel layer (top), a microfluidic channel layer (middle), and a channel array layer (bottom)-that enable selective injection of assay solutions into a target compartment and dynamic formation of the microchannel array. The pneumatic channel also serves as a nitrogen supply path to the assay area, which reduces photobleaching of fluorescently labelled microtubules and deactivation of kinesin by oxygen radicals. The channel array suppresses cross-contamination of molecules caused by diffusion or pressure-driven flow between compartments, facilitating unidirectional transport of molecular shuttles from one compartment to another. The method demonstrates, for the first time, efficient and unidirectional microtubule transport by eliminating diffusion of target molecules on a chip and thus may constitute one of the key aspects of motor-driven nanosystems.

Colocalization of quantum dots by reactive molecules carried by motor proteins on polarized microtubule arrays.

The field of microfluidics has drastically contributed to downscale the size of benchtop experiments to the dimensions of a chip without compromising results. However, further miniaturization and the ability to directly manipulate individual molecules require a platform that permits organized molecular transport. The motor proteins and microtubules that carry out orderly intracellular transport are ideal for driving in vitro nanotransport. Here, we demonstrate that a reconstruction of the cellular kinesin/dynein-microtubule system in nanotracks provides a molecular total analysis system (MTAS) to control massively parallel chemical reactions. The mobility of kinesin and a microtubule dissociation method enable orientation of a microtubule in an array for directed transport of reactive molecules carried by kinesin or dynein. The binding of glutathione S-transferase (GST) to glutathione (GSH) and the binding of streptavidin to biotin are visualized as colocalizations of quantum dots (Q-dots) when motor motilities bring them into contact. The organized nanotransport demonstrated here suggests the feasibility of using our platform to perform parallel biochemical reactions focused at the molecular level.