Artificial Epithelium Research Articles

Advances in Trans-Epithelial Electrical Resistance (TEER) monitoring integration in an Intestinal Barrier-on-Chip (IBoC) platform with microbubbles-tolerant analytical method

Trans-epithelial resistance (TEER) is one of the most widely performed analysis in vitro methods for monitoring barrier tissue development, formation and functional maturation. It is a basic and important tool for the reproduction of an artificial human epithelium. However, in the design and construction of organ on chip devices, the reliable TEER measurements still remain a challenge, in spite of their efficacy in providing real time information on tissue integrity and function. The observed limitations often concern the nature of the selected electrodes, made with opaque or metal materials that prevent microscopic investigation, their correct and stable reposition that affect the impedance spectroscopy analysis and microbubbles interference. Here, we present a method to perform real time TEER analysis on an Intestinal Barrier-on-Chip (IBoC) using integrated transparent electrodes, by means of impedance spectroscopy analysis and supported by a microbubble-tolerant calculation method. The human Caco-2 cells were used as an in vitro model of the intestinal epithelial cell barrier. Measurements can be carried out even in presence of microbubbles during the analysis, thus allowing application of this analytical method in microfluidic organ on chip devices that must perform in continuum for a long time.

Oscillation Characteristics of an Artificial Cochlear Sensory Epithelium Optimized for a Micrometer-Scale Curved Structure.

Based on the modern microelectromechanical systems technology, we present a revolutionary miniaturized artificial cochlear sensory epithelium for future implantation tests on guinea pigs. The device was curved to fit the spiral structure of the cochlea and miniaturized to a maximum dimension of <1 mm to be implanted in the cochlea. First, the effect of the curved configuration on the oscillation characteristics of a trapezoidal membrane was evaluated using the relatively larger devices, which had a trapezoidal and a comparable curved shape designed for high-precision in vitro measurements. Both experimental and numerical analyses were used to determine the resonance frequencies and positions, and multiple oscillation modes were clearly observed. Because the maximum oscillation amplitude positions, i.e., the resonance positions, differed depending on the resonance frequencies in both trapezoidal and curved membrane devices, the sound frequency was determined based on the resonance position, thus reproducing the frequency selectivity of the basilar membrane in the organ of Corti. Furthermore, the resonance frequencies and positions of these two devices with different configurations were determined to be quantitatively consistent and similar in terms of mechanical dynamics. This result shows that despite a curved angle of 50–60°, the effect of the curved shape on oscillation characteristics was negligible. Second, the nanometer-scale oscillation of the miniaturized device was successfully measured, and the local resonance frequency in air was varied from 157 to 277 kHz using an experimental system that could measure the amplitude distribution in a two-dimensional (2D) plane with a high accuracy and reproducibility at a high speed. The miniaturized device developed in this study was shown to have frequency selectivity, and when the device was implanted in the cochlea, it was expected to discriminate frequencies in the same manner as the basilar membrane in the biological system. This study established methods for fabricating and evaluating the miniaturized device, and the proposed miniaturized device in a curved shape demonstrated the feasibility of next-generation cochlear implants.

Generation of Alveolar Epithelium Using Reconstituted Basement Membrane and hiPSC-Derived Organoids.

In vitro modeling of alveolar epithelium needs to recapitulate features of both cellular and noncellular components of the lung tissues. Herein, a method is presented to generate alveolar epithelium by using human induced pluripotent stem cells (hiPSCs) and reconstituted or artificial basement membrane (ABM). The ABM is obtained by self-assembling type IV collagen and laminin with a monolayer of crosslinked gelatin nanofibers as backbone and a patterned honeycomb microframe for handling. Alveolar organoids are obtained from hiPSCs and then dissociated into single cells. After replating the alveolar cells on the ABM and a short-period incubation under submerged and air-liquid interface culture conditions, an alveolar epithelium is achieved, showing high-level expressions of both alveolar cell-specific proteins and characteristic tight junctions. Besides, endothelial cells derived from the same hiPSCs are cocultured on the backside of the epithelium, forming an air-blood barrier. The method is generic and can potentially be applied to other types of artificial epithelium and endothelium.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of coxsackievirus and adenovirus receptor.

Porphyromonas gingivalis is a major pathogen of human periodontitis and dysregulates innate immunity at the gingival epithelial surface. We previously reported that the bacterium specifically degrades junctional adhesion molecule 1 (JAM1), causing gingival epithelial barrier breakdown. However, the functions of other JAM family protein(s) in epithelial barrier dysregulation caused by P. gingivalis are not fully understood. The present results show that gingipains, Arg-specific or Lys-specific cysteine proteases produced by P. gingivalis, specifically degrade coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, at R145 and K235 in gingival epithelial cells. In contrast, a gingipain-deficient P. gingivalis strain was found to be impaired in regard to degradation of CXADR. Furthermore, knockdown of CXADR in artificial gingival epithelium increased permeability to dextran 40 kDa, lipopolysaccharide and peptidoglycan, whereas overexpression of CXADR in a gingival epithelial tissue model prevented penetration by those agents following P. gingivalis infection. Together, these results suggest that P. gingivalis gingipains breach the stratified squamous epithelium barrier by degrading CXADR as well as JAM1, which allows for efficient transfer of bacterial virulence factors into subepithelial tissues. TAKEAWAYS: P. gingivalis, a periodontal pathogen, degraded coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, in gingival epithelial tissues. P. gingivalis gingipains, cysteine proteases, degraded CXADR at R145 and K235. CXADR degradation by P. gingivalis caused increased permeability to lipopolysaccharide and peptidoglycan through gingival epithelial tissues.

Effect of position and shape of electrode on resonant frequency of an artificial epithelium membrane for frequency selectivity

Postoperative medical studies by various groups have shown the relevance of electrode position on the hearing outcome of the Cochlear Implant. There are various types of CI in market that are re-evaluated and changed for its functionality. Prior knowledge of the exact position of the electrode within the cochlea is an important parameter of study to improve the electrical stimulation of the hearing nerve for the implants. Similar studies are performed on the artificial epithelium membrane which involves restoring the function of outer and middle ear without bypassing their functionality and providing solution towards a self sustained implantable cochlea. Different shapes of electrodes are evaluated using various structures in dimensional analysis for improving the displacement and the electric stimulation calculated in terms of voltage. Five designs are modelled and optimized for improving the frequency selectivity for its resonance of an epithelium membrane to function as a biomimetic Epithelium membrane. Simulation studies are carried out using FEA analysis tool to introspect and evaluate the tonotopy of the epithelium membrane in terms of electrode shape and position.

Fluid-structure interaction analysis of traveling waves propagating on an artificial cochlear sensory epithelium

In this paper, oscillations of an artificial sensory epithelium located in the cochlea filled with lymph fluids are simulated by a finite element method. The artificial cochlear sensory epithelium is a trapezoidal piezoelectric membrane to mimic functions of the cochlea, and these physical properties using in the numerical analysis are estimated based on experimental results. The artificial cochlear sensory epithelium is set into the model straightened-cochlea filled with a fluid, and an oscillation is induced by mimicking a transmission of the biological cochlea. Normal frequencies and modes of the artificial cochlear sensory epithelium are well simulated to represent the frequency selectivity of the biological basilar membrane. The Euler-Bernoulli theory for transvers vibrations of a beam successfully predicts normal frequencies when the trapezoidal membrane is modeled as a set of fixed-end beams. Moreover, traveling waves propagating on the artificial cochlear sensory epithelium are numerically and successfully observed in the present study. That is, not only structure but also critical feature of the biological cochlea is represented by an artificial mechanical system.

A Preliminary Prototype High-Speed Feedback Control of an Artificial Cochlear Sensory Epithelium Mimicking Function of Outer Hair Cells.

A novel feedback control technique for the local oscillation amplitude in an artificial cochlear sensory epithelium that mimics the functions of the outer hair cells in the cochlea is successfully developed and can be implemented with a control time on the order of hundreds of milliseconds. The prototype artificial cochlear sensory epithelium was improved from that developed in our previous study to enable the instantaneous determination of the local resonance position based on the electrical output from a bimorph piezoelectric membrane. The device contains local patterned electrodes deposited with micro electro mechanical system (MEMS) technology that is used to detect the electrical output and oscillate the device by applying local electrical stimuli. The main feature of the present feedback control system is the principle that the resonance position is recognized by simultaneously measuring the local electrical outputs of all of the electrodes and comparing their magnitudes, which drastically reduces the feedback control time. In this way, it takes 0.8 s to control the local oscillation of the device, representing the speed of control with the order of one hundred times relative to that in the previous study using the mechanical automatic stage to scan the oscillation amplitude at each electrode. Furthermore, the intrinsic difficulties in the experiment such as the electrical measurement against the electromagnetic noise, adhesion of materials, and fatigue failure mechanism of the oscillation system are also shown and discussed in detail based on the many scientific aspects. The basic knowledge of the MEMS fabrication and the experimental measurement would provide useful suggestions for future research. The proposed preliminary prototype high-speed feedback control can aid in the future development of fully implantable cochlear implants with a wider dynamic range.

Reconstituted epithelial tissues and native cornea: A comparison of the influence of surfactants on ocular permeability

The aim of this study was to prepare an artificial rabbit corneal epithelium (RRCE) to compare with a human corneal epithelial model and excised rabbit cornea through permeation studies to investigate differences of surfactants influence on ocular permeability of a lipophilic compound. First solubility assays with different surfactants were performed and the integrity of the RRCE was assessed by measuring transepithelial electrical resistance (TEER). The permeation parameters showed that the RRCE was more sensitive than native cornea and human cornal epithelial model to the effect of permeation enhancers.

Reconstituted epithelial corneal tissues for evaluation of drug delivery

One possible approach to reduce the use of animals in the evaluation of the ocular drug delivery is that to use cell cultures as model of tissues. In this study are compared two different reconstituted epithelial corneal tissues, a homemade artificial corneal epithelium (Reconstituted Rabbit Corneal Epithelium) and a commercial human corneal epithelial model (COR-100 EpiCornealTM, MatTek), for permeation experiments of three drugs with different physical chemical properties: timolol maleate, cyclosporin A, and a newly synthesized compound, MAGL 17.b. The collected data show that corneal epithelial models are not sufficient to simulate the complexity of the corneal barrier and the presence of a layer simulating the stroma may be necessary to approach the structure of native cornea.

Oscillation characteristics and control methods of artificial cochlear sensory epithelia

Oscillation characteristics and control methods of artificial cochlear sensory epithelia

Simultaneous measurement of the oscillation characteristics and electrical voltage output of an artificial cochlear sensory epithelium immersed in a liquid: Theory and experiment

The artificial cochlear sensory epithelium is a biomedical device that mimics the function of the organ of Corti. The device consists of piezoelectric membranes fixed on a trapezoidal slit and has the functions of acoustic/electric conversion and frequency selectivity. In the present study, we report the frequency selectivity of piezoelectric output from an artificial cochlear epithelium immersed in a liquid environment, where silicone oils are used as working fluids. The oscillation amplitude of the piezoelectric membranes, which is proportional to the strain, and the generated electrical output due to the membrane deformation are measured simultaneously and are found to be well correlated in both air and liquid environments for various frequency ranges in the zone of audibility. In addition, the resonant frequencies of the piezoelectric membranes are reduced by immersing the membranes in the liquid due to a higher fluid damping effect. Although most studies on the development of artificial cochlear epitheliums are conducted in air environments, the present study provides fundamental data toward practical applications, where the devices are immersed in body fluids such as perilymph.

Functional Epithelium Remodeling in Response to Applied Stress under In Vitro Conditions.

Mathematical modeling is often used in tissue engineering in order to overcome one of its major challenges: transformation of complex biological and rheological behaviors of cells and tissue in a mathematically predictive and physically manipulative engineering process. The successive accomplishment of this task will greatly help in quantifying and optimizing clinical application of the tissue engineering products. One of the problems emerging in this area is the relation between resting and migrating cell groups, as well as between different configurations of migrating cells and viscoelasticity. A deeper comprehension of the relation between various configurations of migrating cells and viscoelasticity at the supracellular level represents the prerequisite for optimization of the performance of the artificial epithelium. Since resting and migrating cell groups have a considerable difference in stiffness, a change in their mutual volume ratio and distribution may affect the viscoelasticity of multicellular surfaces. If those cell groups are treated as different phases, then an analogous model may be applied to represent such systems. In this work, a two-step Eyring model is developed in order to demonstrate the main mechanical and biochemical factors that influence configurations of migrating cells. This model could be also used for considering the long-time cell rearrangement under various types of applied stress. The results of this theoretical analysis point out the cause-consequence relationship between the configuration of migrating cells and rheological behavior of multicellular surfaces. Configuration of migrating cells is influenced by mechanical and biochemical perturbations, difficult to measure experimentally, which lead to uncorrelated motility. Uncorrelated motility results in (1) decrease of the volume fraction of migrating cells, (2) change of their configuration, and (3) softening of multicellular surfaces.

新規人工聴覚器（人工聴覚上皮）の開発 ～HIBIKIプロジェクト～

新規人工聴覚器（人工聴覚上皮）の開発 ～HIBIKIプロジェクト～

High-speed active feedback systems mimicking functions of outer hair cells for artificial sensory epithelium

High-speed active feedback systems mimicking functions of outer hair cells for artificial sensory epithelium

Artificial Cochlear Sensory Epithelium with Functions of Outer Hair Cells Mimicked Using Feedback Electrical Stimuli.

We report a novel vibration control technique of an artificial auditory cochlear epithelium that mimics the function of outer hair cells in the organ of Corti. The proposed piezoelectric and trapezoidal membrane not only has the acoustic/electric conversion and frequency selectivity of the previous device developed mainly by one of the authors and colleagues, but also has a function to control local vibration according to sound stimuli. Vibration control is achieved by applying local electrical stimuli to patterned electrodes on an epithelium made using micro-electro-mechanical system technology. By choosing appropriate phase differences between sound and electrical stimuli, it is shown that it is possible to both amplify and dampen membrane vibration, realizing better control of the response of the artificial cochlea. To be more specific, amplification and damping are achieved when the phase difference between the membrane vibration by sound stimuli and electrical stimuli is zero and , respectively. We also demonstrate that the developed control system responds automatically to a change in sound frequency. The proposed technique can be applied to mimic the nonlinear response of the outer hair cells in a cochlea, and to realize a high-quality human auditory system.

Optimal design of artificial cochlear sensory epithelium for mimicking outer hair cells

Optimal design of artificial cochlear sensory epithelium for mimicking outer hair cells

Development of vibration measurement system for artificial cochlear epithelium in liquids

Development of vibration measurement system for artificial cochlear epithelium in liquids

The role of apical contractility in determining cell morphology in multilayered epithelial sheets and tubes

A multilayered epithelium is made up of individual cells that are stratified in an orderly fashion, layer by layer. In such tissues, individual cells can adopt a wide range of shapes ranging from columnar to squamous. From histological images, we observe that, in flat epithelia such as the skin, the cells in the top layer are squamous while those in the middle and bottom layers are columnar, whereas in tubular epithelia, the cells in all layers are columnar. We develop a computational model to understand how individual cell shape is governed by the mechanical forces within multilayered flat and curved epithelia. We derive the energy function for an epithelial sheet of cells considering intercellular adhesive and intracellular contractile forces. We determine computationally the cell morphologies that minimize the energy function for a wide range of cellular parameters. Depending on the dominant adhesive and contractile forces, we find four dominant cell morphologies for the multilayered-layered flat sheet and three dominant cell morphologies for the two-layered curved sheet. We study the transitions between the dominant cell morphologies for the two-layered flat sheet and find both continuous and discontinuous transitions and also the presence of multistable states. Matching our computational results with histological images, we conclude that apical contractile forces from the actomyosin belt in the epithelial cells is the dominant force determining cell shape in multilayered epithelia. Our computational model can guide tissue engineers in designing artificial multilayered epithelia, in terms of figuring out the cellular parameters needed to achieve realistic epithelial morphologies.

Evaluation of sensitivity improvement for a piezoelectric-actuator-integrated artificial cochlear epithelium

In this paper, we report the vibration control of an artificial cochlear epithelium. The device is a trapezoidal piezoelectric membrane with patterned Al electrodes. The resonance frequency of the piezoelectric membrane varies locally according to the longitudinal position, and the electric potential difference is produced by the vibration of the membrane induced by sound stimuli. In this study, we use the electrodes to apply electrical stimuli to the piezoelectric membrane. When the frequencies of the electric stimuli are the same as those of the sound stimuli, it is found that the amplification/damping of the local membrane vibration is possible by choosing the appropriate phase difference between the electric stimuli and the sound stimuli. This vibration control technique can be applied to the development of the artificial cochlear epithelium with nonlinear response, which is necessary to mimic the wide dynamic range of human cochlea.

Local amplitude control of artificial cochlear epithelium for sensitivity improvement

Local amplitude control of artificial cochlear epithelium for sensitivity improvement