Recent advances in understanding the role of the skin microbiome in the treatment of atopic dermatitis.

The skin is the largest organ in the human body, and histologically consists of the epidermis, dermis and subcutaneous tissue. Humans maintain a cooperative symbiotic relationship with their skin microbiota, a complex community of bacteria, fungi and viruses that live on the surface of the skin, and which act as a barrier to protect the body from the inside and outside. The skin is a 'habitat' and vast 'ecosystem' inhabited by countless microbes; as such, relationships have been forged through millions of years of coevolution. It is not surprising then that microbes are key participants in shaping and maintaining essential physiological processes. In addition to maintaining barrier function, the unique symbiotic microbiota that colonizes the skin increases the immune response and provides protection against pathogenic microbes. This review examines our current understanding of skin microbes in shaping and enhancing the skin barrier, as well as skin microbiome-host interactions and their roles in skin diseases, such as atopic dermatitis (AD). We also report on the current status of AD therapeutic drugs that target the skin microbiome, related research on current therapeutic strategies, and the limitations and future considerations of skin microbiome research. In particular, as a future strategy, we discuss the need for a skin-on-a-chip-based microphysiological system research model amenable to biomimetic in vitro studies and human skin equivalent models, including skin appendages.

Skin-on-a-chip strategies for human hair follicle regeneration.

The number of hair loss patients increases every year, and hair loss treatment has several limitations, so research on hair is attracting attention recently. However, most current hair follicle research models are limited by their inability to replicate several key functions of the hair follicle microenvironment. To complement this, an in vitro culture system similar to the in vivo environment must be constructed. It is necessary to develop a hair-on-a-chip that implements a fully functional hair follicle model by reproducing the main characteristics of hair follicle morphogenesis and cycle. In this review, we summarize the gradation of hair follicle morphogenesis and the roles and mechanisms of molecular signals involved in the hair follicle cycle. In addition, we discuss research results of various in vitro organoid products and organ-on-a-chip-based hair follicle tissue chips for the treatment of alopecia and present future research and development directions.

Effects of Indole-3-Lactic Acid, a Metabolite of Tryptophan, on IL-4 and IL-13-Induced Human Skin-Equivalent Atopic Dermatitis Models.

Indole-3-lactic acid (I3LA) is a well-known metabolite involved in tryptophan metabolism. Indole derivatives are involved in the differentiation of immune cells and the synthesis of cytokines via the aryl hydrocarbon receptors for modulating immunity, and the indole derivatives may be involved in allergic responses. I3LA was selected as a candidate substance for the treatment of atopic dermatitis (AD), and its inhibitory effect on AD progression was investigated. Full-thickness human skin equivalents (HSEs) consisting of human-derived cells were generated on microfluidic chips and stimulated with major AD-inducing factors. The induced AD-HSEs were treated with I3LA for 7 days, and this affected the AD-associated genetic biomarkers and increased the expression of the major constituent proteins of the skin barrier. After the treatment for 14 days, the surface became rough and sloughed off, and there was no significant difference between the increased AD-related mRNA expression and the skin barrier protein expression. Therefore, the short-term use of I3LA for approximately one week is considered to be effective in suppressing AD.

An Interleukin-4 and Interleukin-13 Induced Atopic Dermatitis Human Skin Equivalent Model by a Skin-On-A-Chip.

Currently, the mechanism of progression of atopic dermatitis (AD) is not well understood because there is no physiologically appropriate disease model in terms of disease complexity and multifactoriality. Type 2 inflammation, mediated by interleukin (IL)-4 and IL-13, plays an important role in AD. In this study, full-thickness human skin equivalents consisting of human-derived cells were fabricated from pumpless microfluidic chips and stimulated with IL-4 and IL-13. The morphological properties, gene expression, cytokine secretion and protein expression of the stimulated human skin equivalent (HSE) epidermis were investigated. The results showed epidermal and spongy formations similar to those observed in lesions in AD, and decreased expression of barrier-related filaggrin, loricrin and involucrin genes and proteins induced by IL-4Rα signaling. In addition, we induced the expression of carbonic anhydrase II (CAII), a gene specifically expressed in the epidermis of patients with AD. Thus, AD human skin equivalents can be used to mimic the key pathological features of atopic dermatitis, overcoming the limitations of existing studies that rely solely on mouse models and have been unable to translate their effects to humans. Our results will be useful for future research on the development of therapeutic agents for atopic dermatitis.

Development of an Aged Full-Thickness Skin Model Using Flexible Skin-on-a-Chip Subjected to Mechanical Stimulus Reflecting the Circadian Rhythm.

The skin is subject to both intrinsic aging caused by metabolic processes in the body and extrinsic aging caused by exposure to environmental factors. Intrinsic aging is an important obstacle to in vitro experimentation as its long-term progression is difficult to replicate. Here, we accelerated aging of a full-thickness skin equivalent by applying periodic mechanical stimulation, replicating the circadian rhythm for 28 days. This aging skin model was developed by culturing a full-thickness, three-dimensional skin equivalent with human fibroblasts and keratinocytes to produce flexible skin-on-a-chip. Accelerated aging associated with periodic compressive stress was evidenced by reductions in the epidermal layer thickness, contraction rate, and secretion of Myb. Increases in ß-galactosidase gene expression and secretion of reactive oxygen species and transforming growth factor-ß1 were also observed. This in vitro aging skin model is expected to greatly accelerate drug development for skin diseases and cosmetics that cannot be tested on animals.

Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model.

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 µM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models.

Coenzyme Q10 Efficacy Test for Human Skin Equivalents Using a Pumpless Skin-On-A-Chip System.

A human skin equivalent (HSE) composed of the epidermis and dermis is cultured using a pumpless skin-on-a-chip system to supply cultures the desired flow rate using gravity flow without a pump or an external tube connection. Coenzyme Q10 efficacy is tested by adjusting its concentration, as it is known to have anti-aging and antioxidant effects in culture solutions. The relationship between the contraction rate of a full-thickness human skin equivalent and secreted transforming growth factor (TGF) ß-1 is analyzed via enzyme-linked immunosorbent assay (ELISA). Following hematoxylin and eosin (H&E) staining, an image of the skin equivalent is analyzed to measure the epidermal layer's thickness. The cell density and differentiation of the dermis layer are investigated. Gene and protein expression in the dermal and epidermal layers are quantitatively analyzed using quantitative real time polymerase chain reaction (qPCR) and immunohistochemical staining. As the coenzyme Q10 treatment concentration increased, the number of cells per unit area and the thickness of the epidermal layer increased, the expression level of filaggrin increased, and the contraction rate of full-thickness HSE was proportional to the amount of TGF ß-1 secreted.

Testing the Effectiveness of Curcuma longa Leaf Extract on a Skin Equivalent Using a Pumpless Skin-on-a-Chip Model.

The in vitro tests in current research employ simple culture methods that fail to mimic the real human tissue. In this study, we report drug testing with a 'pumpless skin-on-a-chip' that mimics the structural and functional responses of human skin. This model is a skin equivalent constituting two layers of the skin, dermis and epidermis, developed using human primary fibroblasts and keratinocytes. Using the gravity flow device system, the medium was rotated at an angle of 15 degrees on both sides so as to circulate through the pumpless skin-on-a-chip microfluidic channel. This pumpless skin-on-a-chip is composed of upper and lower chips, and is manufactured using porous membranes so that medium can be diffused and supplied to the skin equivalent. Drug testing was performed using Curcuma longa leaf extract (CLLE), a natural product cosmetic ingredient, to evaluate the usefulness of the chip and the efficacy of the cosmetic ingredient. It was found that the skin barrier function of the skin epidermis layer is enhanced to exhibit antiaging effects. This result indicates that the pumpless skin-on-a-chip model can be potentially used not only in the cosmetics and pharmaceutical industries but also in clinical applications as an alternative to animal studies.

Parylene-Coated Polytetrafluoroethylene-Membrane-Based Portable Urea Sensor for Real-Time Monitoring of Urea in Peritoneal Dialysate.

A portable urea sensor for use in fast flow conditions was fabricated using porous polytetrafluoroethylene (PTFE) membranes coated with amine-functionalized parylene, parylene-A, by vapor deposition. The urea-hydrolyzing enzyme urease was immobilized on the parylene-A-coated PTFE membranes using glutaraldehyde. The urease-immobilized membranes were assembled in a polydimethylsiloxane (PDMS) fluidic chamber, and a screen-printed carbon three-electrode system was used for electrochemical measurements. The success of urease immobilization was confirmed using scanning electron microscopy, and fourier-transform infrared spectroscopy. The optimum concentration of urease for immobilization on the parylene-A-coated PTFE membranes was determined to be 48 mg/mL, and the optimum number of membranes in the PDMS chamber was found to be eight. Using these optimized conditions, we fabricated the urea biosensor and monitored urea samples under various flow rates ranging from 0.5 to 10 mL/min in the flow condition using chronoamperometry. To test the applicability of the sensor for physiological samples, we used it for monitoring urea concentration in the waste peritoneal dialysate of a patient with chronic renal failure, at a flow rate of 0.5 mL/min. This developed urea biosensor is considered applicable for (portable) applications, such as artificial kidney systems and portable dialysis systems.

Fabrication of a Urea Biosensor for Real-Time Dynamic Fluid Measurement.

In this study, a portable urea sensor that monitors the urea concentration in flow conditions was fabricated. We propose an electrochemical sensor that continually measures the urea concentration of samples flowing through it at a constant flow rate in real time. For the electrochemical sensing, a porous silk fibroin membrane with immobilized urease was mounted in a polydimethylsiloxane (PDMS) sensor housing. The fabricated urea sensor elicited linear current⁻concentration characteristics in the clinically significant concentration range (0.1⁻20 mM) based on peritoneal dialysis. The sensor maintained the linear current⁻concentration characteristics during operation in flow conditions.

Construction of 3D multicellular microfluidic chip for an in vitro skin model.

Current in vitro skin models do not recapitulate the complex architecture and functions of the skin tissue. In particular, on-chip construction of an in vitro model comprising the epidermis and dermis layer with vascular structure for mass transport has not been reported yet. In this study, we aim to develop a microfluidic, three-dimensional (3D) skin chip with fluidic channels using PDMS and hydrogels. Mass transport within the collagen hydrogel matrix was verified with fluorescent model molecules, and a transport-reaction model of oxygen and glucose inside the skin chip was developed to aid the design of the microfluidic skin chip. Comparison of viabilities of dermal fibroblasts and HaCaT cultured in the chip with various culture conditions revealed that the presence of flow plays a crucial role in maintaining the viability, and both cells were viable after 10 days of air exposure culture. Our 3D skin chip with vascular structures can be a valuable in vitro model for reproducing the interaction between different components of the skin tissue, and thus work as a more physiologically realistic platform for testing skin reaction to cosmetic products and drugs.

Experimental verification of overlimiting current by surface conduction and electro-osmotic flow in microchannels.

Direct evidence is provided for the transition from surface conduction (SC) to electro-osmotic flow (EOF) above a critical channel depth (d) of a nanofluidic device. The dependence of the overlimiting conductance (OLC) on d is consistent with theoretical predictions, scaling as d(-1) for SC and d(4/5) for EOF with a minimum around d=8 µm. The propagation of transient deionization shocks is also visualized, revealing complex patterns of EOF vortices and unstable convection with increasing d. This unified picture of surface-driven OLC can guide further advances in electrokinetic theory, as well as engineering applications of ion concentration polarization in microfluidics and porous media.

Plasmonic waveguide ring resonators with 4 nm air gap and λ0(2)/15,000 mode-area fabricated using photolithography.

Plasmonic air-gap disk resonators with 3.5 µm diameter and a 4 nm thick, 40 nm wide air gap for a mode area of only λ0(2)/15,000 were fabricated using photolithography only. The resonant modes were clearly identified using tapered fiber coupling method at the resonant wavelengths of 1280-1620 nm. We also demonstrate the advantage of the air-gap structure by using the resonators as label-free biosensors with a sensitivity of 1.6 THz/nm.

Optimization of hair follicle spheroids for hair-on-a-chip.

Currently, most models for hair follicle research have the limitation of not replicating some key features of the hair follicle microenvironment. To complement this, we transfected various factors for hair growth into dermal papilla cells (DPCs) by electroporation and cultured the spheroids with keratinocytes (KCs). We optimized the cell number and culture period for applying spheroids to hair-on-a-chip. Furthermore, we investigated the expression of hair growth factors in spheroids depending on the presence or absence of human umbilical vein endothelial cells (HUVECs) and transfection. In spheroids in which DPCs, KCs, and HUVECs were co-cultured for 21 days, the expression of lymphoid enhancer factor 1 (LEF1), T-cell factor 1 (TCF1), and keratin 25 (K25) in the center of the spheroid, the expression of keratin 17 (K17) on the outer surface of the spheroid, and the shape of hair extending outward from the spheroid surface were observed. From these results, it is expected that a hair-on-a-chip experiment in which short-term cultured TKH spheroids are injected into the dermis and co-cultured with KC will enable the production of full-thickness skin equivalents containing hair in vitro without transplantation into animals.

Size and surface modification effects on the pH response of Si nanowire field-effect transistors.

We investigated the effects of Si nanowire (SiNW) dimensions and their surface modifications on the pH-dependent electronic transport characteristics of SiNW Electrolyte-insulator-Semiconductor Field-Effect Transistors (EISFETs). The threshold voltages, Vth's, of all devices were extracted from the Id-Vg characteristics with Vg applied to the reference electrode immersed in different pH solutions, and their pH-dependences were analyzed for various devices. We found that our devices produce the systematic pH-dependence of Vth with respect to the SiNW's length and show significant changes in a linear pH region and a pH sensitivity upon the Si surface modifications. Particularly in the case of the APTES-treated surface, the linear variation was observed in the wide region of pH = 2 to approximately 11 with the sensitivity of 54.7 +/- 0.6 mV/pH. Also we compared our data to a theoretical result based on the Gouy-Chapmam-Stern-Graham model and found a reasonable agreement between them.

Fabrication and Evaluation of Tubule-on-a-Chip with RPTEC/HUVEC Co-Culture Using Injection-Molded Polycarbonate Chips.

To simulate the ADME process such as absorption, distribution, metabolism, and excretion in the human body after drug administration and to confirm the applicability of the mass production process, a microfluidic chip injection molded with polycarbonate (injection-molded chip (I-M chip)) was fabricated. Polycarbonate materials were selected to minimize drug absorption. As a first step to evaluate the I-M chip, RPTEC (Human Renal Proximal Tubule Epithelial Cells) and HUVEC (Human Umbilical Vein Endothelial Cells) were co-cultured, and live and dead staining, TEER (trans-epithelial electrical resistance), glucose reabsorption, and permeability were compared using different membrane pore sizes of 0.4 µm and 3 µm. Drug excretion was confirmed through a pharmacokinetic test with metformin and cimetidine, and the gene expression of drug transporters was confirmed. As a result, it was confirmed that the cell viability was higher in the 3 µm pore size than in the 0.4 µm, the cell culture performed better, and the drug secretion was enhanced when the pore size was large. The injection-molded polycarbonate microfluidic chip is anticipated to be commercially viable for drug screening devices, particularly ADME tests.

Population inversion and low cooperative upconversion in Er-doped silicon-rich silicon nitride waveguide.

Single-mode, strip-loaded silicon-rich silicon nitride (SRSN) waveguide with 11 at.% excess Si and 1.7×10(20) cm(-3) Er was fabricated and characterized. By using a 350 nm thick SRSN:Er core layer and a 850 nm wide SiO2 strip, a high core-mode overlap of 0.85 and low transmission loss of 2.9 dB/cm is achieved. Population inversion of 0.73-0.75, close to the theoretical maximum, is estimated to have been achieved via 1480 nm resonant pumping, indicating that nearly all doped Er in SRSN are optically active. Analysis of the pump power dependence of Er3+ luminescence intensity and lifetime indicate that the Er cooperative upconversion coefficient in SRSN:Er is as low as 2.1×10(-18) cm3/sec.

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach.

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen-antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.

Fabrication of carbon capsules with hierarchical pore structures.

Carbon capsules with hierarchical pore structures were fabricated by using core-shell silica nanoparticles as templates and phenolic resin as a carbon precursor. Carbon capsules with hierarchical pore structures were obtained via in-situ polymerization of the phenolic resin on the surface of the silica nanoparticles followed by the carbonization and removal of the silica templates. The hierarchically pored carbon capsules exhibited multimodal porosity with a high specific surface area (approximately 1834 m2/g) and a large pore volume (approximately 1.83 cm3/g).

Response to cardiac markers in human serum analyzed by guided-mode resonance biosensor.

Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed by use of a guided-mode resonance (GMR) biosensor. Cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) were monitored in the serum of both patients and healthy controls. Dose-response curves ranging from 0.05 to 10 ng/mL for cTnI, from 0.1 to 10 ng/mL for CK-MB, and from 0.03 to 1.7 µg/mL for MYO were obtained. The limits of detection (LOD) for cTnI, CK-MB, and MYO were less than 0.05, 0.1, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements. Antibody immobilization and the hydrophilic properties of the guided-mode resonance filter (GMRF) surface were investigated by X-ray photoelectron spectroscopy (XPS) and by monitoring the peak wavelength shift and water contact angle (CA). Both assays used to evaluate the surface density of the immobilized antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) and a sandwich immunogold assay, showed that the antibodies were successfully immobilized and sufficiently aligned to detect the low concentration of biomarkers. Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.