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.

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.

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.

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.

Efficient Portable Urea Biosensor Based on Urease Immobilized Membrane for Monitoring of Physiological Fluids.

Numerous studies have addressed the utilization of glutaraldehyde (GA) as a homobifunctional cross-linker. However, its applicability has been impeded due to several issues, including the tendency of GA molecules to undergo polymerization. Herein, a portable urea biosensor was developed for the real-time monitoring of the flow of physiological fluids; this was achieved by using disuccinimidyl cross-linker-based urease immobilization. Urease was immobilized on a porous polytetrafluoroethylene (PTFE) solid support using different disuccinimidyl cross-linkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and bis-N-succinimidyl-(pentaethylene glycol) ester (BS(PEG)5). A urease activity test revealed that DSS exhibited the highest urease immobilizing efficiency, whereas FT-IR analysis confirmed that urease was immobilized on the PTFE membrane via DSS cross-linking. The membrane was inserted in a polydimethylsiloxane (PDMS) fluidic chamber that generated an electrochemical signal in the presence of a flowing fluid containing urea. Urea samples were allowed to flow into the urea biosensor (1.0 mL/min) and the signal was measured using chronoamperometry. The sensitivity of the DSS urea biosensor was the highest of all the trialed biosensors and was found to be superior to the more commonly used GA cross-linker. To simulate real-time monitoring in a human patient, flowing urea-spiked human serum was measured and the effective urease immobilization of the DSS urea biosensor was confirmed. The repeatability and interference of the urea biosensor were suitable for monitoring urea concentrations typically found in human patients.

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.

Direct Visualization of Perm-Selective Ion Transportation.

Perm-selective ion transportation in a nanoscale structure such as nanochannel, nanoporous membrane or nanojunction has been extensively studied with aids of nanofabrication technology for a decade. While theoretical and experimental advances pushed the phenomenon to seminal innovative applications, its basic observation has relied only on an indirect analysis such as current-voltage relation or fluorescent imaging adjacent to the nanostructures. Here we experimentally, for the first time, demonstrated a direct visualization of perm-selective ion transportation through the nanoscale space using an ionic plasma generation. A micro/nanofluidic device was employed for a micro bubble formation, plasma negation and penetration of the plasma along the nanojunction. The direct observation provided a keen evidence of perm-selectivity, i.e. allowing cationic species and rejecting anionic species. Furthermore, we can capture the plasma of lithium, which has lower mobility than sodium in aqueous state, passed the nanojunction faster than sodium due to the absence of hydrated shells around lithium. This simple, but essential visualization technique would be effective means not only for advancing the fundamental nanoscale electrokinetic study as well as interfacial ion transportation between liquid and plasma but also for providing the insight of new innovative engineering applications.

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.

Recirculating peritoneal dialysis system using urease-fixed silk fibroin membrane filter with spherical carbonaceous adsorbent.

The chronic kidney disease (CKD) patients are undergoing continuous ambulatory peritoneal dialysis (CAPD). However, there are some constraints, the frequent exchange of the dialysate and limitation of outside activity, associated with CAPD remain to be solved. In this study, we designed the wearable artificial kidney (WAK) system for peritoneal dialysis (PD) using urease-immobilized silk fibroin (SF) membrane and polymer-based spherical carbonaceous adsorbent (PSCA). We evaluated this kit's removal abilities of uremic toxins such as urea, creatinine, uric acid, phosphorus, and ß2-microglobulin from the dialysate of end-stage renal disease (ESRD) patients in vitro. The uremic toxins including urea, creatinine, uric acid, and phosphorus were removed about 99% by immobilized SF membrane and PSCA filter after 24 h treatment. However, only 50% of ß2-microglobulin was removed by this filtering system after 24 h treatment. In vivo study result shows that our filtering system has more uremic toxins removal efficiency than exchanged dialysate at every 6 h. We suggest that recirculating PD system using urease-immobilized SF membrane with PSCA could be more efficient than traditional dialysate exchange system for a WAK for PD.

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.

Novel fabrication method of the peritoneal dialysis filter using silk fibroin with urease fixation system.

During the last decade, there has been a great advance in the kidney dialysis system by wearable artificial kidney (WAK) system for end-stage renal disease patients. Uremic solute removal and water regeneration system are the most prerequisite for WAK to work properly. In this study, we designed a filtering membrane system by using immobilized urease silk fibroin filter and evaluated its comparative effectiveness with a PVDF filtering system in peritoneal dialysate regeneration system by urea removal efficacy. We evaluated this membrane's characteristic and performances by conducting SEM-EDX analyze, water-binding abilities and porosity test, removal abilities of urea, cytotoxicity assay and enzyme activity assay. Under the condition for optimization of urease, the percentage removal of urea was about 40% and 60% in 50 mg/dL urea solution by urease immobilized PVDF and silk fibroin scaffolds, respectively. The batch experimental result showed that immobilized filter removed more than 50% of urea in 50 mg/dL urea solution. In addition silk fibroin with urease filter removed 90 percent of urea in the peritoneal dialysate after 24 h filtration. We suggest that silk fibroin with urease fixation filter can be used more effectively for peritoneal dialysate regeneration system, which have hydrophilic property and prolonged enzyme activity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2136-2144, 2017.

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.

Sub-10 nm transparent all-around-gated ambipolar ionic field effect transistor.

In this paper, we developed a versatile ionic field effect transistor (IFET) which has an ambipolar function for manipulating molecules regardless of their polarity and can be operated at a wide range of electrolytic concentrations (10(-5) M-1 M). The IFET has circular nanochannels radially covered by gate electrodes, called "all-around-gate", with an aluminum oxide (Al2O3) oxide layer of a near-zero surface charge. Experimental and numerical validations were conducted for characterizing the IFET. We found that the versatility originated from the zero-charge density of the oxide layer and all-around-gate structure which increased the efficiency of the gate effect 5 times higher than a previously developed planar-gate by capacitance calculations. Our numerical model adapted Poisson-Nernst-Planck-Stokes (PNPS) formulations with additional nonlinear constraints of a fringing field effect and a counter-ion condensation and the experimental and numerical results were well matched. The device can control the transportation of ions at concentrations up to 1 M electrolyte which resembles a backflow of a shale gas extraction process. Furthermore, while traditional IFETs can manipulate either positively or negatively charged species depending on the inherently large surface charge of oxide layer, the presenting device and mechanism provide effective means to control the motion of both negatively and positively charged molecules which is important in biomolecule transport through nanochannels, medical diagnosis system and point-of-care system, etc.

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.