Direct Fabrication of Freestanding and Patterned Nanoporous Junctions in a 3D Micro-Nanofluidic Device for Ion-Selective Transport.

In the field of micro-nanofluidics, a freestanding configuration of a nanoporous junction is highly demanded to increase the design flexibility of the microscale device and the interfacial area between the nanoporous junction and microchannels, thereby improving the functionality and performance. This work first reports direct fabrication and incorporation of a freestanding nanoporous junction in a microfluidic device by performing an electrolyte-assisted electrospinning process to fabricate a freestanding nanofiber membrane and subsequently impregnating the nanofiber membrane with a nanoporous precursor material followed by a solidification process. This process also enables to readily control the geometry of the nanoporous junction depending on its application. By these advantages, vertically stacked 3D micro-nanofluidic devices with complex configurations are easily achieved. To demonstrate the broad applicability of this process in various research fields, a reverse electrodialysis-based energy harvester and an ion concentration polarization-based preconcentrator are produced. The freestanding Nafion-polyvinylidene fluoride nanofiber membrane (F-NPNM) energy harvester generates a high power (59.87 nW) owing to the enlarged interfacial area. Besides, 3D multiplexed and multi-stacked F-NPNM preconcentrators accumulate multiple preconcentrated plugs that can increase the operating sample volume and the degree of freedom of handling. Hence, the proposed process is expected to contribute to numerous research fields related to micro-nanofluidics in the future.

Stabilizing Coacervate by Microfluidic Engulfment Induced by Controlled Interfacial Energy.

Low interfacial energy, an intrinsic property of complex coacervate, enables the complex coacervate to easily encapsulate desired cargo substances, making it widely used in encapsulation applications. Despite this advantage, the low interfacial energy of the complex coacervate makes it unstable against mechanical mixing, and changes in pH and salt concentration. Hence, a chemical cross-linker is usually added to enhance the stability of the complex coacervate at the expense of sacrificing all intrinsic properties of the coacervate, including phase transition of the coacervate from liquid to solid. In this study, we observed an abrupt increase in the interfacial energy of the coacervate phase in mineral oil. By controlling the interfacial energy of the coacervate phase using a microfluidic device, we successfully created double engulfed PEG-diacrylate (PEGDA) coacervate microparticles, named DEPOT, in which the coacervate is engulfed in a cross-linked PEGDA shell. The engulfed coacervate remained as a liquid phase, retained its original low interfacial energy property to encapsulate the desired cargo substances, and infiltrated into the target site by a simple solvent exchange from oil to water.

Role of Grounded Liquid Collectors in Precise Patterning of Electrospun Nanofiber Mats.

Liquid collectors are applicable as ground collectors in electrospinning, which fabricates complex nanofiber architectures. However, the influence of the electrical properties of liquid collectors on the controlled deposition of electrospun nanofiber mats has received little attention. Here, we prepare two types of liquid collectors (electrolyte solutions and dielectric liquids) and newly scrutinize their roles in the patterning of electrospun nanofiber mats in experiments and in numerical simulations. By simulating the concentrations of the electric fields around the liquid collectors, we indirectly evaluated the patternability of the collectors. The patternability trends were verified by the patterning of nanofiber mats on line-array-shaped liquid collectors fabricated by electrospinning. The deposition accuracy of the electrolyte solution collector was very high, equivalent to that of a conventional metal collector even at low salt concentrations (e.g., 0.01 M KCl). However, the nanofiber mats fabricated by electrospinning with the dielectric liquid collector showed retarded patternability.

Wide and fast focus-tunable dielectro-optofluidic lens via pinning of the interface of aqueous and dielectric liquids.

Electrohydrodynamic actuation of dielectric liquid enables the development of an efficient focus-tunable dielectro-optofluidic lens (DOL) by manipulating a liquid-liquid interface. However, practical utilization of the previous DOL is hindered by its narrow and slow focus-tunability due to the direct movement of the interface. Here, we propose pinning the interface to directly change the interface shape while preventing the interface movement. The newly designed DOL exploits sudden changes in the channel diameter and the surface wettability to firmly pin the interface. Our results demonstrate that the tuning range of the DOL from -40 to +35 diopters is achieved in 0.1 s.

Wireless sEMG System with a Microneedle-Based High-Density Electrode Array on a Flexible Substrate.

Surface electromyography (sEMG) signals reflect muscle contraction and hence, can provide information regarding a user's movement intention. High-density sEMG systems have been proposed to measure muscle activity in small areas and to estimate complex motion using spatial patterns. However, conventional systems based on wet electrodes have several limitations. For example, the electrolyte enclosed in wet electrodes restricts spatial resolution, and these conventional bulky systems limit natural movements. In this paper, a microneedle-based high-density electrode array on a circuit integrated flexible substrate for sEMG is proposed. Microneedles allow for high spatial resolution without requiring conductive substances, and flexible substrates guarantee stable skin-electrode contact. Moreover, a compact signal processing system is integrated with the electrode array. Therefore, sEMG measurements are comfortable to the user and do not interfere with the movement. The system performance was demonstrated by testing its operation and estimating motion using a Gaussian mixture model-based, simplified 2D spatial pattern.

Aquatic flower-inspired cell culture platform with simplified medium exchange process for facilitating cell-surface interaction studies.

Establishing fundamentals for regulating cell behavior with engineered physical environments, such as topography and stiffness, requires a large number of cell culture experiments. However, cell culture experiments in cell-surface interaction studies are generally labor-intensive and time-consuming due to many experimental tasks, such as multiple fabrication processes in sample preparation and repetitive medium exchange in cell culture. In this work, a novel aquatic flower-inspired cell culture platform (AFIP) is presented. AFIP aims to facilitate the experiments on the cell-surface interaction studies, especially the medium exchange process. AFIP was devised to capture and dispense cell culture medium based on interactions between an elastic polymer substrate and a liquid medium. Thus, the medium exchange can be performed easily and without the need of other instruments, such as a vacuum suction and pipette. An appropriate design window of AFIP, based on scaling analysis, was identified to provide a criterion for achieving stability in medium exchange as well as various surface characteristics of the petal substrates. The developed AFIP, with physically engineered petal substrates, was also verified to exchange medium reliably and repeatedly. A closed structure capturing the medium was sustained stably during cell culture experiments. NIH3T3 proliferation results also demonstrated that AFIP can be applied to the cell-surface interaction studies as an alternative to the conventional method.

Curved Microneedle Array-Based sEMG Electrode for Robust Long-Term Measurements and High Selectivity.

Surface electromyography is widely used in many fields to infer human intention. However, conventional electrodes are not appropriate for long-term measurements and are easily influenced by the environment, so the range of applications of sEMG is limited. In this paper, we propose a flexible band-integrated, curved microneedle array electrode for robust long-term measurements, high selectivity, and easy applicability. Signal quality, in terms of long-term usability and sensitivity to perspiration, was investigated. Its motion-discriminating performance was also evaluated. The results show that the proposed electrode is robust to perspiration and can maintain a high-quality measuring ability for over 8 h. The proposed electrode also has high selectivity for motion compared with a commercial wet electrode and dry electrode.

A zeta (ζ)-pipet tip to reduce the spontaneously induced electrical charge of a dispensed aqueous droplet.

We report that the zeta potential of a pipet tip's inner surface is one of the crucial parameters for controlling the electrical charge of the dispensed droplet. Since the charge is unexpected and undesirable for most experiments in various fields of science and, thereby, they can cause unsuspected problems, reducing the charge on a dispensed droplet is important for the results of pipetting-based experiments. We fabricate a graphene-based nanocomposite-coated pipet tip, which we called a zeta-pipet tip, as a proof-of-concept example to reduce the zeta potential of the pipet tip's inner surface. The fabricated zeta-pipet tip can successfully mitigate the undesired droplet separation in the droplet merging experiments in an oil bath, which is one of the unexpected effects caused by the electrification. The findings of this study provide helpful guidelines for researchers in many fields of science and technology, who utilize a pipet tip in their respective experiments.

A Perforated Plate-Based Cell Showering Device for Uniform Cell Distribution over Various Culture Substrates.

Cell distribution is one of the primary factors that can affect cell morphology and behaviors, as it determines cell-cell interactions. Despite the importance of cell distribution, the seeding process of in vitro cell culture still highly relies on the traditional method using manual pipetting. Because manual pipetting cannot ensure a uniform cell distribution and has the possibility of compromising experimental reproducibility, an accurate and systemic seeding method that enables uniform cell seeding over versatile culture substrates is required. Here, we developed a perforated plate-based cell seeding device called the CellShower, which enabled uniform cell seeding over a large area of cell culture substrates. The working principles of the CellShower are based on the laminar filling flow and capillary force in microfluidics, and the design of the CellShower was optimized with numerical simulations. The versatility of the CellShower in view of uniform cell seeding was demonstrated by applying it to various types of culture substrates from a conventional culture dish to culture substrates having nanotopography, porous structures, and 3D concave structures. The CellShower and its operating principles are expected to contribute to enhancing the accuracy and reproducibility of biological experiments.

Guidelines for Manufacturing and Application of Organoids: Kidney.

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

Engineered Regenerative Isolated Peripheral Nerve Interface for Targeted Reinnervation.

A regenerative peripheral nerve interface (RPNI) offers a therapeutic solution for nerve injury through reconstruction of the target muscle. However, implanting a transected peripheral nerve into an autologous skeletal muscle graft in RPNI causes donor-site morbidity, highlighting the need for tissue-engineered skeletal muscle constructs. Here, an engineered regenerative isolated peripheral nerve interface (eRIPEN) is developed using 3D skeletal cell printing combined with direct electrospinning to create a nanofiber membrane envelop for host nerve implantation. In this in vivo study, after over 8 months of RPNI surgery, the eRIPEN exhibits a minimum Feret diameter of 15-20 µm with a cross-sectional area of 100-500 µm2, representing the largest distribution of myofibers. Furthermore, neuromuscular junction formation and muscle contraction with a force of ≈28 N are observed. Notably, the decreased hypersensitivity to mechanical/thermal stimuli and an improved tibial functional index from -77 to -56 are found in the eRIPEN group. The present novel concept of eRIPEN paves the way for the utilization and application of tissue-engineered constructs in RPNI, ultimately realizing neuroprosthesis control through synaptic connections.

One-step microfluidic synthesis of Janus microhydrogels with anisotropic thermo-responsive behavior and organophilic/hydrophilic loading capability.

We report one-step microfluidic synthesis and characterization of novel Janus microhydrogels composed entirely of the same base material, N-isopropylacrylamide (NIPAAm). The microhydrogels were fabricated by the microfluidic generation of Janus monomer microdroplets based on separation of a supersaturated aqueous NIPAAm solution into NIPAAm-rich and -poor phases followed by UV irradiation. The resulting Janus microhydrogels exhibited tunable anisotropic thermo-responsive behavior and organophilic/hydrophilic loading capability.

Recent Progress in Fabrication of Electrospun Nanofiber Membranes for Developing Physiological In Vitro Organ/Tissue Models.

Nanofiber membranes (NFMs), which have an extracellular matrix-mimicking structure and unique physical properties, have garnered great attention as biomimetic materials for developing physiologically relevant in vitro organ/tissue models. Recent progress in NFM fabrication techniques immensely contributes to the development of NFM-based cell culture platforms for constructing physiological organ/tissue models. However, despite the significance of the NFM fabrication technique, an in-depth discussion of the fabrication technique and its future aspect is insufficient. This review provides an overview of the current state-of-the-art of NFM fabrication techniques from electrospinning techniques to postprocessing techniques for the fabrication of various types of NFM-based cell culture platforms. Moreover, the advantages of the NFM-based culture platforms in the construction of organ/tissue models are discussed especially for tissue barrier models, spheroids/organoids, and biomimetic organ/tissue constructs. Finally, the review concludes with perspectives on challenges and future directions for fabrication and utilization of NFMs.

Human iPS-derived blood-brain barrier model exhibiting enhanced barrier properties empowered by engineered basement membrane.

The basement membrane (BM) of the blood-brain barrier (BBB), a thin extracellular matrix (ECM) sheet underneath the brain microvascular endothelial cells (BMECs), plays crucial roles in regulating the unique physiological barrier function of the BBB, which represents a major obstacle for brain drug delivery. Owing to the difficulty in mimicking the unique biophysical and chemical features of BM in in vitro systems, current in vitro BBB models have suffered from poor physiological relevance. Here, we describe a highly ameliorated human BBB model accomplished by an ultra-thin ECM hydrogel-based engineered basement membrane (nEBM), which is supported by a sparse electrospun nanofiber scaffold that offers in vivo BM-like microenvironment to BMECs. BBB model reconstituted on a nEBM recapitulates the physical barrier function of the in vivo human BBB through ECM mechano-response to physiological relevant stiffness (∼500 kPa) and exhibits high efflux pump activity. These features of the proposed BBB model enable modelling of ischemic stroke, reproducing the dynamic changes of BBB, immune cell infiltration, and drug response. Therefore, the proposed BBB model represents a powerful tool for predicting the BBB permeation of drugs and developing therapeutic strategies for brain diseases.

A Liquid Triboelectric Series.

The triboelectric series is a generally accepted method for describing the triboelectric effect. It provides a way to control the double face of the ubiquitous triboelectric effect: causes of unpredictable accidents and the resultant surface charge as energy sources. However, previous studies have been biased in solids despite being observed in liquids (liquid-solid contact electrification). Therefore, a liquid triboelectric series is necessary to be established to manipulate the liquid triboelectric effect according to the appropriate goal. In this study, a liquid triboelectric series is first established to describe the triboelectric properties of each liquid when contact electrification occurs with a solid surface. The liquid triboelectric series covers electrolytes, organic solvents, oxidants, and higher sugar alcohols. Common chemical groups can be derived from the liquid triboelectric series that hydroxyl groups enhance, and benzene groups suppress the liquid triboelectric effect. The results are demonstrated by the amplified efficiency of an energy harvester and particle contamination after surface washing. This study will play a pivotal role in understanding the liquid-solid contact electrification phenomenon and providing new perspectives on the applications of the liquid triboelectric effect.

Curcumin Enhances the Anticancer Effects of Binimetinib on Melanoma Cells by Inducing Mitochondrial Dysfunction and Cell Apoptosis with Necroptosis.

BACKGROUND: Recent studies suggest that MEK1/2 inhibitors, including binimetinib, significantly improve malignant melanoma (MM) patient survival. Growing evidence suggests that phytochemicals, especially curcumin, can overcome drug resistance in cancer cells through a variety of mechanisms. OBJECTIVE: This study aims to examine curcumin's efficacy in vitro combined with binimetinib in human MM cells. METHODS: We used 2D monolayer and 3D spheroid human epidermal melanocyte culture models, HEMn-MP (human epidermal melanocytes, neonatal, moderately pigmented), and two human MM cell lines, G361 and SK-MEL-2, to evaluate cell viability, proliferation, migration, death, and reactive oxygen species (ROS) production following single therapy treatment, with either curcumin or binimetinib, or a combination of both. RESULTS: Compared to MM cells treated with single therapy, those with combination therapy showed significantly decreased cell viability and increased ROS production. We observed apoptosis following both single and combination therapies. However only those who had had combination therapy had necroptosis. CONCLUSION: Collectively, our data demonstrates that curcumin exerts significant synergistic anticancer effects on MM cells by inducing ROS and necroptosis when combined with binimetinib. Therefore, a strategy of adding curcumin to conventional anticancer agents holds promise for treating MM.

Facile and adhesive-free method for bonding nanofiber membrane onto thermoplastic polystyrene substrate to fabricate 3D cell culture platforms.

Nanofiber (NF) membranes have been highlighted as functional materials for biomedical applications owing to their high surface-to-volume ratios, high permeabilities, and extracellular matrix-like biomimetic structures. Because many in vitro platforms for biomedical applications are made of thermoplastic polymers (TP), a simple and leak-free method for bonding NF membranes onto TP platforms is essential. Here, we propose a facile but leak-free localized thermal bonding method for integrating 2D or 3D-structured NF membrane onto a TP supporting substrate while preserving the pristine nanofibrous structure of the membrane, based on localized preheating of the substrate. A methodology for determining the optimal preheating temperature was devised based on a numerical simulation model considering the melting temperature of the NF material and was experimentally validated by evaluating bonding stability and durability under cell culture conditions. The thermally-bonded interface between the NF membrane and TP substrate was maintained stably for 3 weeks allowing the successful construction of an intestinal barrier model. The applicability of the localized thermal bonding method was also demonstrated on various combinations of TP materials (e.g., polystyrene and polymethylmethacrylate) and geometries of the supporting substrate, including a culture insert and microfluidic chip. We expect the proposed localized thermal bonding method to contribute toward broadening and realizing the practical applications of functional NF membranes in various biomedical fields.

Overexpression of Dock180 and Elmo1 in Melanoma is Associated with Cell Survival and Migration.

BACKGROUND: Melanoma is one of the most aggressive and metastatic skin cancers. Although overexpression of Dock180 and Elmo1 has been identified in various cancers, including glioma, ovarian cancer, and breast cancer, their expression and functions in melanoma remain unknown. OBJECTIVE: This study aims to confirm the expression of Dock180 and Elmo1, their underlying mechanisms, and roles in melanoma. METHODS: Both immunohistochemical staining and Western blotting were used to confirm expression of Dock180 and Elmo1 in human melanoma. To identify roles of Dock180 and Elmo1 in cell survival, apoptosis and migration, downregulation of Dock180 or Elmo1 in melanoma cells with small interfering RNA (siRNA) was performed. RESULTS: We identified overexpression of Dock180 and Elmo1 in human melanoma compared to normal skin ex vivo. Inhibition of Dock180 or Elmo1 following siRNA in melanoma cells reduced cell viability and increased apoptosis as supported by increased proportion of cells with Annexin V-PE (+) staining and sub-G0/G1 peak in cell cycle analysis. Moreover, inhibition of Dock180 or Elmo1 regulated apoptosis-related proteins, showing downregulation of Bcl-2, caspase-3, and PARP and upregulation of Bax, PUMA, cleaved caspase-3, and cleaved PARP. Furthermore, knockdown of Dock180 and Elmo1 in melanoma cells reduced cell migration and changed cellular signaling pathways including ERK and AKT. Vemurafenib decreased cell viability in concentration-dependent manner, while transfection with Dock180- or Elmo1-specific siRNA in melanoma cells significantly reduced cell viability. CONCLUSION: Our results suggest that both Dock180 and Elmo1 may be associated with cancer progression, and can be potential targets for treatment of melanoma.

Engineering porous membranes mimicking in vivo basement membrane for organ-on-chips applications.

Porous membrane-based microfluidic chips are frequently used for developing in vitro tissue-barrier models, the so-called tissue barriers-on-chips (TBoCs). The porous membrane in a TBoC plays a crucial role as an alternative to an in vivo basement membrane (BM). To improve the physiological relevance of an artificial porous membrane, it should possess complex BM-like characteristics from both biophysical and biochemical perspectives. For practical use, artificial membranes should have high mechanical robustness, and their fabrication processes should be conducive to mass production. There have been numerous approaches to accomplishing these requirements in BM-like porous membranes. Extracellular matrix (ECM) hydrogels have emerged as physiologically relevant materials for developing artificial BMs; they remarkably improve the phenotypes and functions of both cells and their layers when compared to previous synthetic porous membranes. However, for practical use, the poor mechanical robustness of ECM membranes needs to be improved. Recently, an advanced ECM membrane reinforced with a nanofiber scaffold has been introduced that possesses both BM-like characteristics and practical applicability. This advanced ECM membrane is expected to promote not only in vivo-like cellular functions but also cellular responses to drugs, which in turn further facilitates the practical applications of TBoCs.

Thin and stretchable extracellular matrix (ECM) membrane reinforced by nanofiber scaffolds for developingin vitrobarrier models.

An extracellular matrix (ECM) membrane made up of ECM hydrogels has great potentials to develop a physiologically relevant organ-on-a-chip because of its biochemical and biophysical similarity toin vivobasement membranes (BMs). However, the limited mechanical stability of the ECM hydrogels makes it difficult to utilize the ECM membrane in long-term and dynamic cell/tissue cultures. This study proposes a thin but robust and transparent ECM membrane reinforced with silk fibroin (SF)/polycaprolactone (PCL) nanofibers, which is achieved byin situself-assembly throughout a freestanding SF/PCL nanofiber scaffold. The SF/PCL nanofiber-reinforced ECM (NaRE) membrane shows biophysical characteristics reminiscent of native BMs, including small thickness (<5µm), high permeability (<9 × 10-5cm s-1), and nanofibrillar architecture (∼10-100 nm). With the BM-like characteristics, the nanofiber reinforcement ensured that the NaRE membrane stably supported the construction of various types ofin vitrobarrier models, from epithelial or endothelial barrier models to complex co-culture models, even over two weeks of cell culture periods. Furthermore, the stretchability of the NaRE membrane allowed emulating the native organ-like cyclic stretching motions (10%-15%) and was demonstrated to manipulate the cell and tissue-level functions of thein vitrobarrier model.