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Abstract Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.

Abstract SARS-CoV-2 provokes devastating tissue damage by cytokine release syndrome and leads to multi-organ failure. Modeling the process of immune cell activation and subsequent tissue damage is a significant task. Organoids from human tissues advanced our understanding of SARS-CoV-2 infection mechanisms though, they are missing crucial components: immune cells and endothelial cells. This study aims to generate organoids with these components. We established vascular immune organoids from human pluripotent stem cells and examined the effect of SARS-CoV-2 infection. We demonstrated that infections activated inflammatory macrophages. Notably, the upregulation of interferon signaling supports macrophages’ role in cytokine release syndrome. We propose vascular immune organoids are a useful platform to model and discover factors that ameliorate SARS-CoV-2-mediated cytokine release syndrome.

A bstract Background Clonorchis sinensis infection is one of the risk factors that provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis and even cholangiocarcinoma (CCA). Disrupted or aberrant intercellular communication among liver-constituting cells leads to pathological states that cause various hepatic diseases. This study was designed to investigate the pathological changes caused by C. sinensis excretory-secretory products (ESPs) in non-cancerous human cell lines (cholangiocytes [H69 cell line] and human hepatic stellate cells [LX2 cell line]) and their intercellular crosstalk, as well the pathological changes in infected mouse liver tissues. Methods The cells were treated with ESPs, following which transforming growth factor beta 1 (TGF-β1) and interleukin-6 (IL-6) secretion levels and epithelial-mesenchymal transition (EMT)- and fibrosis-related protein expression were measured. The ESP-mediated cellular motility (migration/invasion) between two cells was assessed using the Transwell and three-dimensional microfluidic assay models. The livers of C. sinensis-infected mice were stained using EMT and fibrotic marker proteins. Results Treatment of cells with ESPs increased TGF-β1 and IL-6 secretion and the expression of EMT- and fibrosis-related proteins. The ESP-mediated mutual cell interaction further affected the cytokine secretion and protein expression levels and promoted cellular motility. N-cadherin overexpression and collagen fiber deposition were observed in the livers of C. sinensis-infected mice. Conclusions These findings suggest that EMT and biliary fibrosis occur through intercellular communication between cholangiocytes and hepatic stellate cells during C. sinensis infection, promoting malignant transformation and advanced hepatobiliary abnormalities. Graphical Abstract

Abstract The human cutaneous lymphatic system strictly controls lymphatic functions by coordinating with skin cells. The lymphatic system plays important roles in removing cell waste, residual proteins, various antigens, and immune cells from tissues to maintain homeostasis and activate the immune system through the drainage of interstitial fluid1,2. The skin protects our body from external stimuli such as pathogens through the cutaneous lymphatic system3,4. Herein, to develop an in vitro human cutaneous lymphatic model, we present two 3D microfluidic platforms: a lymphangiogenesis model with a precollecting lymphatic vessel-like structure and an advanced lymphangiogenesis model with a functional cutaneous barrier and a precollecting lymphatic vessel-like structure. In addition, we rapidly analyzed prolymphangiogenic effects using methods that incorporate a high-speed image processing system and a deep learning-based vascular network analysis algorithm by 12 indices. Using these platforms, we evaluated the pro-lymphangiogenic effect of Lymphanax, a natural product derived from fresh ginseng. As a result, we demonstrated that Lymphanax induces robust lymphangiogenesis without any structural abnormalities. In conclusion, we suggest that these innovative platforms are useful for studying the interaction between the skin and lymphatic system as well as evaluating the prolymphangiogenic effects of drugs and cosmetics.

Abstract Prominent techniques such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and rapid kits are currently being explored to both enhance sensitivity and reduce assay time for diagnostic tests. Existing commercial molecular methods typically take several hours, while immunoassays can range from several hours to tens of minutes. Rapid diagnostics are crucial in Point-of-Care Testing (POCT). We propose an approach that integrates a time-series deep learning architecture and AI-based verification, for the enhanced result analysis of lateral flow assays. This approach is applicable to both infectious diseases and non-infectious biomarkers. In blind tests using clinical samples, our method achieved diagnostic times as short as 2 minutes, exceeding the accuracy of human analysis at 15 minutes. Furthermore, our technique significantly reduces assay time to just 1-2 minutes in the POCT setting. This advancement has the potential to greatly enhance POCT diagnostics, enabling both healthcare professionals and non-experts to make rapid, accurate decisions.

Abstract Background Recently, natural killer (NK) cells emerged as a treatment option for various solid tumors. However, the immunosuppressive tumor immune microenvironment (TIME) can reduce the cytotoxic ability of NK cells in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts within the tumor stroma can suppress immune surveillance by dysregulating factors involved in the cellular activity of NK cells. Herein, the effect of activated pancreatic stellate cells (aPSCs) on NK cell-mediated anticancer efficacy under three-dimensional (3D) coculture conditions was investigated. Methods 3D cocultures of PANC-1 tumor spheroids (TSs) with aPSCs and NK-92 cells in a collagen matrix were optimized to identify the occurring cellular interactions and differential cytokine profiles in conditioned media using microchannel chips. PANC-1 TSs and aPSCs were indirectly cocultured, whereas NK-92 cells were allowed to infiltrate the TS channel using convective medium flow. Results Coculture with aPSCs promoted PANC-1 TSs growth and suppressed the antitumor cytotoxic effects of NK-92 cells. Mutual inhibition of cellular activity without compromising migration ability was observed between aPSCs and NK-92 cells. Moreover, the reduced killing activity of NK-92 cells was found to be related with reduced granzyme B expression in NK cells. Conclusions Herein, a novel TIME-on-chip model based on the coculture of PANC-1 TSs, aPSCs, and NK-92 cells was described. This model may be useful for studying the detailed mechanisms underlying NK cells dysregulation and for exploring future therapeutic interventions to restore NK cell activity in the tumor microenvironment.

Abstract Palladium is the most prominent material in both scientific and industrial research on gas storage, purification, detection, and catalysis due to its unique properties as a catalyst and hydrogen absorber. Advancing the dynamic optical phenomena of palladium reacting with hydrogen, transduction of the gas-matter reaction into light-matter interaction is attempted to visualize the dynamic surface chemistry and reaction behaviors. The simple geometry of the metal-dielectric-metal structure, Fabry–Perot etalon, is employed for a colorimetric reactor, to display the catalytic reaction of the exposed gas via water-film/bubble formation at the dielectric/palladium interface. The adsorption/desorption behavior and catalytic reaction of hydrogen and oxygen on the palladium surface display highly repeatable and dramatic color changes based on two distinct water formation trends: the foggy effect by water bubbles and the whiteout effect by water film formation. Simulations and experiments demonstrate the robustness of the proposed Fabry–Perot etalon as an excellent platform for monitoring the opto-physical phenomena driven by heterogeneous catalysis.

COVID-19: Corticosteroids counteract inflammation by interferon The ability of corticosteroid drugs to alleviate extreme inflammation in severe cases of COVID-19 is linked to their reduction of the gene expression that is stimulated by interferon, an immunological cytokine protein. Interferon is crucial for a healthy immune response, but when produced in excess it can play a central role in causing damaging inflammation. Hyun-Woo Jeong at the Max Planck Institute for Molecular Biomedicine, Münster, Germany, and colleagues in South Korea compared the activity of genes involved in the inflammatory response in blood cells from COVID-19 patients and healthy donors. The results provided insights into the role of altered gene activity in both aggravation and improvement of COVID-19. Of particular interest was the ability of corticosteroids to downregulate the activity of a gene called STAT1, involved in mediating interferon signals.

Skin-nerve crosstalk is a major element of skin physiological pathology. Here the authors report a 3D innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte coculture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization.

Radiation treatment is one of the most frequently used therapies in patients with cancer, employed in approximately half of all patients. However, the use of radiation therapy is limited by acute or chronic adverse effects and the failure to consider the tumor microenvironment. Blood vessels substantially contribute to radiation responses in both normal and tumor tissues. The present study employed a three-dimensional (3D) microvasculature-on-a-chip that mimics physiological blood vessels to determine the effect of radiation on blood vessels. This model represents radiation-induced pathophysiological effects on blood vessels in terms of cellular damage and structural and functional changes. DNA double-strand breaks (DSBs), apoptosis, and cell viability indicate cellular damage. Radiation-induced damage leads to a reduction in vascular structures, such as vascular area, branch length, branch number, junction number, and branch diameter; this phenomenon occurs in the mature vascular network and during neovascularization. Additionally, vasculature regression was demonstrated by staining the basement membrane and microfilaments. Radiation exposure could increase the blockage and permeability of the vascular network, indicating that radiation alters the function of blood vessels. Radiation suppressed blood vessel recovery and induced a loss of angiogenic ability, resulting in a network of irradiated vessels that failed to recover, deteriorating gradually. These findings demonstrate that this model is valuable for assessing radiation-induced vascular dysfunction and acute and chronic effects and can potentially improve radiotherapy efficiency.

Abstract In vivo, the membrane potential of the excitable cell working by ion gradients plays a significant role in bioelectricity generation and nervous system operation. Conventional bioinspired power systems generally have adopted ion gradients, but overlook the functions of ion channels and Donnan effect to generate efficient ion flow in the cell. Here, cell‐inspired ionic power device implementing the Donnan effect using multi‐ions and monovalent ion exchange membranes as artificial ion channels is realized. Different ion‐rich electrolytes on either side of the selective membrane generate the ion gradient potentials with high ionic currents and reduce the osmotic imbalance of the membrane. Based on this device, the artificial neuronal signaling is presented by the mechanical switching system of the ion selectivity like mechanosensitive ion channels in a sensory neuron. Compared with reverse electrodialysis, which requires a low concentration, a high‐power device with ten times the current and 8.5 times the power density is fabricated. This device activates grown muscle cells by increasing power through serial connection like an electric eel, and shows the possibility of an ion‐based artificial nervous system.

Abstract The protozoan parasite Toxoplasma gondii (T. gondii) causes one of the most common human zoonotic diseases and infects approximately one-third of the global population. T. gondii infects nearly every cell type and causes severe symptoms in susceptible populations. In previous laboratory animal studies, T. gondii movement and transmission were not analyzed in real time. In a three-dimensional (3D) microfluidic assay, we successfully supported the complex lytic cycle of T. gondii in situ by generating a stable microvasculature. The physiology of the T. gondii-infected microvasculature was monitored in order to investigate the growth, paracellular and transcellular migration, and transmission of T. gondii, as well as the efficacy of T. gondii drugs.

Abstract Although numerous organ‐on‐a‐chips have been developed, bone‐on‐a‐chip platforms have rarely been reported because of the high complexity of the bone microenvironment. With an increase in the elderly population, a high‐risk group for bone‐related diseases such as osteoporosis, it is essential to develop a precise bone‐mimicking model for efficient drug screening and accurate evaluation in preclinical studies. Here, we developed a high‐throughput biomimetic bone‐on‐a‐chip platform combined with an artificial intelligence (AI)‐based image analysis system. To recapitulate the key aspects of natural bone microenvironment, mouse osteocytes (IDG‐SW3) and osteoblasts (MC3T3‐E1) were cocultured within the osteoblast‐derived decellularized extracellular matrix (OB‐dECM) built in a well plate‐based three‐dimensional gel unit. This platform spatiotemporally and configurationally mimics the characteristics of the structural bone unit, known as the osteon. Combinations of native and bioactive ingredients obtained from the OB‐dECM and coculture of two types of bone cells synergistically enhanced osteogenic functions such as osteocyte differentiation and osteoblast maturation. This platform provides a uniform and transparent imaging window that facilitates the observation of cell–cell interactions and features high‐throughput bone units in a well plate that is compatible with a high‐content screening system, enabling fast and easy drug tests. The drug efficacy of anti‐SOST antibody, which is a newly developed osteoporosis drug for bone formation, was tested via β‐catenin translocation analysis, and the performance of the platform was evaluated using AI‐based deep learning analysis. This platform could be a cutting‐edge translational tool for bone‐related diseases and an efficient alternative to bone models for the development of promising drugs.

Abstract During radiotherapy, microenvironments neighboring the tumor are also exposed to gamma irradiation; this results in unexpected side effects. Blood vessels can serve as microenvironments for tumors and they play an important role in providing nutrients to tumors. This is mostly related to tumor progression, metastasis, and relapse after therapy. Many studies have been performed to obtain a better understanding of tumor vasculature after radiotherapy with in vitro models. However, compared to 3-D models, 2-D in vitro endothelial monolayers cannot physiologically reflect in vivo blood vessels. We previously remodeled the extracellular matrix (ECM) hydrogel that enhanced the tight barrier formation of 3-D blood vessels and the vascular endothelial growth factor (VEGF) gradient induced angiogenesis in a microfluidic device. In this study, the blood vessel model is further introduced to understand how gamma irradiation affects the endothelial monolayer. After the gamma irradiation exposure, we observed a collapsed endothelial barrier and a reduced angiogenic potential. Changes in the cell behaviors of the tip and stalk cells were also detected in the angiogenesis model after irradiation, which is difficult to observe in 2-D monolayer models. Therefore, the 3-D in vitro blood vessel model can be used to understand radiation-induced endothelial injuries.

Abstract Tertiary lymphoid structures (TLSs) provide specialized niches for immune cells, resulting in improved prognoses for patients undergoing cancer immunotherapy. Shaping TLS‐like niches may improve anti‐cancer immunity and overcome the current limitations of immune cell‐based immunotherapy. Here, it is shown that stromal vascular fraction (SVF) from adipose tissues can enhance dendritic cell (DC)‐mediated T cell immunity by inducing ectopic T lymphocyte clusters. SVF cells expanded ex vivo have phenotypes and functions similar to those of fibroblastic reticular cells in a secondary lymphoid organ, and their properties can be modulated using three‐dimensional spheroid culture and coculture with DCs spiked with antigen‐loaded iron oxide–zinc oxide core‐shell nanoparticles. Thereby, the combination of SVF spheroids and mature DCs significantly augments T cell recruitment and retention at the injection site. This strategy elicits enhanced antigen‐specific immune response and anti‐tumoral immunity in mice, illustrating the potential for a novel immunotherapeutic design using SVF as a structural scaffold for TLS.

Although the proportion of ulcer patients with medical problems among the elderly has increased with the extension of human life expectancy, treatment efficiency is drastically low, incurring substantial social costs. MSCs have independent regeneration potential, making them useful in clinical trials of difficult-to-treat diseases. In particular, ADMSCs are promising in the stem cell therapy industry as they can be obtained in vast amounts using non-invasive methods. Furthermore, studies are underway to enhance the regeneration potential of ADMSCs using cytokines, growth factors, and gene delivery to generate highly functional ADMSCs. In this study, key regulators of wound healing, SOCS-1, -3, and -5, were combined to maximize the regenerative potential of ADMSCs in pressure ulcer treatments. After transfecting SOCS-1, -3, -5, and SOCS-com into ADMSCs using a non-viral method, the expression of the inflammatory factors TNF-alpha, INF-gamma, and IL-10 was confirmed. ADMSCs transfected with SOCS-com showed decreased overall expression of inflammatory factors and increased expression of anti-inflammatory factors. Based on these results, we implanted ADMSCs transfected with SOCS-com into a pressure ulcer mouse model to observe their subsequent wound-healing effects. Notably, SOCS-com improved wound closure in ulcers, and reconstruction of the epidermis and dermis was observed. The healing mechanism of ADMSCs transfected with SOCS-com was examined by RNA sequencing. Gene analysis results confirmed that expression changes occurred in genes of key regulators of wound healing, such as chemokines, MMP-1, 9, CSF-2, and IL-33, and that such genetic changes enhanced wound healing in ulcers. Based on these results, we demonstrate the potential of ADMSCs transfected with SOCS-com as an ulcer treatment tool.

Abstract PTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

Abstract Background Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation. Methods A three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection. Results Microarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq. Conclusions These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer. Graphic abstract

Abstract Water, abundant and ubiquitous in nature, is an easy yet powerful resource for the creatures to survive by putting together with their topologies interfacing their living environment. Here, a slippery, water‐infusing surface (SWIS) that retains a thick and stable water layer on the membrane is presented, robustly maintaining the oil repellency against the pressure and friction of immiscible liquids. Inspired by the plant trichome structures and their function, grooved nanotrichome, formed on the fibrous membrane by the oxygen plasma etching, induces robust water lubrication on the SWIS. SWIS membrane repels and separates highly viscous and adhesive oils in air and underwater by preventing oils from adhering to the lubricating surface. Repeated tests both in air and underwater confirm the antiadhesion and self‐cleaning properties of the SWIS. The SWIS oil scooper, fixed on a frame with a handle, successfully collects spilled oil on a pilot‐scale oil spill site and a real ocean oil spill site by simply scooping and recovering the oil. In addition, SWIS membrane is expected to help protect environments with further applications such as oil‐wastewater treatment and oil separation in food.

Abstract Background Glioblastoma (GBM) is a complex disease with extensive molecular and transcriptional heterogeneity. GBM can be subcategorized into four distinct subtypes; tumors that shift towards the mesenchymal phenotype upon recurrence are generally associated with treatment resistance, unfavorable prognosis, and the infiltration of pro-tumorigenic macrophages. Results We explore the transcriptional regulatory networks of mesenchymal-associated tumor-associated macrophages (MA-TAMs), which drive the malignant phenotypic state of GBM, and identify macrophage receptor with collagenous structure (MARCO) as the most highly differentially expressed gene. MARCOhigh TAMs induce a phenotypic shift towards mesenchymal cellular state of glioma stem cells, promoting both invasive and proliferative activities, as well as therapeutic resistance to irradiation. MARCOhigh TAMs also significantly accelerate tumor engraftment and growth in vivo. Moreover, both MA-TAM master regulators and their target genes are significantly correlated with poor clinical outcomes and are often associated with genomic aberrations in neurofibromin 1 (NF1) and phosphoinositide 3-kinases/mammalian target of rapamycin/Akt pathway (PI3K-mTOR-AKT)-related genes. We further demonstrate the origination of MA-TAMs from peripheral blood, as well as their potential association with tumor-induced polarization states and immunosuppressive environments. Conclusions Collectively, our study characterizes the global transcriptional profile of TAMs driving mesenchymal GBM pathogenesis, providing potential therapeutic targets for improving the effectiveness of GBM immunotherapy.

Lymphangiogenesis is a stage of new lymphatic vessel formation in development and pathology, such as inflammation and tumor metastasis. Physiologically relevant models of lymphatic vessels have been in demand because studies on lymphatic vessels are required for understanding the mechanism of tumor metastasis. In this study, a new three-dimensional lymphangiogenesis model in a tumor microenvironment is proposed, using a newly designed macrofluidic platform. It is verified that controllable biochemical and biomechanical cues, which contribute to lymphangiogenesis, can be applied in this platform. In particular, this model demonstrates that a reconstituted lymphatic vessel has an in vivo–like lymphatic vessel in both physical and biochemical aspects. Since biomechanical stress with a biochemical factor influences robust directional lymphatic sprouting, whether our model closely approximates in vivo, the initial lymphatics in terms of the morphological and genetic signatures is investigated. Furthermore, attempting an incorporation with a tumor spheroid, this study successfully develops a complex tumor microenvironment model for use in lymphangiogenesis and reveals the microenvironment factors that contribute to tumor metastasis. As a first attempt at a coculture model, this reconstituted model is a novel system with a fully three-dimensional structure and can be a powerful tool for pathological drug screening or disease model.

Abstract Blood vessels in central nervous system act as a great hurdle for drug delivery to the human brain. They only allow passage of water, some gases, lipid molecules, glucose and amino acid by selective transporter while restricting most of solutes and pathogens to protect brain. A lot of studies have tried to overcome this hurdle by discovering and optimizing brain deliverable drugs however the platforms used for preclinical stage are still limited. In this study, we constructed an in vitro 3-dimensional model for brain endothelial monolayer using hydrogel incorporated microfluidic device that provides an 3D extracellular matrix scaffold. We confirmed the stable endothelial barrier by staining a tight junction marker, VE-Cadherin, and strong block ability by comparing permeability with normal endothelial cells. Also, we succeed in verifying the strong permeability of angiopep-2 using our device that is known as a brain permeable peptide by utilizing receptor-mediated transcytosis. We propose our microfluidic device as an in vitro platform for evaluating various brain drugs or drug carrier candidates.

Isolation of spheroid-forming cells is important to investigate cancer stem cell (CSC) characteristics. However, conventional tumor spheroid culture methods have not proven suitable because the aggregated spheroids generally maintain their original heterogeneity and harbor multiple cells with various characteristics. Here we cultured spheroids using a polydimethylsiloxane microwell-based method and a limiting dilution protocol. We then isolated and enriched for CSCs that formed single cell-derived spheroids from gastric cancer cell lines. Cells from the microwell demonstrated higher self-renewal, increased expression of stem cell markers and resistance to apoptosis compared with spheroid cells made by the traditional method. This novel approach allows efficient cancer stem cell isolation and represents a step forward in cancer stem cell studies.

Abstract Background Pancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance. Methods A 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay. Results PANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of α-SMA. Expression of EMT-related markers, such as vimentin and TGF-β, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs. Conclusions We demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.

Gelatin has excellent biological properties, but its poor physical properties are a major obstacle to its use as a biomaterial ink. These disadvantages not only worsen the printability of gelatin biomaterial ink, but also reduce the dimensional stability of its 3D scaffolds and limit its application in the tissue engineering field. Herein, biodegradable suture fibers were added into a gelatin biomaterial ink to improve the printability, mechanical strength, and dimensional stability of the 3D printed scaffolds. The suture fiber reinforced gelatin 3D scaffolds were fabricated using the thermo-responsive properties of gelatin under optimized 3D printing conditions (−10 °C cryogenic plate, 40–80 kPa pneumatic pressure, and 9 mm/s printing speed), and were crosslinked using EDC/NHS to maintain their 3D structures. Scanning electron microscopy images revealed that the morphologies of the 3D printed scaffolds maintained their 3D structure after crosslinking. The addition of 0.5% (w/v) of suture fibers increased the printing accuracy of the 3D printed scaffolds to 97%. The suture fibers also increased the mechanical strength of the 3D printed scaffolds by up to 6-fold, and the degradation rate could be controlled by the suture fiber content. In in vitro cell studies, DNA assay results showed that human dermal fibroblasts’ proliferation rate of a 3D printed scaffold containing 0.5% suture fiber was 10% higher than that of a 3D printed scaffold without suture fibers after 14 days of culture. Interestingly, the supplement of suture fibers into gelatin biomaterial ink was able to minimize the cell-mediated contraction of the cell cultured 3D scaffolds over the cell culture period. These results show that advanced biomaterial inks can be developed by supplementing biodegradable fibers to improve the poor physical properties of natural polymer-based biomaterial inks.

Ion concentration polarization (ICP) has been widely applied in microfluidic systems in pre-concentration, particle separation, and desalination applications. General ICP microfluidic systems have three components (i.e., source, ion-exchange, and buffer), which allow selective ion transport. Recently developed trials to eliminate one of the three components to simplify the system have suffered from decreased performance by the accumulation of unwanted ions. In this paper, we presented a new ICP microfluidic system with only an ion-exchange membrane-coated channel. Numerical investigation on hydrodynamic flow and electric fields with a series of coupled governing equations enabled a strong correlation to experimental investigations on electroconvective vortices and the trajectory of charged particles. This study has significant implications for the development and optimization of ICP microfluidic and electrochemical systems for biomarker concentration and separation to improve sensing reliability and detection limits in analytic chemistry.

Abstract Harmful materials in the blood are prevented from entering the healthy brain by a highly selective blood–brain barrier (BBB), and impairment of barrier function has been associated with a variety of neurological diseases. In Alzheimer's disease (AD), BBB breakdown has been shown to occur even before cognitive decline and brain pathology. To investigate the role of the cerebral vasculature in AD, a physiologically relevant 3D human neural cell culture microfluidic model is developed having a brain endothelial cell monolayer with a BBB‐like phenotype. This model is shown to recapitulate several key aspects of BBB dysfunction observed in AD patients: increased BBB permeability, decreased expression of claudin‐1, claudin‐5, and VE‐cadherin, increased expression of matrix‐metalloproteinase‐2 and reactive oxygen species, and deposition of β‐amyloid (Aβ) peptides at the vascular endothelium. Thus, it provides a well‐controlled platform for investigating BBB function as well as for screening of new drugs that need to pass the BBB to gain access to neural tissues.

Hemodynamic and biochemical factors play important roles in critical steps of angiogenesis. In particular, interstitial flow has attracted attention as an important hemodynamic factor controlling the angiogenic process. Here, we applied a wide range of interstitial flow magnitudes to an in vitro three-dimensional (3D) angiogenesis model in a microfluidic device. This study aimed to investigate the effect of interstitial flow magnitude in combination with the vascular endothelial growth factor (VEGF) concentration on 3D microvascular network formation. Human umbilical vein endothelial cells (HUVECs) were cultured in a series of interstitial flow generated by 2, 8, and 25 mmH2O. Our findings indicated that interstitial flow significantly enhanced vascular sprout formation, network extension, and the development of branching networks in a magnitude-dependent manner. Furthermore, we demonstrated that the proangiogenic effect of interstitial flow application could not be substituted by the increased VEGF concentration. In addition, we found that HUVECs near vascular sprouts significantly elongated in >8 mmH2O conditions, while activation of Src was detected even in 2 mmH2O conditions. Our results suggest that the balance between the interstitial flow magnitude and the VEGF concentration plays an important role in the regulation of 3D microvascular network formation in vitro.

Abstract The primed microenvironment of future metastatic sites, called the pre‐metastatic niche, is a prerequisite for overt metastasis. However, a mechanistic understanding of the contributions of recruited cells to the niche is hindered by complex in vivo systems. Herein, a microfluidic platform that incorporates endothelial cells and extracellular matrix (ECM) scaffolds is developed, and the distinct role of recruited monocytes and macrophages in establishing pre‐metastatic niches is delineated. It is observed that monocyte‐derived matrix metalloproteinase 9 facilitates cancer cell extravasation through destruction of endothelial tight junctions. Furthermore, subsequent cancer cell invasiveness is significantly enhanced. Close examination of ECM structures reveals that cancer cells move within characteristic “microtracks” generated by macrophages, suggesting that macrophages could serve as a compensatory mechanism for the reduced migratory capacity of cancer cells. Thus, the first evidence of monocyte/macrophage‐induced remodeling is shown, and these findings will open up new horizons for improving characterization of the pre‐metastatic niche and corresponding immunotherapies.

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer.

Blood plasma is a source of biomarkers in blood and a simple, fast, and easy extraction method is highly required for point-of-care testing (POCT) applications. This paper proposes a membrane filter integrated microfluidic device to extract blood plasma from whole blood, without any external instrumentation. A commercially available membrane filter was integrated with a newly designed dual-cover microfluidic device to avoid leakage of the extracted plasma and remaining blood cells. Nano-interstices installed on both sides of the microfluidic channels actively draw the extracted plasma from the membrane. The developed device successfully supplied 20 μL of extracted plasma with a high extraction yield (~45%) in 16 min.

Tissue engineering applications call for controllable micro-structural units as a platform, but their fabrication remains challenging. Here, Jeong et al. show a method that enables soft materials to self-organize into highly packed micro-honeycomb structures with aspect ratios up to 500, and tunable shapes.

Massive efforts to develop neural interfaces have been made for controlling prosthetic limbs according to the will of the patient, with the ultimate goal being long-term implantation. One of the major struggles is that the electrode’s performance degrades over time due to scar formation. Herein, we have developed peripheral nerve electrodes with a cone-shaped flexible artificial conduit capable of protecting wire electrodes from scar formation. The wire electrodes, which are composed of biocompatible alloy materials, were embedded in the conduit where the inside was filled with collagen to allow the damaged nerves to regenerate into the conduit and interface with the wire electrodes. After implanting the wire electrodes into the sciatic nerve of a rat, we successfully recorded the peripheral neural signals while providing mechanical stimulation. Remarkably, we observed the external stimuli-induced nerve signals at 19 weeks after implantation. This is possibly due to axon regeneration inside our platform. To verify the tissue response of our electrodes to the sciatic nerve, we performed immunohistochemistry (IHC) and observed axon regeneration without scar tissue forming inside the conduit. Thus, our strategy has proven that our neural interface can play a significant role in the long-term monitoring of the peripheral nerve signal.

There is a demand for three-dimensional (3D) angiogenesis model including endothelial cells (ECs) and mesenchymal stem cells (MSCs), which are known to differentiate into pericytes, to construct stabilized and matured microvascular networks in vitro. However, it remains to be elucidated how MSCs affected on ECs in the process of 3D angiogenesis. In this study, we utilized a microfluidic device to develop a 3D coculture system including human umbilical vein ECs and human MSCs, which allowed us to investigate the effects of MSCs on ECs in the context of 3D angiogenesis. A series of EC:MSC ratio was tested in the EC-MSC coculture. First, we confirmed that MSCs differentiated into pericytes by direct EC-MSC contacts. Next, we found that MSCs attenuated vascular sprout formation of ECs regardless of EC:MSC ratio in the early stage of 3D angiogenesis as well as extension of microvascular networks in the later stage. ECs and MSCs were also cultured under interstitial flow to enhance angiogenesis. However, the stabilization effects of MSCs on the extension of capillary structures were dominant over the promotion effects of the interstitial flow. These results indicate the stabilization effect of MSCs on the formation of microvascular networks in vitro. Although some HMSCs differentiated into pericytes and located around microvascular networks, vascular structures became thick over time in coculture. The 3D EC-MSC coculture model described in this study is useful to further investigate culture microenvironments for constructing stabilized and matured microvascular networks with aligning pericytes.

The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells.

Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a 'more clinically relevant' tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation.

Bacterial biofilms—aggregations of bacterial cells and extracellular polymeric substrates (EPS)—are an important subject of research in the fields of biology and medical science. Under aquatic conditions, bacterial cells form biofilms as a mechanism for improving survival and dispersion. In this review, we discuss bacterial biofilm development as a structurally and dynamically complex biological system and propose microfluidic approaches for the study of bacterial biofilms. Biofilms develop through a series of steps as bacteria interact with their environment. Gene expression and environmental conditions, including surface properties, hydrodynamic conditions, quorum sensing signals, and the characteristics of the medium, can have positive or negative influences on bacterial biofilm formation. The influences of each factor and the combined effects of multiple factors may be addressed using microfluidic approaches, which provide a promising means for controlling the hydrodynamic conditions, establishing stable chemical gradients, performing measurement in a high-throughput manner, providing real-time monitoring, and providing in vivo-like in vitro culture devices. An increased understanding of biofilms derived from microfluidic approaches may be relevant to improving our understanding of the contributions of determinants to bacterial biofilm development.

Numerous experimental and epidemiological studies have demonstrated a correlation between Clonorchis sinensis (C. sinensis) infestation and cholangiocarcinoma (CCA). However, the role of C. sinensis in the increased invasiveness and proliferation involved in the malignancy of CCA has not been addressed yet. Here, we investigated the possibility that C. sinensis infestation promotes expression of focal and cell-cell adhesion proteins in CCA cells and secretion of matrix metalloproteinases (MMPs). Adhesion proteins help maintain cell aggregates, and MMPs promote the three-dimensional invasion of cells into the neighboring extracellular matrix (ECM). Using a novel microfluidic assay, we quantitatively addressed the role of excretory-secretory products (ESPs) gradients from C. sinensis in promoting the invasion of cells into the neighboring ECM.

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.