Angio-Net: deep learning-based label-free detection and morphometric analysis of in vitro angiogenesis.

Despite significant advancements in three-dimensional (3D) cell culture technology and the acquisition of extensive data, there is an ongoing need for more effective and dependable data analysis methods. These concerns arise from the continued reliance on manual quantification techniques. In this study, we introduce a microphysiological system (MPS) that seamlessly integrates 3D cell culture to acquire large-scale imaging data and employs deep learning-based virtual staining for quantitative angiogenesis analysis. We utilize a standardized microfluidic device to obtain comprehensive angiogenesis data. Introducing Angio-Net, a novel solution that replaces conventional immunocytochemistry, we convert brightfield images into label-free virtual fluorescence images through the fusion of SegNet and cGAN. Moreover, we develop a tool capable of extracting morphological blood vessel features and automating their measurement, facilitating precise quantitative analysis. This integrated system proves to be invaluable for evaluating drug efficacy, including the assessment of anticancer drugs on targets such as the tumor microenvironment. Additionally, its unique ability to enable live cell imaging without the need for cell fixation promises to broaden the horizons of pharmaceutical and biological research. Our study pioneers a powerful approach to high-throughput angiogenesis analysis, marking a significant advancement in MPS.

U-IMPACT: a universal 3D microfluidic cell culture platform.

The development of organs-on-a-chip has resulted in advances in the reconstruction of 3D cellular microenvironments. However, there remain limitations regarding applicability and manufacturability. Here, we present an injection-molded plastic array 3D universal culture platform (U-IMPACT) for various biological applications in a single platform, such as cocultures of various cell types, and spheroids (e.g., tumor spheroids, neurospheres) and tissues (e.g., microvessels). The U-IMPACT consists of three channels and a spheroid zone with a 96-well plate form factor. Specifically, organoids or spheroids (~500 µm) can be located in designated areas, while cell suspensions or cell-laden hydrogels can be selectively placed in three channels. For stable multichannel patterning, we developed a new patterning method based on capillary action, utilizing capillary channels and the native contact angle of the materials without any modification. We derived the optimal material hydrophilicity (contact angle of the body, 45-90°; substrate, <30°) for robust patterning through experiments and theoretical calculations. We demonstrated that the U-IMPACT can implement 3D tumor microenvironments for angiogenesis, vascularization, and tumor cell migration. Furthermore, we cultured neurospheres from induced neural stem cells. The U-IMPACT can serve as a multifunctional organ-on-a-chip platform for high-content and high-throughput screening.

Properties and anti-inflammatory effects of Sargassum muticum enzymatic extracts decomposed using crude enzyme from Shewanella oneidensis PKA1008.

This study was conducted to investigate the general properties of an enzymatic extract of Sargassum muticum (SM) produced using a crude enzyme from Shewanella oneidensis PKA 1008 and their anti-inflammatory activities. The SM was mixed with crude enzymes from S. oneidensis PKA 1008 (1:1 (v/v)) and incubated at 30 °C for 0, 3, 6, 12, 24, 48, and 60 h. S. oneidensis PKA 1008 crude enzyme showed the highest SM enzymatic extracts degradation ability when reacted with SM for 48 h. These evaluations demonstrated a 134.25% increase in reducing sugar content and a 14.90% reduction in viscosity at 48 h. The pH, lightness (L*) and yellowness (b*) of the SM enzymatic extracts decreased significantly with increasing reaction time. Moreover, the SM enzymatic extracts demonstrated significant anti-inflammatory activity. These results indicate that the crude enzyme from S. oneidensis PKA 1008 can be used to enhance the polysaccharide degradation of SM, and the resultant oligosaccharides may have an anti-inflammatory effect.

All-in-one microfluidic design to integrate vascularized tumor spheroid into high-throughput platform.

The development of a scalable and highly reproducible in vitro tumor microenvironment (TME) platform still sheds light on new insights into cancer metastasis mechanisms and anticancer therapeutic strategies. Here, we present an all-in-one injection molded plastic array three-dimensional culture platform (All-in-One-IMPACT) that integrates vascularized tumor spheroids for highly reproducible, high-throughput experimentation. This device allows the formation of self-assembled cell spheroids on a chip by applying the hanging drop method to the cell culture channel. Then, when the hydrogel containing endothelial cells and fibroblasts is injected, the spheroid inside the droplet can be patterned together in three dimensions along the culture channel. In just two steps above, we can build a vascularized TME within a defined area. This process does not require specialized user skill and minimizes error-inducing steps, enabling both reproducibility and high throughput of the experiment. We have successfully demonstrated the process, from spheroid formation to tumor vascularization, using patient-derived cancer cells (PDCs) as well as various cancer cell lines. Furthermore, we performed combination therapies with Taxol (paclitaxel) and Avastin (bevacizumab), which are used in standard care for metastatic cancer. The All-in-One IMPACT is a powerful tool for establishing various anticancer treatment strategies through the development of a complex TME for use in high-throughput experiments.

Effects of Gamma Irradiation on Inhibition of Urease Activity and Fishy Smell in Mackerel (Scomber japonicus) during Refrigerated Storage.

In this study, gamma-irradiated mackerel (Scomber japonicus) meat was stored in a refrigerator for 20 days to examine the physicochemical changes related to fishy smell. The effect of gamma irradiation on the inhibition of the activity of crude urease extracted from Vibrio parahaemolyticus was also evaluated. Increased levels of trimethylamine (TMA) and volatile basic nitrogen (VBN) content, which are the main components causing fishy smell, were significantly reduced by day 20 of storage after gamma irradiation, indicating that freshness was maintained during storage. The ammonia nitrogen contents of 3, 7, 10, and 20 kGy gamma-irradiated groups were significantly decreased by 6.5, 15.2, 17.4, and 23.9%, respectively, compared to non-irradiated groups on day 20 of storage. In addition, urease activity decreased in a gamma irradiation intensity-dependent manner. Volatile organic compounds (VOCs) were measured during the storage of gamma-irradiated mackerel meat. The contents of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavors due to spoilage of fish, were significantly reduced by day 20 of storage. Therefore, gamma irradiation can be considered useful for inhibiting urease activity and reducing fishy smell during fish storage.

Perfusable micro-vascularized 3D tissue array for high-throughput vascular phenotypic screening.

Microfluidic organ-on-a-chip technologies have enabled construction of biomimetic physiologically and pathologically relevant models. This paper describes an injection molded microfluidic platform that utilizes a novel sequential edge-guided patterning method based on spontaneous capillary flow to realize three-dimensional co-culture models and form an array of micro-vascularized tissues (28 per 1 × 2-inch slide format). The MicroVascular Injection-Molded Plastic Array 3D Culture (MV-IMPACT) platform is fabricated by injection molding, resulting in devices that are reliable and easy to use. By patterning hydrogels containing human umbilical endothelial cells and fibroblasts in close proximity and allowing them to form vasculogenic networks, an array of perfusable vascularized micro-tissues can be formed in a highly efficient manner. The high-throughput generation of angiogenic sprouts was quantified and their uniformity was characterized. Due to its compact design (half the size of a 96-well microtiter plate), it requires small amount of reagents and cells per device. In addition, the device design is compatible with a high content imaging machine such as Yokogawa CQ-1. Furthermore, we demonstrated the potential of our platform for high-throughput phenotypic screening by testing the effect of DAPT, a chemical known to affect angiogenesis. The MV-IMPACT represent a significant improvement over our previous PDMS-based devices in terms of molding 3D co-culture conditions at much higher throughput with added reliability and robustness in obtaining vascular micro-tissues and will provide a platform for developing applications in drug screening and development.

Effect of High Hydrostatic Pressure Treatment on Urease Activity and Inhibition of Fishy Smell in Mackerel (Scomber japonicus) during Storage.

In this study, the physicochemical changes related to fishy smell were determined by storing high hydrostatic pressure (HHP)-treated mackerel (Scomber japonicus) meat in a refrigerator for 20 days. The inhibition of crude urease activity from Vibrio parahaemolyticus using HHP treatment was also investigated. The mackerel meat storage experiment demonstrated that production of trimethylamine (TMA) and volatile basic nitrogen (VBN), the main components of fishy smell, was significantly reduced on the 20th day of storage after the HHP treatment compared to the untreated mackerels. The results demonstrated that the increased ammonia nitrogen rates in the 2000, 3000, and 4000 bar, HHP-treated groups decreased by 23.8%, 23.8%, and 31.0%, respectively, compared to the untreated groups. The enzyme activity of crude urease was significantly reduced in the HHP-treated group compared to that in the untreated group. Measurement of the volatile organic compounds (VOCs) in mackerel meat during storage indicated that the content of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to spoilage, were significantly reduced by HHP treatment. Collectively, our results suggested that HHP treatment would be useful for inhibiting the activity of urease, thereby reducing the fishy smells from fish and shellfish.

High-throughput injection molded microfluidic device for single-cell analysis of spatiotemporal dynamics.

Single-cell level analysis of various cellular behaviors has been aided by recent developments in microfluidic technology. Polydimethylsiloxane (PDMS)-based microfluidic devices have been widely used to elucidate cell differentiation and migration under spatiotemporal stimulation. However, microfluidic devices fabricated with PDMS have inherent limitations due to material issues and non-scalable fabrication process. In this study, we designed and fabricated an injection molded microfluidic device that enables real-time chemical profile control. This device is made of polystyrene (PS), engineered with channel dimensions optimized for injection molding to achieve functionality and compatibility with single cell observation. We demonstrated the spatiotemporal dynamics in the device with computational simulation and experiments. In temporal dynamics, we observed extracellular signal-regulated kinase (ERK) activation of PC12 cells by stimulating the cells with growth factors (GFs). Also, we confirmed yes-associated protein (YAP) phase separation of HEK293 cells under stimulation using sorbitol. In spatial dynamics, we observed the migration of NIH 3T3 cells (transfected with Lifeact-GFP) under different spatiotemporal stimulations of PDGF. Using the injection molded plastic devices, we obtained comprehensive data more easily than before while using less time compared to previous PDMS models. This easy-to-use plastic microfluidic device promises to open a new approach for investigating the mechanisms of cell behavior at the single-cell level.

Anchor-IMPACT: A standardized microfluidic platform for high-throughput antiangiogenic drug screening.

In vitro models are becoming more advanced to truly present physiological systems while enabling high-throughput screening and analysis. Organ-on-a-chip devices provide remarkable results through the reconstruction of a three-dimensional (3D) cellular microenvironment although they need to be further developed in terms of multiple liquid patterning principle, material properties, and scalability. Here we present a 3D anchor-based microfluidic injection-molded plastic array culture platform (Anchor-IMPACT) that enables selective, space-intensive patterning of hydrogels using anchor-island for high-throughput angiogenesis evaluation model. Anchor-IMPACT consists of a central channel and an anchor-island, integrating the array into an abbreviated 96-well plate format with a standard microscope slide size. The anchor-island enables selective 3D cell patterning without channel-to-channel contact or any hydrogel injection port using an anchor structure unlike conventional culture compartment. The hydrogel was patterned into defined regions by spontaneous capillary flow under hydrophilic conditions. We configured multiple cell patterning structures to investigate the angiogenic potency of colorectal cancer cells in Anchor-IMPACT and the morphological properties of the angiogenesis induced by the paracrine effect were evaluated. In addition, the efficacy of anticancer drugs against angiogenic sprouts was verified by following dose-dependent responses. Our results indicate that Anchor-IMPACT offers not only a model of high-throughput experimentation but also an advanced 3D cell culture platform and can significantly improve current in vitro models while providing the basis for developing predictive preclinical models for biopharmaceutical applications.

A 3D disease and regeneration model of peripheral nervous system-on-a-chip.

Demyelinating diseases involve loss of myelin sheaths and eventually lead to neurological problems. Unfortunately, the precise mechanisms remain unknown, and there are no effective therapies. To overcome these limitations, a reliable and physiologically relevant in vitro model is required. Here, we present a three-dimensional peripheral nervous system (PNS) microfluidic platform that recapitulates the full spectrum of myelination, demyelination, and remyelination using primary Schwann cells (SCs) and motor neurons (MNs). The platform enables reproducible hydrogel patterning and long-term stable coculture of MNs and SCs over 40 days in vitro based on three distinct design factors. Furthermore, the on-demand detachable substrate allows in-depth biological analysis. We demonstrated the possibility of mimicking segmental demyelination by lysophosphatidylcholine, and recovery of myelin structure by application of two drugs: benzatropine or methylcobalamin. This 3D PNS disease-on-a-chip may serve as a potential platform for understanding the pathophysiology of demyelination and screening drugs for remyelination.

Tumor spheroid-on-a-chip: a standardized microfluidic culture platform for investigating tumor angiogenesis.

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

Development of a functional airway-on-a-chip by 3D cell printing.

We used 3D cell printing to emulate an airway coupled with a naturally-derived blood vessel network in vitro. Decellularized extracellular matrix bioink derived from porcine tracheal mucosa (tmdECM) was used to encapsulate and print endothelial cells and fibroblasts within a designated polycarprolactone (PCL) frame. Providing a niche that emulates conditions in vivo, tmdECM gradually drives endothelial re-orientation, which leads to the formation of a lumen and blood vessel network. A fully-differentiated in vitro airway model was assembled with the printed vascular platform, and collectively reproduced a functional interface between the airway epithelium and the vascular network. The model presented respiratory symptoms including asthmatic airway inflammation and allergen-induced asthma exacerbation in physiological context. Because of the adaptable and automated nature of direct 3D cell printing, we expect that this will have relevance in vivo and high reproducibility for production of high-content platforms for preclinical trials in biomedical research.

The 5-item Alcohol Use Disorders Identification Test (AUDIT-5): an effective brief screening test for problem drinking, alcohol use disorders and alcohol dependence.

AIMS: To identify an optimal brief version of the Alcohol Use Disorders Identification Test (AUDIT) and to evaluate its effectiveness as a screening test for problem drinking (PD), alcohol use disorders (AUD) and alcohol dependence (AD). METHODS: A multicenter trial was conducted from March, 2010, to January, 2012, in 101 normal comparison, 203 risky drinking, 92 alcohol abuse and 101 AD men aged below 65 years of age in a Korean population. For the purposes of screening, risky drinking, alcohol abuse and AD were grouped: all the three grouped as PD and the latter two grouped as AUD. Logistic regression analysis was used to determine the items among the 10-item AUDIT that provided information predictive of PD, AUD and AD. Receiver operating characteristic (ROC) curve analysis was performed to investigate the discrimination ability of the brief versions of AUDIT, 10-item AUDIT and Cut-down, Annoyed, Guilt, Eye-opener as a screen for PD, AUD and AD. Areas under the ROC curve were compared between tests according to the method suggested by Hanley and McNeil. RESULTS: The 5-item AUDIT (AUDIT-5: AUDIT items 2, 4, 5, 9 and 10) was obtained by stepwise multiple regression analyses for each screening. AUDIT-5 exhibited an AUD screening accuracy significantly superior to that of the 10-item AUDIT, but other brief versions of AUDIT and CAGE did not. Furthermore, AUDIT-5 had a high PD and AD screening accuracy equivalent to that of the 10-item AUDIT. CONCLUSION: These results strongly support the usefulness of AUDIT-5 for screening of PD, AUD and AD in clinical settings in Korean male populations.