A long-term storable gel-laden chip composite built in a multi-well plate enablingin situcell encapsulation for high-throughput liver model.

Hydrogels are widely used as scaffold materials for constructingin vitrothree-dimensional microphysiological systems. However, their high sensitivity to various external cues hinders the development of hydrogel-laden, microscale, and high-throughput chips. Here, we have developed a long-term storable gel-laden chip composite built in a multi-well plate, which enablesin situcell encapsulation and facilitates high-throughput analysis. Through optimized chemical crosslinking and freeze-drying method (C/FD), we have achieved a high-quality of gel-laden chip composite with excellent transparency, uniform porosity, and appropriate swelling and mechanical characteristics. Besides collagen, decellularized extracellular matrix with tissue-specific biochemical compound has been applied as chip composite. As a ready-to-use platform,in situcell encapsulation within the gel has been achieved through capillary force generated during gel reswelling. The liver-mimetic chip composite, comprising HepG2 cells or primary hepatocytes, has demonstrated favorable hepatic functionality and high sensitivity in drug testing. The developed fabrication process with improved stability of gels and storability allows chip composites to be stored at a wide range of temperatures for up to 28 d without any deformation, demonstrating off-the-shelf products. Consequently, this provides an exceptionally simple and long-term storable platform that can be utilized for an efficient tissue-specific modeling and various biomedical applications.

Codonopsis lanceolata Extract Restores Smooth Muscle Vasorelaxation in Rat Carotid Arteries Even under High Extracellular K+ Concentrations.

Recent studies showed that Codonopsis lanceolata (CL) has antihypertensive effects. However, to date, no study has examined the effects of CL on vascular tone under a high extracellular K+ concentration ([K+]o). Thus, the present study examined the effect of an extract of Codonopsis lanceolata (ECL) on the vascular tension of rat carotid arteries exposed to high [K+]o. We used myography to investigate the effect of an ECL on the vascular tension of rat carotid arteries exposed to high [K+]o and the underlying mechanism of action. In arteries with intact endothelia, the ECL (250 µg/mL) had no effect on vascular tension in arteries exposed to normal or high [K+]o. In contrast, the ECL significantly increased vasorelaxation in endothelium-impaired arteries exposed to a physiologically normal or high [K+]o compared with control arteries exposed to the same [K+]o conditions in the absence of ECL. This vasorelaxing action was unaffected by a broad-spectrum K+ channel blocker and an ATP-sensitive K+ channel blocker. The ECL significantly inhibited the vasoconstriction induced by Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) but not Ca2+ influx induced via receptor-operated Ca2+ channels or the release of Ca2+ from the sarcoplasmic reticulum in the vascular smooth muscle. In summary, our study reveals that the ECL acts through VDCCs in vascular smooth muscle to promote the recovery of vasorelaxation even in arteries exposed to high [K+]o in the context of endothelial dysfunction and provides further evidence of the vascular-protective effects of ECL.

Ginsenoside Rb1 Reduces Hyper-Vasoconstriction Induced by High Glucose and Endothelial Dysfunction in Rat Aorta.

Acute hyperglycemia induces oxidative damage and inflammation, leading to vascular dysfunction. Ginsenoside Rb1 (Rb1) is a major component of red ginseng with anti-diabetic, anti-oxidant and anti-inflammatory properties. Here, we investigated the beneficial effects and the underlying mechanisms of Rb1 on hypercontraction induced by high glucose (HG) and endothelial dysfunction (ED). The isometric tension of aortic rings was measured by myography. The rings were treated with NG-nitro-L-arginine methyl ester (L-NAME) to induce chemical destruction of the endothelium, and Rb1 was added after HG induction. The agonist-induced vasoconstriction was significantly higher in the aortic rings treated with L-NAME + HG50 than in those treated with HG50 or L-NAME (p = 0.011) alone. Rb1 significantly reduced the hypercontraction in the aortic rings treated with L-NAME + HG50 (p = 0.004). The ATP-sensitive K+ channel (KATP) blocker glibenclamide tended to increase the Rb1-associated reduction in the agonist-induced vasoconstriction in the rings treated with L-NAME + HG50. The effect of Rb1 in the aortic rings treated with L-NAME + HG50 resulted from a decrease in extracellular Ca2+ influx through the receptor-operated Ca2+ channel (ROCC, 10-6-10-4 M CaCl2, p < 0.001; 10-3-2.5 × 10-3 M CaCl2, p = 0.001) and the voltage-gated Ca2+ channel (VGCC, 10-6 M CaCl2, p = 0.003; 10-5-10-2 M CaCl2, p < 0.001), whereas Rb1 did not interfere with Ca2+ release from the sarcoplasmic reticulum. In conclusion, we found that Rb1 reduced hyper-vasoconstriction induced by HG and ED by inhibiting the ROCC and the VGCC, and possibly by activating the KATP in rat aorta. This study provides further evidence that Rb1 could be developed as a therapeutic target for ED in diabetes.

A high-throughput biomimetic bone-on-a-chip platform with artificial intelligence-assisted image analysis for osteoporosis drug testing.

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 ß-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.

Inhibition of tumor progression and M2 microglial polarization by extracellular vesicle-mediated microRNA-124 in a 3D microfluidic glioblastoma microenvironment.

Background: Glioblastoma (GBM) is one of the most aggressive types of brain cancer. GBM progression is closely associated with microglia activation; therefore, understanding the regulation of the crosstalk between human GBM and microglia may help develop effective therapeutic strategies. Elucidation of efficient delivery of microRNA (miRNA) via extracellular vesicles (EVs) and their intracellular communications is required for therapeutic applications in GBM treatment. Methods: We used human GBM cells (U373MG) and human microglia. MiRNA-124 was loaded into HEK293T-derived EVs (miR-124 EVs). Various anti-tumor effects (proliferation, metastasis, chemosensitivity, M1/M2 microglial polarization, and cytokine profile) were investigated in U373MG and microglia. Anti-tumor effect of miR-124 EVs was also investigated in five different patient-derived GBM cell lines (SNU-201, SNU-466, SNU-489, SNU-626, and SNU-1105). A three-dimensional (3D) microfluidic device was used to investigate the interactive microenvironment of the tumor and microglia. Results: MiR-124 EVs showed highly efficient anti-tumor effects both in GBM cells and microglia. The mRNA expression levels of tumor progression and M2 microglial polarization markers were decreased in response to miR-124 EVs. The events were closely related to signal transducer and activator of transcription (STAT) 3 signaling in both GBM and microglia. In 3D microfluidic experiments, both U373MG and microglia migrated to a lesser extent and showed less-elongated morphology in the presence of miR-124 EVs compared to the control. Analyses of changes in cytokine levels in the microfluidic GBM-microglia environment showed that the treatment with miR-124 EVs led to tumor suppression and anti-cancer immunity, thereby recruiting natural killer (NK) cells into the tumor. Conclusions: In this study, we demonstrated that EV-mediated miR-124 delivery exerted synergistic anti-tumor effects by suppressing the growth of human GBM cells and inhibiting M2 microglial polarization. These findings provide new insights toward a better understanding of the GBM microenvironment and provide substantial evidence for the development of potential therapeutic strategies using miRNA-loaded EVs.