We present a microfluidic device generating three-dimensional (3D) coaxial flow by the addition of a simple hillock to produce an alginate core-shell microcapsule for the efficient formation of a cell spheroid. A hillock tapered at downstream of the two-dimensional focusing channel enables outside flow to enclose the core flow. The aqueous solution in the core flow was focused and surrounded by 1.8% alginate solution to be solidified as a shell. The double-layered coaxial flow (aqueous phase) was broken up into a droplet by the shear flow of oleic acid (oil phase) containing calcium chloride for the polymerization of the alginate shell. The droplet generated from the laminar coaxial flow maintained a double-layer structure and gelation of the alginate solution made a core-shell microcapsule. The shell-thickness of the microcapsule was adjusted from 8–21 μm by the variation of two aqueous flow rates. The inner shape of the shell was almost spherical when the ratio of the water–glycol mixture in the core flow exceeded 20%. The microcapsule was used to form a spheroid of embryonic carcinoma cells (embryoid body; EB) by injecting a cell suspension into the core flow. The cells inside the microcapsule aggregated into an EB within 2 days and the EB formation rate was more than 80% with strong compaction. The microcapsule formed single spherical EBs without small satellite clusters or a bumpy shape as observed in solid microbeads. The microfluidic chip for encapsulation of cells could generate a number of EBs with high rate of EB formation when compared with the conventional hanging drop method. The core-shell microcapsule generated by 3D focusing in the microchannel was effective in forming large number of spherical cell clusters and the encapsulation of cells in the microcapsule is expected to be useful in the transplantation of islet cells or cancer stem cell enrichment.

We present an engineered three-dimensional (3D) in vitro brain microvasculature system embedded within the bulk of a collagen matrix. To create a hydrogel template for the functional brain microvascular structure, we fabricated an array of microchannels made of collagen I using microneedles and a 3D printed frame. By culturing mouse brain endothelial cells (bEnd.3) on the luminal surface of cylindrical collagen microchannels, we reconstructed an array of brain microvasculature in vitro with circular cross-sections. We characterized the barrier function of our brain microvasculature by measuring transendothelial permeability of 40 kDa fluorescein isothiocyanate-dextran (Stoke's radius of ∼4.5 nm), based on an analytical model. The transendothelial permeability decreased significantly over 3 weeks of culture. We also present the disruption of the barrier function with a hyperosmotic mannitol as well as a subsequent recovery over 4 days. Our brain microvasculature model in vitro, consisting of system-in-hydrogel combined with the widely emerging 3D printing technique, can serve as a useful tool not only for fundamental studies associated with blood-brain barrier in physiological and pathological settings but also for pharmaceutical applications.

In this paper, we propose a serial dilution microfluidic chip which is able to generate logarithmic or linear step-wise concentrations. These concentrations were generated via adjustments in the flow rate of two converging fluids at the channel junctions of the ladder network. The desired dilution ratios are almost independent of the flow rate or diffusion length of molecules, as the dilution device is influenced only by the ratio of volumetric flow rates. Given a set of necessary dilution ratios, whether linear or logarithmic, a serial dilution chip can be constructed via the modification of a microfluidic resistance network. The design principle was suggested and both the logarithmic and linear dilution chips were fabricated in order to verify their performance in accordance with the fluorescence intensity. The diluted concentrations of a fluorescein solution in the microfluidic device evidenced relatively high linearity, and the cytotoxicity test of MCF-7 breast cancer cellsvia the logarithmic dilution chip was generally consistent with the results generated with manual dilution.

EVs/exosomes are considered as the next generation of biomarkers, including for liquid biopsies. Consequently, the quantification of EVs/exosomes is crucial for facilitating EV/exosome research and applications. Paper-based enzyme-linked immunosorbent assay (p-ELISA) is a portable diagnostic system with low cost that is simple and easy to use; however, it shows low sensitivity and linearity. In this study, we develop p-ELISA for targeting EVs/exosomes by using streptavidin agarose resin-based immobilization (SARBI). This method reduces assay preparation times, provides strong binding, and retains good sensitivity and linearity. The time required for the total assay, including preparation steps and surface immobilization, was shortened to ∼2 h. We evaluated SARBI p-ELISA systems with/without CD63 capture Ab and then with fetal bovine serum (FBS) and EVs/exosome-depleted fetal bovine serum (dFBS). The results provide evidence supporting the selective capture ability of SARBI p-ELISA. We obtain semiquantitative p-ELISA results using an exosome standard (ES) and human serum (HS), with R² values of 0.95 and 0.92, respectively.

This paper proposes a new cytotoxicity assay in a microfluidic device with microwells and a distributive microfluidic channel network for the formation of cancer cell spheroids. The assay can generate rapid and uniform cell clusters in microwells and test in situ cytotoxicity of anticancer drugs including sequential drug treatments, long term culture of spheroids and cell viability assays. Inlet ports are connected to the microwells by a hydraulic resistance network. This uniform distribution of cell suspensions results in regular spheroid dimensions. Injected cancer cells were trapped in microwells, and aggregated into tumor spheroids within 3 days. A cytotoxicity test of the spheroids in microwells was subsequently processed in the same device without the extraction of cells. The in situ cytotoxicity assay of tumor spheroids in microwells was comparable with the MTT assay on hanging drop spheroids using a conventional 96-well plate. It was observed that the inhibition rate of the spheroids was less than that in the 2D culture dish and the effect on tumor spheroids was different depending on the anticancer drug. This device could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids, offering more information than the conventional 2D culture plate.