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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.

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In this paper, we propose a generalized serial dilution module for universal microfluidic concentration gradient generators including N cascaded-mixing stages in a stepwise manner. Desired concentrations were generated by means of controlled volumetric mixing ratios of two merging solutions in each stage. The flow rates were adjusted by controlling channel length, which is proportional to fluidic resistance in each channel. A generalized mathematical model for generating any complex concentration and output flow rate gradients is presented based on the fact that there is an analogy between microfluidic circuits and electrical circuits. The pressure drop corresponds to a voltage drop, the flow rate to an electrical current, and the flow resistance to an electrical resistance. A simple equivalent electrical circuit model was generalized, and in the model each channel segment was represented by an electrical resistance. As a result of the mathematical modelling, the only variable parameter in the generalized serial dilution module was the channel length. By the use of the generalized serial dilution module with N = 4, three types of microfluidic gradient generators for linear, logarithmic and Gaussian gradients were successfully designed and tested. The proposed strategy is capable of generating universal monotonic gradients with a single module or arbitrary gradients with multiple modules ranging from linear to complex non-linear shapes of concentration gradients as well as arbitrary output flow rate gradients in a stepwise manner. The simple universal gradient generation technology using the generalized serial dilution module will find widespread use in the greater chemical and biological community, and address many challenges of gradient-dependent phenomena.

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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.

An innovative polymer lab-on-a-chip (LOC) for reverse transcription (RT)-polymerase chain reaction (PCR) has been designed, fabricated, and characterized for point-of-care testing (POCT) clinical diagnostics. In addition, a portable analyzer that consists of a non-contact infrared (IR) based temperature control system for RT-PCR process and an optical detection system for on-chip detection, has also been developed and used to monitor the RT-PCR LOC. The newly developed LOC and analyzer have been interfaced and optimized for performing RT-PCR procedures and chemiluminescence assays in sequence. As a clinical diagnostic application, human immunodeficiency virus (HIV) for the early diagnosis of acquired immune deficiency syndrome (AIDS) has been successfully detected and analyzed using the newly developed LOC and analyzer, where the primer sets for p24 and gp120 were used as the makers for HIV. The developed polymer LOC and analyzer for RT-PCR can be used for POCT for the analysis of HIV with the on-chip RT-PCR and chemiluminescence assays in shorter than one hour with minimized cross-contamination.