Semibulk RNA-seq analysis as a convenient method for measuring gene expression statuses in a local cellular environment.

When biologically interpretation of the data obtained from the single-cell RNA sequencing (scRNA-seq) analysis is attempted, additional information on the location of the single cells, behavior of the surrounding cells, and the microenvironment they generate, would be very important. We developed an inexpensive, high throughput application while preserving spatial organization, named "semibulk RNA-seq" (sbRNA-seq). We utilized a microfluidic device specifically designed for the experiments to encapsulate both a barcoded bead and a cell aggregate (a semibulk) into a single droplet. Using sbRNA-seq, we firstly analyzed mouse kidney specimens. In the mouse model, we could associate the pathological information with the gene expression information. We validated the results using spatial transcriptome analysis and found them highly consistent. When we applied the sbRNA-seq analysis to the human breast cancer specimens, we identified spatial interactions between a particular population of immune cells and that of cancer-associated fibroblast cells, which were not precisely represented solely by the single-cell analysis. Semibulk analysis may provide a convenient and versatile method, compared to a standard spatial transcriptome sequencing platform, to associate spatial information with transcriptome information.

Rapid Prototyping of a Nanoparticle Concentrator Using a Hydrogel Molding Method.

Nanoparticle (NP) concentration is crucial for liquid biopsies and analysis, and various NP concentrators (NPCs) have been developed. Methods using ion concentration polarization (ICP), an electrochemical phenomenon based on NPCs consisting of microchannels, have attracted attention because samples can be non-invasively concentrated using devices with simple structures. The fabrication of such NPCs is limited by the need for lithography, requiring special equipment and time. To overcome this, we reported a rapid prototyping method for NPCs by extending the previously developed hydrogel molding method, a microchannel fabrication method using hydrogel as a mold. With this, we fabricated NPCs with both straight and branched channels, typical NPC configurations. The generation of ICP was verified, and an NP concentration test was performed using dispersions of negatively and positively charged NPs. In the straight-channel NPC, negatively and positively charged NPs were concentrated >50-fold and >25-fold the original concentration, respectively. To our knowledge, this is the first report of NP concentration via ICP in a straight-channel NPC. Using a branched-channel NPC, maximum concentration rates of 2.0-fold and 1.7-fold were obtained with negatively and positively charged NPs, respectively, similar to those obtained with NPCs fabricated through conventional lithography. This rapid prototyping method is expected to promote the development of NPCs for liquid biopsy and analysis.

One-to-one encapsulation based on alternating droplet generation.

This paper reports the preparation of encapsulated particles as models of cells using an alternating droplet generation encapsulation method in which the number of particles in a droplet is controlled by a microchannel to achieve one-to-one encapsulation. Using a microchannel in which wettability is treated locally, the fluorescent particles used as models of cells were successfully encapsulated in uniform water-in-oil-in-water (W/O/W) emulsion droplets. Furthermore, 20% of the particle-containing droplets contained one particle. Additionally, when a surfactant with the appropriate properties was used, the fluorescent particles within each inner aqueous droplet were enclosed in the merged droplet by spontaneous droplet coalescence. This one-to-one encapsulation method based on alternating droplet generation could be used for a variety of applications, such as high-throughput single-cell assays, gene transfection into cells or one-to-one cell fusion.

Fabrication of Microfluidic Valves Using a Hydrogel Molding Method.

In this paper, a method for fabricating a microfluidic valve made of polydimethylsiloxane (PDMS) using a rapid prototyping method for microchannels through hydrogel cast molding is discussed. Currently, the valves in microchannels play an important role in various microfluidic devices. The technology to prototype microfluidic valves rapidly is actively being developed. For the rapid prototyping of PDMS microchannels, a method that uses a hydrogel as the casting mold has been recently developed. This technique can be used to prepare a three-dimensional structure through simple and uncomplicated methods. In this study, we were able to fabricate microfluidic valves easily using this rapid prototyping method that utilizes hydrogel cast molding. In addition, we confirmed that the valve displacement could be predicted within a range of constant pressures. Moreover, because microfluidic valves fabricated using this method can be directly observed from a cross-sectional direction, we anticipate that this technology will significantly contribute to clarifying fluid behavior and other phenomena in microchannels and microfluidic valves with complex structures.

Hyper alginate gel microbead formation by molecular diffusion at the hydrogel/droplet interface.

We report a simple method for forming monodispersed, uniformly shaped gel microbeads with precisely controlled sizes. The basis of our method is the placement of monodispersed sodium alginate droplets, formed by a microfluidic device, on an agarose slab gel containing a high-osmotic-pressure gelation agent (CaCl(2) aq.): (1) the droplets are cross-linked (gelated) due to the diffusion of the gelation agent from the agarose slab gel to the sodium alginate droplets and (2) the droplets simultaneously shrink to a fraction of their original size (<100 µm in diameter) due to the diffusion of water molecules from the sodium alginate droplets to the agarose slab gel. We verified the mass transfer mechanism between the droplet and the agarose slab gel. This method circumvents the limitations of gel microbead formation, such as the need to prepare microchannels of various sizes, microchannel clogging, and the deformation of the produced gel microbeads.

A lithography-free procedure for fabricating three-dimensional microchannels using hydrogel molds.

We present a lithography-free procedure for fabricating intrinsically three-dimensional smooth-walled microchannels within poly(dimethylsiloxane) (PDMS) elastomer using hydrogel molds. In the fabrication process, small pieces of agarose gel ("wires" or "chips") are embedded in uncured PDMS composite, arranged in the shape of the desired microchannels, and used as molds to form the microchannels. The point of the process is that molds for creating junctions of microchannels such as T-junctions or cross-junctions can be robustly formed by simply grafting gel wires in uncured PDMS composite without using adhesive agents. The technical advantage of this method is that three-dimensional microstructures such as microchannels with circular cross sections, three-dimensionally arranged junctions or interchanges of microchannels can be flexibly designed and fabricated with a straightforward procedure without the need for any specialized equipment or layer-by-layer assemblage processes. This method provides a low-cost, green procedure for fabricating microfluidic devices and promises to make microfluidic processes more accessible and easy to implement in a variety of scientific fields.

Droplet handling.

When quantitative analysis or quantitative chemical synthesis is performed using a micrototal analysis system (microTAS), the technologies for precise metering, transporting, and mixing of droplets are required. In this chapter, several technologies for the handling of droplets are described. For metering, dispensing and transporting of droplets, pneumatic and electrokinetic forces are used. Separation of cells and particles is also performed by electrical operation. Other handling technique, such as ultrasonic or centrifugal force applications, are also reviewed. Robotic synthesis devices or high throughput screening devices are promising applications for these technologies.

Controlled formulation of monodisperse double emulsions in a multiple-phase microfluidic system.

This paper gives an overview of our recent work on the use of microfluidic devices to formulate double emulsions. Key issues in the controlled encapsulation of highly monodisperse drops include: (a) regular periodicity in the formation of micro droplets due to the interplay between viscous shearing and interfacial tension in low Reynolds number streams; (b) serially connected hydrophobic and hydrophilic microchannels to form aqueous and organic drops consecutively. Water-in-oil-in-water emulsions and oil-in-water-in-oil emulsions can both be produced by reversing the order of hydrophobic and hydrophilic junctions. Alternating formation of aqueous droplets at a cross junction has enabled the production of organic droplets that encase two aqueous droplets of differing compositions.

Controlled production of monodisperse double emulsions by two-step droplet breakup in microfluidic devices.

A microfluidic device having both hydrophobic and hydrophilic components is exploited for production of multiple-phase emulsions. For producing water-in-oil-in-water (W/O/W) dispersions, aqueous droplets ruptured at the upstream hydrophobic junction are enclosed within organic droplets formed at the downstream hydrophilic junction. Droplets produced at each junction could have narrow size distributions with coefficients of variation in diameter of less than 3%. Control of the flow conditions produces variations in internal/external droplet sizes and in the internal droplet number. Both W/O/W emulsions (with two types of internal droplets) and oil-in-water-in-oil emulsions were prepared by varying geometry and wettability in microchannels.

Chemical reactions in microdroplets by electrostatic manipulation of droplets in liquid media.

A microchemical reaction method involving microdroplets is proposed. Microdroplets are formed in a chemically stable medium on electric panel devices. These devices are substrates which have electrode arrays or electrode dots, and its surfaces are coated by an insulating film (such as Teflon or polypropylene) to prevent discharge and electrolysis of solutions. Microdroplets can be separately manipulated by a traveling electric field, which arises on applying a sequential voltage to the electrodes. Droplets moved smoothly at 1 Hz and voltage 400 V(0-p). Reagents were then put in droplets that were collided and coalesced, resulting in chemical reactions that included alkalization of phenolphthalein and the luciferin-luciferase reaction.

Droplet formation in a microchannel network.

A method is given for generating droplets in a microchannel network. With oil as the continuous phase and water as the dispersed phase, pico/nanoliter-sized water droplets can be generated in a continuous phase flow at a -junction. The channel for the dispersed phase is 100 microm wide and 100 microm deep, whereas the channel for the continuous phase is 500 microm wide and 100 microm deep. For given experimental parameters, regular-sized droplets are reproducibly formed at a uniform speed. The diameter of these droplets is controllable in the range from 100-380 microm as the flow velocity of the continuous phase is varied from 0.01 m s(-1) to 0.15 m s(-1).