Minoru Seki

Microgap channels, created by sandwiching a polymeric sheet with thermally-nanoimprinted microcone arrays between two flat plates, enable selective and highly efficient immunocapture of cancer cells in blood samples.

A multiscale microfluidic system was developed that connects porous PDMS microchambers and microfluidic channels to generate a vertical flow of medium for spheroid culture.

Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.

Collagen-based hydrogels are widely used for three-dimensional (3D) culture of mammalian cells because of their high cell-activating characteristis. However, techniques for preparing of cell-embedding collagen hydrogels with micrometer-size precision in perfusable, microfluidic devices have not been fully developed. In this study, we propose a facile strategy enabling microfabrication of collagen hydrogels in microfluidic devices. We used phosphate particle-embedding polydimethylsiloxane (PP-PDMS) as a gelation agent, which neutralizes the acidic collagen soltuion. The collagen solution near the surface of the PP-PDMS is selectively gelled. We fabricated micropatterns and tubular structures made of collagen hydrogel, both of which were used for perfusion culture of mammalian cells encapsulated in the hydrogel matrix. The presented approach would be applicable to various types of cell culture experiments.

Although many types of technologies for hydrogel-based cell cultivation have recently been developed, strategies to integrate cell-adhesive micrometer-sized supports with bulk-scale hydrogel platforms have not been fully established. Here, we present a highly unique approach to produce cell-adhesive, protein-based microfibers assisted by the sacrificial template of alginate; we applied these fibers as microengineered scaffolds for hydrogel-based cell encapsulation. Two types of microfluidic devices were designed and fabricated: a single-layered device for producing relatively thick (Φ of 10−60 μm) alginate–protein composite fibers with a uniform cross-sectional morphology and a four-layered device for preparing thinner (Φ of ∼4 μm) ones through the formation of patterned microfibers with eight distinct alginate–protein composite regions. Following chemical cross-linking of protein molecules and the subsequent removal of the sacrificial alginate from the double-network matrices, microfibers composed only of cross-linked proteins were obtained. We used gelatin, albumin, and hemoglobin as the protein material, and the gelatin-based cell-adhesive fibers were further encapsulated in hydrogels together with the mammalian cells. We clarified that the thinner fibers were especially effective in promoting cell proliferation, and the shape of the constructs was maintained even after removing the hydrogel matrices. The presented approach offers cells with biocompatible solid supports that enhance cell adhesion and proliferation, paving the way for the next generation of techniques for tissue engineering and multicellular organoid formation.

Visualization and quantification of intracellular molecules of mammalian cells are crucial steps in clinical diagnosis, drug development, and basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium and have limited reproducibility and recovery efficiency. Here we present a microfluidic cell processor that can perform four-step exchange of carrier medium, simply by introducing a cell suspension and fluid reagents into the device. The reaction time period for each reaction step, including fixation, membrane permeabilization, and staining, was tunable in the range of 2 to 15 min by adjusting the volume of the reaction tube connecting the neighboring exchanger modules. We double-stained the cell nucleus and cytoskeleton (F-actin) using the presented device with an overall reaction period of ~30 min, achieving a high recovery ratio and high staining efficiency. Additionally, intracellular cytokine (IL-2) was visualized for T cells to demonstrate the feasibility of the device as a pretreatment system for downstream flow-cytometric analysis. The presented approach would facilitate the development of labor-less, automated microfluidic systems that integrate cell processing and analysis operations, and would pave a new path to high-throughput biological experiments.

Lateral flow immunoassay devices have revolutionized the style of on-site disease detection and point-of-care testing in the past few decades. The surface nanotopography of a solid substrate is a dominant parameter in the efficiency of antibody immobilization, but precise control over surface roughness has not been fully investigated. Here we presented lateral flow immunoassay platforms with nanometer-scale surface roughness reproducibly engineered using thermal nano-imprinting lithography, and investigated the effects of surface nanotopography on immunoadsorption and immunoassay performance. We fabricated three types of imprinted polycarbonate sheets with microcone array structures having different degrees of surface roughness using three types of molds fabricated by micromachining or laser ablation. The structures fabricated by laser-ablated nickel mold exhibited numerous bumps measuring several tens of nanometers, which enhanced antibody adsorption. We performed sandwich immunoassays of C-reactive protein in serum samples and achieved highly sensitive detection with a detection limit of ~0.01 µg mL-1 and a broad dynamic range. The present results provide useful information on the remarkable effect of nano-engineered surfaces on biomolecule adsorption, and the platforms presented here will widen the applicability and versatility of lateral flow immunoassay devices.

We herein propose a new hydrodynamic mechanism of particle separation using dual-depth, lattice-patterned asymmetric microchannel networks. This mechanism utilizes three-dimensional (3D) laminar flow profiles formed at intersections of lattice channels. Large particles, primarily flowing near the bottom surface, frequently enter the shallower channels (separation channels), whereas smaller particles flowing near the microchannel ceiling primarily flow along the deeper channels (main channels). Consequently, size-based continuous particle separation was achieved in the lateral direction in the lattice area. We confirmed that the depth of the main channel was a critical factor dominating the particle separation efficiencies, and the combination of 15-μm-deep separation channels and 40-μm-deep main channels demonstrated the good separation ability for 3–10-μm particles. We prepared several types of microchannels and successfully tuned the particle separation size. Furthermore, the input position of the particle suspension was controlled by adjusting the input flow rates and/or using a Y-shaped inlet connector that resulted in a significant improvement in the separation precision. The presented concept is a good example of a new type of microfluidic particle separation mechanism using 3D flows and may potentially be applicable to the sorting of various types of micrometer-sized objects, including living cells and synthetic microparticles.

A numbering-up strategy of hydrodynamic filters was presented to dramatically increase the throughput of cell/particle sorting up to ∼15 mL min ⁻¹ .