Shintaro Takahashi

Preparation of coculture system with three extracellular matrices using capillary force lithography and layer-by-layer deposition.

Micropatterned cocultures were fabricated with 3 extracellular matrices, hyaluronic acid (HA), fibronectin, and collagen. The feature of the fabrication processes is to avoid the use of potentially cytotoxic materials and utilize capillary force of the solution and interactions between the extracellular matrix components. The coculture system can be used to investigate the effects of heterocellular interactions on cellular fate. Direct heterocellular connections between hepatocytes and fibroblasts were visualized by the transcellular diffusion of fluorescein in this coculture system. The interactions between hepatocytes and fibroblasts were crucial for the maintenance of albumin synthesis by hepatocytes. The coculture system was also beneficial for investigating the effects of cell-cell interactions on the induction of embryonic stem (ES) cell differentiation. In cocultures grown in a sea-island pattern, ES cells formed isolated colonies surrounded by PA6 cells and differentiated into neurons with branched neurites that extended from the colonies. This versatile and biocompatible coculture system could potentially be a powerful tool for investigating cell-cell interaction and for tissue engineering applications.

Processing of nanolitre liquid plugs for microfluidic cell-based assays

Abstract Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.

Flatbed epi relief-contrast cellular monitoring system for stable cell culture

Consistent cell preparation is a fundamental preliminary step for understanding complex cellular mechanisms in various cell-based research fields, including basic cell biology, cancer research, and tissue engineering. However, certain elusive factors, such as cellular de-differentiation and contamination with mycoplasma or other types of cells, have compromised the reproducibility and reliability of cell-based approaches. Here, we propose an epi relief-contrast cellular monitoring system (eRC-CMS) that allows images of cells in a typical culture plate to be acquired, stored, and analysed for daily cell quality control. Due to its full flatbed nature and automated system, cells placed at any location on the stage can be analysed without special attention. Using this system, changes in the size, circularity, and proliferation of endothelial cells in subculture were recorded. Analyses of images of ~9,930,000 individual cells revealed that the growth activity and cell circularity in subcultures were closely correlated with their angiogenic activity in a subsequent hydrogel assay, demonstrating that eRC-CMS is useful for assessing cell quality in advance. We further demonstrated that eRC-CMS was feasible for the imaging of neurite elongation and spheroid formation. This system may provide a robust and versatile approach for daily cell preparation to facilitate reliable and reproducible cell-based studies.