Serial Cultivation of an MSC-Like Cell Line with Enzyme-Free Passaging Using a Microporous Titanium Scaffold.

In vitro studies on adherent cells require a process of passage to dissociate the cells from the culture substrate using enzymes or other chemical agents to maintain cellular activity. However, these proteolytic enzymes have a negative influence on the viability and phenotype of cells. The mesenchymal stem cell (MSC)-like cell line, C3H10T1/2, adhered, migrated, and proliferated to the same extent on newly designed microporous titanium (Ti) membrane and conventional culture dish, and spontaneous transfer to another substrate without enzymatic or chemical dissociation was achieved. The present study pierced a 10 µm-thick pure Ti sheet with 25 µm square holes at 75 µm intervals to create a dense porous structure with biomimetic topography. The pathway of machined holes allowed the cells to access both sides of the membrane frequently. In a culture with Ti membranes stacked above- and below-seeded cells, cell migration between the neighboring membranes was confirmed using the through-holes of the membrane and contact between the membranes as migration routes. Furthermore, the cells on each membrane migrated onto the conventional culture vessel. Therefore, a cell culture system with enzyme-free passaging was developed.

A cell culture device equipped with a micro-needle electrode array fabricated using backside exposure mold and resin casting.

A cell culture device equipped with a micro-needle electrode array was fabricated for the signal analysis of cell spheroids, cell masses, and cell sheets. For the analysis, sharp needle electrodes with a high aspect ratio for facilitating easy penetration into the cell mass and a small pitch for fine spatial resolution were required. Microelectromechanical systems (MEMS) technology is one of the common solutions for the fabrication of devices. However, an additional process, such as anisotropic etching or electro-polishing, is required for fabricating sharp needles. Tapered needles were fabricated using backside exposure for coating a layer of thick resist film on a glass substrate. The incident beam from mask apertures were diffracted and attenuated in the medium, resulting in tapered intensity distribution. A needle-like shape was obtained after performing resist development without using additional MEMS process. In this study, the theoretical analysis of optical intensity distribution and design and fabrication process of the device were described. Finally, the effectiveness of the device was evaluated by adding cultured cell mass on the needle array. Signals with spikes and fluctuations were observed in the electrode covered with cell mass, whereas only noise was observed on the non-covered electrode, demonstrating the signal pick-up ability of the device during cell culture.

Capillary-assisted fabrication of biconcave polymeric microlenses from microfluidic ternary emulsion droplets.

In this study, a simple capillary-based approach for producing biconcave polymeric microlenses with uniform size and shape from ternary emulsion droplets is presented. Monodisperse ternary emulsion droplets (0.6-4.0 nL) are produced which contain a photocurable segment of an acrylate monomer and two non-curable segments of silicone oil (SO) by using a microfluidic sheath-flowing droplet generator on a glass chip. The curvature radius of the interfaces separating the droplet segments, as well as the droplet size, and production rate can be flexibly varied by changing the flow conditions of the organic and aqueous phases. Subsequently, off-chip suspension photopolymerization yields non-spherical polymeric microparticles with two spherical concave surfaces templated by two SO segments at random positions. By ultraviolet light irradiation of ternary droplets with two SO segments trapped by the interior wall of a cylindrical microcapillary (internal diameter: 130 µm), biconcave microlenses can be produced with two spherical concave surfaces with a common lens axis. The produced lenses are suitable for use as optical diverging lenses.

High-volume production of single and compound emulsions in a microfluidic parallelization arrangement coupled with coaxial annular world-to-chip interfaces.

This study describes a microfluidic platform with coaxial annular world-to-chip interfaces for high-throughput production of single and compound emulsion droplets, having controlled sizes and internal compositions. The production module consists of two distinct elements: a planar square chip on which many copies of a microfluidic droplet generator (MFDG) are arranged circularly, and a cubic supporting module with coaxial annular channels for supplying fluids evenly to the inlets of the mounted chip, assembled from blocks with cylinders and holes. Three-dimensional flow was simulated to evaluate the distribution of flow velocity in the coaxial multiple annular channels. By coupling a 1.5 cm × 1.5 cm microfluidic chip with parallelized 144 MFDGs and a supporting module with two annular channels, for example, we could produce simple oil-in-water (O/W) emulsion droplets having a mean diameter of 90.7 µm and a coefficient of variation (CV) of 2.2% at a throughput of 180.0 mL h(-1). Furthermore, we successfully demonstrated high-throughput production of Janus droplets, double emulsions and triple emulsions, by coupling 1.5 cm × 1.5 cm - 4.5 cm × 4.5 cm microfluidic chips with parallelized 32-128 MFDGs of various geometries and supporting modules with 3-4 annular channels.

Fabrication of core-shell structured nanoparticle layer substrate for excitation of localized surface plasmon resonance and its optical response for DNA in aqueous conditions.

LSPR from nanostructured noble metals such as gold and silver offers great potential for biosensing applications. In this study, a core-shell structured nanoparticle layer substrate was fabricated and the localized surface plasmon resonance (LSPR) optical characteristics were investigated for DNA in aqueous conditions. Factors such as DNA length dependence, concentration dependence, and the monitoring of DNA aspects (ssDNA or dsDNA) were measured. Different lengths and concentrations of DNA solutions were introduced onto the surface of the substrate and the changes in the LSPR optical characteristics were measured. In addition, to monitor the changes in LSPR optical characteristics for different DNA aspects, a DNA solutions denatured by means of heat or alkali were introduced onto the surface, after which optical characterization of the core-shell structured nanoparticle substrate was carried out. With this core-shell structured nanoparticle layer for the excitation of LSPR, the dependence upon specific DNA conditions (length, concentration, and aspect) could be monitored. In particular, the core-shell structured nanoparticle layer substrate could detect DNA of length 100-5000 bp and 400-bp DNA at a concentration of 4.08 ng mL(-1) (1 x 10(7) DNA molecules mL(-1)). Furthermore, the changes in LSPR optical characteristics with DNA aspect could be monitored. Thus, LSPR-based optical detection using a core-shell structured nanoparticle layer substrate can be used to determine the kinetics of biomolecular interactions in a wide range of practical applications such as medicine, drug delivery, and food control.

Stimuli-responsive hydrogel-silver nanoparticles composite for development of localized surface plasmon resonance-based optical biosensor.

In this paper, the development of a localized surface plasmon resonance (LSPR)-based optical enzyme biosensor using stimuli-responsive hydrogel-silver nanoparticles composite is described. This optical enzyme biosensor was constructed by immobilizing glucose oxidase (GOx) into the stimuli-responsive hydrogel. When a sample solution such as glucose was applied to the surface of this optical enzyme biosensor, the interparticle distances of the silver nanoparticles present in the stimuli-responsive hydrogel were increased, and thus the absorbance strength of the LSPR was decreased. Furthermore, hydrogen peroxide, which was produced by the enzymatic reaction, induced the degradation of highly clustered silver nanoparticles by the decomposition of hydrogen peroxide. Hence, a drastic LSPR absorbance change, which depends on the glucose concentrations, could be observed. On the basis of the abovementioned mechanism, the characterization of the LSPR-based optical enzyme biosensor was carried out. It was found that the LSPR-based optical enzyme biosensor could be used to specifically determine glucose concentrations. Furthermore, the detection limit of this biosensor was 10 pM. Therefore, this LSPR-based optical enzyme biosensor has the potential to be applied in cost-effective, highly simplified, and highly sensitive test kits for medical applications.