Kayoko Hirayama

Cellular building unit integrated with microstrand-shaped bacterial cellulose

In bottom-up tissue engineering, a method to integrate a pathway of nutrition and oxygen into the resulting macroscopic tissue has been highly desired, but yet to be established. This paper presents a cellular building unit made from microstrand-shaped bacterial cellulose (BC microstrand) covered with mammalian cells. The BC microstrands are fabricated by encapsulating Acetobacter xylinum with a calcium alginate hydrogel microtube using a double co-axial microfluidic device. The mechanical strength and porous property of the BC microstrands can be regulated by changing the initial density of the bacteria. By folding or reeling the building unit, we demonstrated the multiple shapes of millimeter-scale cellular constructs such as coiled and ball-of-yarn-shaped structures. Histological analysis of the cellular constructs indicated that the BC microstrand served as a pathway of nutrition and oxygen to feed the cells in the central region. These findings suggest that our approach facilitates creating functional macroscopic tissue used in various fields such as drug screening, wound healing, and plastic surgery.

Purification and Characterization of Termite Endogenous β-1,4-Endoglucanases Produced in Aspergillus oryzae

Although termites are known to have a highly efficient lignocellulose-digesting system, mass production of native endogenous cellulases of termites has failed in Escherchia coli, and in Saccharomyces cerevisiae, and it has not been accomplished. Here we report the successful production, purification, and characterization of two termite endogenous β-1,4-endoglucanases, RsEG and NtEG, from the salivary gland of Reticulitermes speratus and the midgut of Nasutitermes takasagoensis respectively, using Aspergillus oryzae as host. Thin-layer chromatography analysis showed that both enzymes hydrolyzed the β-1,4-cellulosic linkage of cellodextrin into cellobiose and glucose. Kinetic studies indicated that the specific activity and Vmax values of the two enzymes were significantly higher than those of previously reported fungal and bacterial endoglucanases.

Measuring the vibration of cells subjected to ultrasound using a MEMS-based force sensor array

This paper reports on a method to measure vibration occurring on the membrane of a mammalian cell which is subjected to ultrasound. The measurement is based on an array of piezoresistive MEMS force sensors. Experimental results with the fabricated sensors showed a change in frequency response to the ultrasound-induced vibration of NIH3T3 cells adhered to the fabricated sensor.

Biofilms in hydrogel core-shell fibers

This paper describes a hydrogel microfiber encapsulating bacteria using an axisymmetric microfluidic device that creates co-axial laminar flow. Since the encapsulated bacteria proliferate at high density in the core of the microfiber, they easily form biofilms; these films are suggested to be important for the cell viability and high-level bacterial secretion. Here, we succeeded in preparing densely packed bacteria in the hydrogel microfiber, and found they proliferated and formed biofilm. We believe this microfiber will be a useful material in the field of bioreactors and microbial sensors.

Micro pillars with thin hydrophobic layer formed on the side walls to prevent cell protrusion toward side wall

The paper reports on micro pillars with thin fluorocarbon layer formed onto their side walls and the surface of the substrate where the pillars are standing. Since the cell cannot adhere to the fluorocarbon layer, the cultured cells will adhere only to the top of the pillars. Therefore, the traction forces of the cells might be measured without considering their adhering positions. We fabricated micro pillars and formed 60 nm thick CYTOP layer around its side walls and to the surface of silicon substrate. By forming the CYTOP layer around micro pillars, we demonstrated that the numbers of the protruded cells to the gaps around the pillar became a half compared to those without CYTOP.

Micropillar type three-axis force sensor for measurement of cellular force

This paper reports a three-axis force sensor that can measure cellular forces in real time with high sensitivity. The sensor features a photoresist micropillar fabricated on a flexible cross-shaped Si structure. The three dimensional forces acting on the micropillar can be detected from the resistance changes of three piezoresistors designed on the Si structure. Due to the flexibility of the Si beams, a sensing resolution on the order of several nN was obtained for both shear forces and normal force. Moreover, in our sensor design, the sensing beams are covered by a photoresist cap that prevents cells from attaching to the piezoresistors while it maintains the space for the beams and the micropillar to deform. We confirmed that our sensor can detect the normal and shear forces acting on the micropillar caused by an osteosarcoma cell during its detachment from the surrounding walls.

Micropatterning of bacterial cellulose as degradable substrate for cell culture

This paper describes microfabrication of bacterial cellulose membrane. Bacterial cellulose is a nanofibrous cellulosic material produced by the bacteria called Acetobacter xylinum. We micropatterned a bacterial cellulose membrane by utilizing MEMS process. This patterned bacterial cellulose worked as a scaffold for mouse embryonic fibroblast cells: The cells attached and grew on the patterned bacterial cellulose membrane. Moreover, formation of cell cluster was observed by the treatment of cellulose degrading enzyme. We believe that this micropatterned cellulose membrane would be useful as degradable microscaffolds for cell culture.

3D microfluidics formed with hydrogel sacrificial structures

This paper describes a fast method to fabricate 3D microchannel. We use alginate gel tubes to mold PDMS microchannel. Our method does not need any of those complicated processes such as photolithography or bonding. It consists of three simple steps. (1) Arrangement of the hydrogel tubes according to the objective design, (2) Molding the tubes into PDMS, (3) Dissolution and extrusion of the hydrogel tubes after curing process of the PDMS. The curing process can be performed at normal condition (i.e. 75 degC, 1.5 h). The hollow-centered structure of hydrogel tube reduces fluidic resistance when we dissolved Buried hydrogels in microchannel (Figure 2D). Our method allows us to fabricate complex 3D microfluidics which was difficult to fabricate in the conventional methods using 2D molds fabricated by photolithography.

Piezoresistive cantilever integrated microfluidic channel for measuring cellular properties

We propose a microfluidic device that has piezoresistive cantilever on the bottom wall of its microchannel to measure cellular mechanical property. This sensor allows us to measure force applied to the wall of microfluidic channel directly with high sensitivity and high temporal resolution. Moreover, feedback from the sensor can be used to control liquid flow above the cantilever. We demonstrate that our device is able to detect the force acting on the channel bottom wall during the passing-through of mammalian cells by the piezoresistive cantilever.

Formation of cross-shaped Escherichia coli

This paper describes preliminary results of a method to regulate the shapes of a single bacterial cell by confining the cell into a micro chamber. The cell wall of Escherichia coli determines the rod-shape (0.5 μm in width, 2 μm in length) of the bacteria and protects from outer stress. We removed its cell wall by enzyme treatment and confined the cell into cross-shaped microchambers formed on the surface of 2% agar contained the growth media. We believe that our method could contribute to the comprehensive understanding of the shape regulation mechanism of E. coli. This paper focuses on the fabrication of cross-shaped microchambers formed on agar and confinement of the bacteria into the microchambers.