Yukiko Tsuda

Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering.

Reconstructing a vascular network is a common task for three-dimensional (3-D) tissue engineering. Three-dimensional stratified tissues were created by stacking cell sheets, and the co-culture with endothelial cells (ECs) in the tissues was found to lead to in vitro pre-vascular network formation and promoted in vivo neovascularization after their transplantation. In this study, to clarify the effect of tissue fabrication process on a pre-vascular network formation, human origin ECs were introduced into the stratified tissue in several different ways, and the behavior of ECs in various positions of the 3-D tissue were compared each other. Human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), and their mixture were harvested as an intact cell sheet from temperature-responsive culture dish at low-temperature (<20 degrees C). Single mono-culture EC sheet was stacked with two NHDF-sheets in different orders, and 3 co-cultured cell sheets were layered by a cell sheet collecting device. Morphological analyses revealed that pre-vascular networks composing of HUVECs were formed in all the triple layer constructs. Confocal microscope observation showed that the pre-vascular networks formed tube structures like a native microvasculature. These data indicate that the primary EC positioning in 3-D tissues may be critical for vascular formation.

Monodisperse cell-encapsulating peptide microgel beads for 3D cell culture.

This paper describes a method to produce monodisperse cell-encapsulating microgel beads composed of a self-assembling peptide gel for three-dimensional (3D) cell culture. We used a 3D microfluidic axisymmetric flow-focusing device with an external gelation method. The finely powdered salts were dispersed into a continuous phase, and the salts induced the gelation when in contact with the peptide solution. Over 93% of the cells survived after the encapsulation, and the cells migrated and grew within the gels. Applications of our cell-encapsulating beads include bead-based cell assays in drug testing and engineering tissue constructs.

A neurospheroid network-stamping method for neural transplantation to the brain.

Neural transplantation therapy using neural stem cells has received as potential treatments for neurodegenerative diseases. Indeed, this therapy is thought to be effective for replacement of degenerating neurons in restricted anatomical region. However, because injected neural stem cells integrate randomly into the host neural network, another approach is needed to establish a neural pathway between selective areas of the brain or treat widespread degeneration across multiple brain regions. One of the promising approaches might be a therapy using pre-made neural network in vitro by the tissue engineering technique. In this study, we engineered a three-dimensional (3D) tissue with a neuronal network that can be easily manipulated and transplanted onto the host brain tissue in vivo. A polydimethylsiloxane microchamber array facilitated the formation of multiple neurospheroids, which in turn interconnected via neuronal processes to form a centimeter-sized neurospheroid network (NSN). The NSN was transferable onto the cortical surface of the brain without damage of the neuronal network. After transfer onto the cortical tissue, the NSN showed neural activity for more than 8 days. Moreover, neurons of the transplanted NSN extended their axons into the host cortical tissue and established synaptic connections with host neurons. Our findings suggest that this method could lay the foundation for treating severe degenerative brain disease.

Core-shell gel wires for the construction of large area heterogeneous structures with biomaterials

This paper describes core-shell hydrogel wires for constructing 3D heterogeneous hydrogel microstructures containing biomaterials. A core hydrogel layer contains biomaterials such as cells, and a shell hydrogel layer covers the core to realize a mechanically-robust hydrogel wire. Using this core-shell gel wires, we demonstrate a woven sheet with heterogeneous gel wires by using our stereolithographically-made weaving machine. We also fabricate a reeled-up tube-like cell-containing structure by a glass tube spindle, inspired by tools from fiber industry. We show that our core-shell gel wires and weaving methods are powerful approach to arrange heterogeneous biomaterials in three dimensions.

Transplantation of a neurospheroid network onto the rat brain

This paper describes a method to transplant neurospheroid network onto the rat brain. We first patterned the uniform sized neurospheroids on the PDMS microchamber, and cultured for 1–2 weeks. After 2-week culture, the neurospheroids tightly connected each other with extending their neuronal processes (e.g. dendrite, axons) and formed neuronal network. We realized that the neurospheroid network can be transferred from the PDMS microchamber onto the glass plate or the rat brain. These transferred neurospheroid network had also neuronal activities. We believe that this transfer method of neurospheroid network should be a useful model for the tissue engineering and medical transplantation.

Size-Controlled Islet-Cell Spheroids for Geometric Analysis of Insulin Secretion

We developed a method to prepare size- and shape-controlled islet-cell spheroids. We cultured ß-cell line (MIN6-m9) in PDMS-microwells and enabled to construct their spheroids with the various sizes corresponding to those of the microwells. We demonstrated the geometric analysis of insulin secretion of the cells in the spheroids. We found that these spheroids respond to glucose and subsequently secrete insulin. Also, immunocytochemistry and the real-time live cell imaging of the Ca2+ oscillation revealed for the first time that the only cells approximately 36 ¿m apart from the periphery showed higher insulin secretion in the spheroid. This result indicates that our MEMS technique controlling the size and the shape of the spheroids is useful to construct islet-like tissue that functions efficiently and is applicable to transplantation therapy targeted to insulin dependent diabetes mellitus.

A micro-engineered three-dimensional neuronal network

Developing neuronal networks generate various patterns of synchronized oscillatory activity that is believed to participate in circuit refinement. In the immature cortex and hippocampus, two major forms of the synchronized activity have been well documented, i.e., early network oscillations and giant depolarizing potentials. However, the neuronal activity patterns in other memory-relevant networks such as the amygdala still remain unclear. Using a functional multineuron calcium imaging technique that monitors the activity of thousands of cells, we revealed that synchronized waves propagated from the cortex to the lateral/basolateral amygdala in acute horizontal slice of developing rat. This cortico-amygdalar waves occasionally activated the central amygdala and terminated within the amygdala. The origin of the synchronized waves was likely located in the entorhinal and perirhinal cortex, which correspond to anatomically important networks that connect the hippocampus and amygdala with large parts of the neocortex. To the best of our knowledge, these findings demonstrate the first report showing the early amygdalar network oscillations.

Implantable fluorescent hydrogel for continous blood glucose monitoring

We present a continuous glucose monitoring sensors using glucose-sensitive fluorescent dyes. We prepared microgels by copolymerizing acrylamide with the glucose sensitive fluorescent dyes. This fluorescent gel shows a high quantifiability and rapid response in the physiological glucose concentration range of 62.5 to 1000 mg/dL. Our in vivo study revealed that fluorescent intensity of the gels changed in response to in vivo blood-glucose levels. We expect this method to be useful in the development of implantable continuous monitoring systems based on fluorescent gels.

A selective release method using electrolytically generated bubbles for cell array applications

This paper describes a method of selective release of particles/cells by bubbles generated by electrolysis. The particles/cells trapped into microchambers that were produced with SU-8 on the top of an electrode of indium tin oxide (ITO) patterned on glass. The bubbles of oxygen and chlorine (or hydrogen) were selectively generated in a target chamber by applying voltage through the electrodes. We successfully released a microbead (100 µm in diameter) confined in the microchamber. Since this method is gentle enough to maintain the cellular activity, we believe that this method will be useful for quantitative analysis of cells in a microarray.