Yukiko T. Matsunaga

Metre-long cell-laden microfibres exhibit tissue morphologies and functions

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

Molding Cell Beads for Rapid Construction of Macroscopic 3D Tissue Architecture

A microfluidic system was used to prepare a large number of size‐controlled collagen gel beads to form microtissue units, “cell beads”, as tissue building blocks. By stacking cell beads into a doll‐shaped silicone chamber, millimeter‐thick tissue with uniform cell dens...

Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose monitoring.

Fluorescent microbeads hold great promise for in vivo continuous glucose monitoring with wireless transdermal transmission and long-lasting activity. The full potential of fluorescent microbeads has yet to be realized due to insufficient intensity for transdermal transmission and material toxicity. This paper illustrates the highly-sensitive, biostable, long-lasting, and injectable fluorescent microbeads for in vivo continuous glucose monitoring. We synthesized a fluorescent monomer composed of glucose-recognition sites, a fluorogenic site, spacers, and polymerization sites. The spacers are designed to be long and hydrophilic for increasing opportunities to bind glucose molecules; consequently, the fluorescent monomers enable high-intensive responsiveness to glucose. We then fabricated injectable-sized fluorescent polyacrylamide hydrogel beads with high uniformity and high throughput. We found that our fluorescent beads provide sufficient intensity to transdermally monitor glucose concentrations in vivo. The fluorescence intensity successfully traced the blood glucose concentration fluctuation, indicating our method has potential uses in highly-sensitive and minimally invasive continuous blood glucose monitoring.

Bio-inspired smart hydrogel with temperature-dependent properties and enhanced cell attachment

Stimuli-responsive smart hydrogels have been exploited for various applications, including as biomaterials with environment-dependent changes in hydrophobicity, stiffness or volume. In this study, we report the functionalisation of a temperature-responsive poly(N-isopropylacryamide) (PNIPAAm) smart hydrogel with catechol groups to enhance its stiffness and cell attachment. To introduce biomimetic adhesive catechol group, which is derived from mussel feet, a photo-crosslinkable 3-hydroxytyramine hydrochloride-derived dopamine methacrylamide (DMA) monomer was synthesised. Then, temperature-responsive smart copolymer hydrogels were successfully fabricated by photo-polymerisation of the DMA with N-isopropylacryamide (NIPAAm). The fabricated smart hydrogels demonstrated temperature-dependent properties, and the DMA affected the swelling behaviour and compressive mechanical strength. In vitro cell culture experiments showed that the catechol groups in the smart hydrogels promoted cell attachment and spreading. These smart hydrogels will be useful as biomaterials as tissue scaffolds with controllable properties.

Living cell fabric

This paper describes a centimeter-scale living cell fabric made of cell-containing core-shell hydrogel fibers, “cell fiber.” We improved core-shell fiber applicable to various types of cells, and precisely characterized their biofunctions and mechanical properties. Using these cell fibers, we demonstrate a centimeter-scale living cell fabric woven by our micro weaving machine. We believe that our weaving approach using cell fibers would be a powerful method for constructing large-scale 3D-patterned functional tissues.

TFT display panel technology as a base for biological cells electrical manipulation - application to dielectrophoresis

This paper reports for the first time the use of TFT (Thin Film Transistor) technology of display panels for biological cells electrical manipulation. This technology allows to have high density distributed transparent micro-electrodes, independently controllable, covering centimeter-size glass substrates. This technology is much superior to usual micro-technology used for Multielectrode Arrays (MEAs), which allows only millimeter size surface with micro-electrodes, and with a limited number of 64 micro-electrodes maximum. The chosen application, to demonstrate the capability of such technology, is dielectrophoresis on micro-beads and yeast cells.

Visualizing dynamics of angiogenic sprouting from a three-dimensional microvasculature model using stage-top optical coherence tomography

Three-dimensional (3D) in vitro microvasculature in a polydimethylsiloxane-based microdevice was developed as a physiologically relevant model of angiogenesis. The angiogenic process is monitored using stage-top optical coherence tomography (OCT). OCT allows non-invasive monitoring of the 3D structures of the prepared host microvasculature and sprouted neovasculature without fluorescence staining. OCT monitoring takes only a few minutes to scan through the several-millimetre scale range, which provides the advantage of rapid observation of living samples. The obtained OCT cross-sectional images capture 3D features of the angiogenic sprouting process and provide information on the dynamics of luminal formation. The stage-top system used in this study enables the observer to visualize the in vitro dynamics of 3D cultured cells simply and conveniently, offering an alternative monitoring method for studies on angiogenesis and providing quantitative information about vascular morphological changes.

A Vascular Endothelial Growth Factor-Dependent Sprouting Angiogenesis Assay Based on an In Vitro Human Blood Vessel Model for the Study of Anti-Angiogenic Drugs

Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vasculature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogenesis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelial growth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulating angiogenesis, and have been the targets for the development of drugs. However, most experimental assays assessing angiogenesis rely on animal models. We report an in vitro model using a microvessel-on-a-chip. It mimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a single angiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observed in vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, as demonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2 pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstrates that sorafenib impairs the endothelial barrier function, while sunitinib does not. Such in vitro human model provides a significant complimentary approach to animal models for the development of effective therapies.

A 3D in vitro pericyte-supported microvessel model: visualisation and quantitative characterisation of multistep angiogenesis

Angiogenesis, which refers to the formation of new blood vessels from already existing vessels, is a promising therapeutic target and a complex multistep process involving many different factors. Pericytes (PCs) are attracting attention as they are considered to make significant contributions to the maturation and stabilisation of newly formed vessels, although not much is known about the precise mechanisms involved. Since there is no single specific marker for pericytes, in vivo models may complicate PC identification and the study of PCs in angiogenesis would benefit from in vitro models recapitulating the interactions between PCs and endothelial cells (ECs) in a three-dimensional (3D) configuration. In this study, a 3D in vitro co-culture microvessel model incorporating ECs and PCs was constructed by bottom-up tissue engineering. Angiogenesis was induced in the manner of sprout formation by the addition of a vascular endothelial cell growth factor. It was found that the incorporation of PCs prevented expansion of the parent vessel diameter and enhanced sprout formation and elongation. Physical interactions between ECs and PCs were visualised by immunostaining and it disclosed that PCs covered the EC monolayer from its basal side in the parent vessel as well as angiogenic sprouts. Furthermore, the microvessels were visualized in 3D by using a non-invasive optical coherence tomography (OCT) imaging system and sprout features were quantitatively assessed. It revealed that the sprouts in EC-PC co-culture vessels were longer and tighter than those in EC mono-culture vessels. The combination of the microvessel model and the OCT system analysis can be useful for the visualisation and demonstration of the multistep process of angiogenesis, which incorporates PCs.

Bundle Gel Fibers with a Tunable Microenvironment for in Vitro Neuron Cell Guiding

As scaffolds for neuron cell guiding in vitro, gel fibers with a bundle structure, comprising multiple microfibrils, were fabricated using a microfluidic device system by casting a phase-separating polymer blend solution comprising hydroxypropyl cellulose (HPC) and sodium alginate (Na-Alg). The topology and stiffness of the obtained bundle gel fibers depended on their microstructure derived by the polymer blend ratio of HPC and Na-Alg. High concentrations of Na-Alg led to the formation of small microfibrils in a one-bundle gel fiber and stiff characteristics. These bundle gel fibers permitted for the elongation of the neuron cells along their axon orientation with the long axis of fibers. In addition, human-induced pluripotent-stem-cell-derived dopaminergic neuron progenitor cells were differentiated into neuronal cells on the bundle gels. The bundle gel fibers demonstrated an enormous potential as cell culture scaffold materials with an optimal microenvironment for guiding neuron cells.