Published papers

© 2018 Elsevier Inc. Every organ demonstrates specific vascular characteristics and functions maintained by interactions of endothelial cells (ECs) and parenchymal cells. Particularly, brain ECs play a central role in the formation of a functional blood brain barrier (BBB). Organ-specific ECs have their own morphological features, and organ specificity must be considered when investigating interactions between ECs and other cell types constituting a target organ. Here we constructed angiogenesis-based microvascular networks with perivascular cells in a microfluidic device setting by coculturing ECs and mesenchymal stem cells (MSCs). Furthermore, we analyzed endothelial barrier functions as well as fundamental morphology, an essential step to build an in vitro BBB model. In particular, we used both brain microvascular ECs (BMECs) and human umbilical vein ECs (HUVECs) to test if organ specificity of ECs affects the formation processes and endothelial barrier functions of an engineered microvascular network. We found that microvascular formation processes differed by the source of ECs. HUVECs formed more extensive microvascular networks compared to BMECs while no differences were observed between BMECs and HUVECs in terms of both the microvascular diameter and the number of pericytes peripherally associated with the microvasculatures. To compare the endothelial barrier functions of each type of EC, we performed fluorescence dextran perfusion on constructed microvasculatures. The permeability coefficient of BMEC microvasculatures was significantly lower than that of HUVEC microvasculatures. In addition, there were significant differences in terms of tight junction protein expression. These results suggest that the organ source of ECs influences the properties of engineered microvasculature and thus is a factor to be considered in the design of organ-specific cell culture models.

© Copyright 2019, Mary Ann Liebert, Inc., publishers 2019. Construction of small and continuous capillary networks is a fundamental challenge for the development of three-dimensional (3D) tissue engineering. In particular, to construct mature and stable capillary networks, it is important to consider interactions between endothelial cells and pericytes. This study aimed to construct stable capillary networks covered by pericyte-like perivascular cells, which maintain the lumen of small diameter similar to that of capillary structures in vivo. Vascular sprouting, capillary extension, and stabilization were investigated using a 3D angiogenesis model containing human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) in a microfluidic device. A series of HUVEC:MSC ratios was tested; the ratio was found to be an important factor in the construction of capillary structures. We found that stable capillary networks that were covered by MSC-derived perivascular cells can be constructed at 1:1 HUVEC:MSC ratio. The constructed capillary networks had continuous lumens with <10-μm diameter, which were maintained for at least 21 days. This angiogenic process and basement membrane formation were regulated by HUVEC-MSC interactions. Construction of capillary networks is a fundamental challenge for the development of three-dimensional (3D) tissue engineering. However, it is not well understood how to construct stable capillary networks that maintain a luminal size similar to that of capillary structures in vivo (i.e., <10 μm diameter). In this study, we demonstrated the construction of stable capillary networks covered by pericyte-like perivascular cells using an in vitro 3D angiogenesis model by optimizing interactions between endothelial cells and perivascular cells. Our 3D angiogenesis model can be combined with 3D culture of epithelial cells in the context of vascularization of 3D tissue-engineered constructs.

© 2019 IOP Publishing Ltd. Recent advances in microfabrication technologies have enabled us to construct collagen gel microbeads, which can be cultured with hepatocytes. However, little is known about the hepatocyte-collagen gel microbead interactions. Here, we aimed to clarify the effects of the balance between cell-cell and cell-collagen gel microbead interactions on hepatocyte morphogenesis and functions. The magnitude of cell-microbead interactions was controlled by changing the size of the microbeads, which were smaller than, comparable to, and larger than hepatocytes. These small, medium, and large microbeads were cultured separately with primary hepatocytes. Phase-contrast and time-lapse imaging revealed that the medium microbeads significantly induced the construction of 3D structures composed of the microbeads and hepatocytes in a self-organizing manner, whereas hepatocytes formed 2D monolayers with the small or large microbeads. These results suggest that only the medium microbeads induced the 3D tissue formation of hepatocytes. Furthermore, liver-specific functions, such as albumin secretion and ammonia clearance, were significantly upregulated in the 3D structures. These findings are critical to understand how to control the construction of 3D hepatocyte tissues with hydrogel microbeads in the context of biofabrication.

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor. A deeper mechanistic understanding of the invasion of heterogeneous GBM cell populations is crucial to develop therapeutic strategies. A key regulator of GBM cell invasion is interstitial flow. However, the effect of an interstitial flow on the invasion of heterogeneous GBM cell populations composed of glioma initiating cells (GICs) and relatively differentiated progeny cells remains unclear. In the present study, we investigated how GICs invade three-dimensional (3D) hydrogels in response to an interstitial flow with respect to their differentiation status. Microfluidic culture systems were used to apply an interstitial flow to the cells migrating from the cell aggregates into the 3D hydrogel. Phase-contrast microscopy revealed that the invasion and protrusion formation of the GICs in differentiated cell conditions were significantly enhanced by a forward interstitial flow, whose direction was the same as that of the cell invasion, whereas those in stem cell conditions were not enhanced by the interstitial flow. The mechanism of flow-induced invasion was further investigated by focusing on differentiated cell conditions. Immunofluorescence images revealed that the expression of cell-extracellular matrix adhesion-associated molecules, such as integrin β1, focal adhesion kinase, and phosphorylated Src, was upregulated in forward interstitial flow conditions. We then confirmed that cell invasion and protrusion formation were significantly inhibited by PP2, a Src inhibitor. Finally, we observed that the flow-induced cell invasion was preceded by nestin-positive immature GICs at the invasion front and followed by tubulin β3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion. A mechanistic understanding of heterogeneous glioblastoma cell invasion is crucial for developing therapeutic strategies. We observed that the invasion and protrusion formation of glioma initiating cells (GICs) were significantly enhanced by forward interstitial flow in differentiated cell conditions. The expression of integrin β1, focal adhesion kinase, and phosphorylated Src was upregulated, and the flow-induced invasion was significantly inhibited by a Src inhibitor. The flow-induced heterogeneous cell invasion was preceded by nestin-positive GICs at the invasion front and followed by tubulin β3-positive differentiated cells. Our findings provide insights into the development of novel therapeutic strategies to inhibit flow-induced glioma invasion.

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

Hemodynamic and biochemical factors play important roles in critical steps of angiogenesis. In particular, interstitial flow has attracted attention as an important hemodynamic factor controlling the angiogenic process. Here, we applied a wide range of interstitial flow magnitudes to an in vitro three-dimensional (3D) angiogenesis model in a microfluidic device. This study aimed to investigate the effect of interstitial flow magnitude in combination with the vascular endothelial growth factor (VEGF) concentration on 3D microvascular network formation. Human umbilical vein endothelial cells (HUVECs) were cultured in a series of interstitial flow generated by 2, 8, and 25 mmH2O. Our findings indicated that interstitial flow significantly enhanced vascular sprout formation, network extension, and the development of branching networks in a magnitude-dependent manner. Furthermore, we demonstrated that the proangiogenic effect of interstitial flow application could not be substituted by the increased VEGF concentration. In addition, we found that HUVECs near vascular sprouts significantly elongated in >8 mmH2O conditions, while activation of Src was detected even in 2 mmH2O conditions. Our results suggest that the balance between the interstitial flow magnitude and the VEGF concentration plays an important role in the regulation of 3D microvascular network formation in vitro.

© 2019 Acta Materialia Inc. There is a great deal of demand for the construction of transplantable liver grafts. Over the last decade, decellularization techniques have been developed to construct whole liver tissue grafts as potential biomaterials. However, the lack of intact vascular networks, especially sinusoids, in recellularized liver scaffolds leads to hemorrhage and thrombosis after transplantation, which is a major obstacle to the development of transplantable liver grafts. In the present study, we hypothesized that both mechanical (e.g., fluid shear stress) and chemical factors (e.g., fibronectin coating) can enhance the formation of hierarchical vascular networks including sinusoid-scale microvessels. We demonstrated that perfusion culture promoted formation of sinusoid-scale microvessels in recellularized liver scaffolds, which was not observed in static culture. In particular, perfusion culture at 4.7 ml/min promoted the formation of sinusoid-scale microvessels compared to perfusion culture at 2.4 and 9.4 ml/min. In addition, well-aligned endothelium was observed in perfusion culture, suggesting that endothelial cells sensed the flow-induced shear stress. Moreover, fibronectin coating of decellularized liver scaffolds enhanced the formation of sinusoid-scale microvessels in perfusion culture at 4.7 ml/min. This study represents a critical step in the development of functional recellularized liver scaffolds, which can be used not only for transplantation but also for drug screening and disease-modeling studies. Statement of Significance: Decellularized liver scaffolds are promising biomaterials that allow production of large-scale tissue-engineered liver grafts. However, it is difficult to maintain recellularized liver grafts after transplantation due to hemorrhage and thrombosis. To overcome this obstacle, construction of an intact vascular network including sinusoid-scale microvessels is essential. In the present study, we succeeded in constructing sinusoid-scale microvessels in decellularized liver scaffolds via a combination of perfusion culture and surface coating. We further confirmed that endothelial cells in decellularized liver scaffolds responded to flow-derived mechanical stress by aligning actin filaments. Our strategy to construct sinusoid-scale microvessels is critical for the development of intact vascular networks, and addresses the limitations of recellularized liver scaffolds after transplantation.

Formation of vascular anastomoses is critical for the development of transplantable tissue-engineered grafts, because rapid blood perfusion is required for the maintenance of implanted tissue grafts. However, the process of vascular anastomosis remains unclear due to difficulties in observing vascular anastomosis after transplantation. Although several groups have developed in vitro models of vascular anastomosis, there is a lack of a suitable in vitro anastomosis model that includes perivascular cells. Therefore, we aimed to establish an in vitro vascular anastomosis model containing perivascular cells by a combination of human umbilical vein endothelial cell (HUVEC) monoculture and HUVEC-mesenchymal stem cell (MSC) coculture in a microfluidic device. We found that vascular formation was inhibited when HUVECs were seeded on both sides of gel scaffolds, but HUVECs formed vascular networks when they were seeded on one side only. Next, we tested a series of HUVEC:MSC ratios to induce vascular anastomoses. The results demonstrated that addition of MSCs induced vascular anastomosis. In particular, the number of vascular anastomoses was significantly increased at a HUVEC:MSC ratio of 2:8. The process of vascular anastomosis was further investigated by live-cell imaging of green fluorescent protein-expressing HUVECs, which revealed that vascular anastomoses with continuous lumens were constructed during days 8-10. Computational simulation of VEGF concentrations suggested that local VEGF gradients play important roles in vascular formation while the addition of MSCs was critical for anastomosis. This anastomosis model will provide insights for both the development of tissue-engineered grafts and for the construction of large tissues by assembling multiple tissue-engineered constructs.

We report on a novel microdevice to tune the curvature of a cell-adhering surface by controlling the air-pressure and micro-slit. Human aortic smooth muscle cells were cultured on demi-cylindrical concaves formed on a microdevice. Their shape-adapting behavior could be tracked when the groove direction was changed to the orthogonal direction. This microdevice demonstrated live observation of cells responding to dynamic changes of the anisotropic curvature of the adhering surface and could serve as a new platform to pursue mechanobiology on curved surfaces.

Organ transplantation is the most effective therapy for end-stage organ failure. However, the demand for lifesaving organ transplants far exceeds the supply of available organs owing to organ shortage. To address this problem, tissue engineering has offered potential strategies for in vitro construction of organs as medical and clinical applications. However, tissue-engineered organs are difficult to construct owing to the lack of functional vascular networks because avascular organs lead to tissue dysfunctions, such as hypoxia and clot formation. Therefore, establishing functional vascular networks is required for the construction and maintenance of organs in terms of morphology and function. Recent advances in tissue engineering have allowed the in vitro construction of a wide range of functional vascular networks, ranging from microvessels to organ-scale vascular networks, using self-organization and pre-designed approaches. In particular, various new models have been developed utilizing microfluidics, 3D bioprinting, and organ decellularization. These models have enabled the in vitro recapitulation of key features of physiological vascular networks, such as morphology (e.g., network formation, luminal structure, and perivascular cell coverage) and function (e.g., barrier and antithrombogenic functions). In this review, we summarize the progress and challenges in vascular tissue engineering based on two distinct categories: self-organization and pre-designed approaches. In addition, the advantages and limitations of these models are highlighted, and future perspectives are discussed. These models will provide useful insights for the construction of vascularized functional tissues and organs and can contribute to development in tissue engineering and regenerative medicine.

Vascular networks consist of hierarchical structures of various diameters and are necessary for efficient blood distribution. Recent advances in vascular tissue engineering and bioprinting have allowed us to construct large vessels, such as arteries, small vessels, such as capillaries and microvessels, and intermediate-scale vessels, such as arterioles, individually. However, little is known about the control of vessel diameters between small vessels and intermediate-scale vessels. Here, we focus on vascular remodeling, which creates lasting structural changes in the vessel wall in response to hemodynamic stimuli, to regulate vessel diameters in vitro. The purpose of this study is to control the vessel diameter at an intermediate scale by inducing outward remodeling of microvessels in vitro. Human umbilical vein endothelial cells and mesenchymal stem cells were cocultured in a microfluidic device to construct microvessels, which were then perfused with a culture medium to induce outward vascular remodeling. We successfully constructed vessels with diameters of 40-150 μm in perfusion culture, whereas vessels with diameters of <20 μm were maintained in static culture. We also revealed that the in vitro vascular remodeling was mediated by NO pathways and MMP-9. These findings provide insight into the regulation of diameters of tissue-engineered blood vessels. This is an important step toward the construction of hierarchical vascular networks within biofabricated three-dimensional systems.

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.

Vascular remodeling is a crucial process for the effective delivery of oxygen and nutrients to the entire body during vascular formation. However, detailed mechanisms underlying vascular remodeling are not yet fully understood owing to the absence of an appropriate experimental model. To address this, in this study, we utilized a microfluidic vascular model with perivascular cells to investigate the mechanism of vascular remodeling by culturing human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) in a microfluidic device. We compared two different cell culture conditions: culturing HUVECs and MSCs (1) separately in different channels and (2) in the same channel. In both conditions, microvascular networks covered with perivascular cells were formed. Interestingly, a significant inward vascular remodeling occurred over time when HUVECs and MSCs were cultured in different channels. This remodeling was mediated by direct endothelial–perivascular crosstalk through α6 integrin. Furthermore, computational fluid analysis revealed that hypothetical shear stress on the luminal surface of microvessels was attenuated during inward vascular remodeling, suggesting that the remodeling might be an adaptive change. Our findings and the microfluidic model will be useful not only for further elucidation of mechanisms underlying physiological and pathological vascular remodeling but also for constructing functional vascularized tissues and organs by controlling vascular remodeling.

Tissue engineers have utilised a variety of three-dimensional (3D) scaffolds for controlling multicellular dynamics and the resulting tissue microstructures. In particular, cutting-edge microfabrication technologies, such as 3D bioprinting, provide increasingly complex structures. However, unpredictable microtissue detachment from scaffolds, which ruins desired tissue structures, is becoming an evident problem. To overcome this issue, we elucidated the mechanism underlying collective cellular detachment by combining a new computational simulation method with quantitative tissue-culture experiments. We first quantified the stochastic processes of cellular detachment shown by vascular smooth muscle cells on model curved scaffolds and found that microtissue morphologies vary drastically depending on cell contractility, substrate curvature, and cell-substrate adhesion strength. To explore this mechanism, we developed a new particle-based model that explicitly describes stochastic processes of multicellular dynamics, such as adhesion, rupture, and large deformation of microtissues on structured surfaces. Computational simulations using the developed model successfully reproduced characteristic detachment processes observed in experiments. Crucially, simulations revealed that cellular contractility-induced stress is locally concentrated at the cell-substrate interface, subsequently inducing a catastrophic process of collective cellular detachment, which can be suppressed by modulating cell contractility, substrate curvature, and cell-substrate adhesion. These results show that the developed computational method is useful for predicting engineered tissue dynamics as a platform for prediction-guided scaffold design. Statement of significance: Microfabrication technologies aiming to control multicellular dynamics by engineering 3D scaffolds are attracting increasing attention for modelling in cell biology and regenerative medicine. However, obtaining microtissues with the desired 3D structures is made considerably more difficult by microtissue detachments from scaffolds. This study reveals a key mechanism behind this detachment by developing a novel computational method for simulating multicellular dynamics on designed scaffolds. This method enabled us to predict microtissue dynamics on structured surfaces, based on cell mechanics, substrate geometry, and cell-substrate interaction. This study provides a platform for the physics-based design of micro-engineered scaffolds and thus contributes to prediction-guided biomaterials design in the future.