論文

Organs-on-Chips (OoCs) are 3D models aiming to faithfully replicate in vitro specific functions of human organs or tissues. While promising as an alternative to traditional 2D cell culture and animal models in drug development, controlled realization of complex microvasculature within OoC remains a significant challenge. Here, we demonstrate how femtosecond laser patterning allows to produce hollow microvascular-like channels inside a collagen-based matrix directly within a microfluidic chip. The hydrogel preparation protocol was optimized to maintain structural stability, facilitating successful endothelialization of produced channels. The resulting microvascular structures exhibit notable physiological relevance, as evidenced by the expression of key endothelial markers (ZO-1, and VE-cadherin) and the successful reproduction of the barrier function. Furthermore, tumor necrosis factor-alpha (TNF-α) exposure induces a concentration-dependent increase in vascular permeability and expression of intercellular adhesion molecule-1 (ICAM-1). The proposed method holds the potential to control and faithfully reproduce the vascularization process in OoC platforms, in both physiological and inflammatory conditions.

Primary hepatocytes are widely recognized for their ability to accurately represent the in vivo hepatocyte phenotype. However, traditional avascular primary hepatocyte culture models are limited by inadequate mass transfer, which leads to a rapid decline in hepatocyte function and survival. To address these challenges, vascularization of hepatic spheroids is crucial for enhancing oxygen and nutrient supply, thereby enabling the construction of larger and more complex hepatic tissues in vitro. In this study, we achieved vascularization of hepatic spheroids containing freshly isolated primary hepatocytes by incorporating fibroblasts as a source of paracrine factors to induce angiogenesis. Multicellular spheroids composed of primary hepatocytes and fibroblasts were formed in non-adhesive concave wells, and one of the spheroids was subsequently embedded in a fibrin-collagen hydrogel within a microfluidic device. Endothelial cells were then seeded onto adjacent microfluidic channels. They formed microvascular networks that extended toward and penetrated the hepatic spheroid. The vascularized hepatic spheroid closely mimicked hepatic sinusoids, with hepatocytes in close contact with microvessels. Moreover, the vascularized spheroid exhibited significantly enhanced hepatic function, specifically albumin secretion and urea synthesis. Our findings provide insights into the establishment of highly vascularized hepatic spheroids in vitro, which is crucial for constructing scalable hepatic tissues in the context of biofabrication.

Advances in tissue engineering have enabled the construction of two-dimensional tissues such as skin and cornea, as well as avascular three-dimensional (3D) tissues such as cartilage. However, building fully functional 3D tissues and organs remains a major challenge. In 3D constructs, microvascular networks are indispensable for delivering oxygen and nutrients to the inner regions; thus, the formation of capillaries and microvessels and their integration with 3D parenchymal tissues––so-called vascularization––are of critical importance. In this review, we discuss the current status and future perspectives of in vitro tissue engineering from a bioengineering standpoint, with a particular focus on the vascularization of 3D tissues. We first summarize recent progress in the in vitro construction of capillaries and microvessels, including microfluidic- and bioprinting-based approaches. We then focus on the liver as a representative parenchymal tissue, given its vital metabolic functions and the potential of human cell-derived liver models for drug discovery and as alternatives to animal testing. Finally, we highlight emerging strategies for vascularizing liver and other organ models and outline future directions in this evolving field. We conclude that bioengineering approaches for designing biomimetic systems, when integrated with vascular biology, organ-specific cell and stem cell biology, pathology, multiphoton lithography, and materials science, will enable the construction of in vitro 3D tissues with stable vasculature and functional integration with parenchymal structures, opening new avenues for drug discovery, disease modeling, and regenerative medicine.

There is a great demand for development of advanced in vitro liver models to predict the efficacy and safety of drug candidates accurately in the preclinical drug development. Despite the great efforts to develop biomimetic models, it remains challenging to precisely mimic a functional unit of the liver (i.e., hepatic lobule) with a continuous microvascular network. Recent progress in laser patterning has allowed us to create arbitrary biomimetic structures with high resolution. Here, we propose an advanced liver-on-chip model mimicking the hepatic lobule with a continuous microvascular network, ranging from the microvessels to the central vein of the liver, utilizing femtosecond laser patterning. Firstly, we optimize the laser power to pattern microchannels mimicking the microvessel and central vein of the hepatic lobule by using a femtosecond laser within a collagen-based hydrogel containing hepatic cells. Secondly, we construct continuous microvessels with luminal structures by comparing different microchannel sizes in diameter. Finally, we assemble a millimeter-scale hepatic lobule-like structure with multiple layers of microvascular networks in the liver-on-chip. Furthermore, our liver-on-chip model exhibits major liver functions and drug-induced hepatotoxicity, as evidenced by albumin and urea productions and by a toxic response to acetaminophen, respectively. Our approach provides valuable strategies for the development of advanced physiological and pathological liver-on-chip models for pharmaceutical and toxicological studies.

Forming capillary networks with predefined geometries is a critical challenge in engineering complex three-dimensional tissues in vitro. While bioprinting and microfluidic technologies have enabled vascular tissue fabrication, precise control over capillary-scale vascularization remains limited. In this study, we investigated vascular formation process along hydrogel microchannels to elucidate mechanisms governing capillary-scale lumen formation. Microchannels were fabricated by femtosecond laser ablation in collagen and fibrin hydrogels. We optimized multiphoton lithography parameters to fabricate microchannels within these hydrogels and analyzed vascular formation along the channels. Luminal vascular structures formed readily in 50-μm channels, while vascular formation failed in 20-μm channels under static conditions, suggesting a significant shift in endothelial organization at the cellular scale. Flow stimulation significantly promoted vessel formation through collective endothelial cell migration and adhesion, whereas static conditions induced endothelial-to-mesenchymal transition. These findings provide key insights into capillary-scale vascularization and contribute to the development of more complex architectures with predefined shapes.

Donor organ shortages drive an urgent need for engineered hepatocyte tissues; however, functional vascular integration remains a major bottleneck in liver tissue engineering. Current vascularization strategies struggle to achieve perfusable microvessels that penetrate hepatocyte tissue. Furthermore, recapitulating hepatic regeneration in vitro remains challenging. This study presents a paracrine factor local gradient (PFLG)-generating system that constructs vascularized, perfusable hepatocyte tissues and recapitulates the perfusion-mediated proliferative activity of primary hepatocytes. The PFLG-generating platform integrates fibroblast-loaded cryogels with a microfluidic device to direct angiogenesis prior to hepatocyte seeding, thereby enabling microvessels to penetrate 3D hepatocyte tissue. Within the vascularized constructs, microvessels directly penetrated the hepatocyte parenchyma, recapitulating the intimate hepatocyte–microvessel contacts observed in vivo. These constructs enhanced hepatocyte polarity and promoted functional bile canaliculi formation. Importantly, perfusion culture induced robust hepatocyte proliferative activity, as evidenced by a significant increase in Ki67-positive hepatocytes, including mitotic cells, without loss of cellular polarity. By contrast, this proliferative response was minimal under static conditions. Time-lapse imaging and functional assays confirmed perfusion through penetrating microvessels. These findings demonstrate that perfusion-mediated cues are essential for inducing hepatocyte cell-cycle re-entry while maintaining functional polarity. This modular and programmable culture platform lays the foundation for scaling-up toward transplantable liver tissues.

This study presents a method for applying multi-photon absorption to fabricate micro-scale scaffold structures for blood vessel formation within hydrogels, using a femtosecond laser. The structures were fabricated via femtosecond laser ablation and cavitation enabling the replication of complex micro-scale microvascular systems. A pressure control system was designed to reproduce variations in channel diameter observed in natural blood vessels. By adjusting the pressure, changes in diameter were confirmed demonstrating the influence of pressure on cavitation. Fabricated structures using the two modes were also evaluated by seeding endothelial cells and subsequent blood vessel formation was observed.

Construction of three-dimensional (3D) hepatic tissue structures is important for in vitro tissue engineering of the liver, because 3D culture of hepatocytes is critical for the maintenance of liver-specific functions. Although conventional 3D culture methods are useful for constructing 3D hepatic tissue structures, the precise control of culture microenvironments is required to construct more physiological tissues in vitro. Recent advances in microfluidics technologies have allowed us to utilize microfluidic devices for hepatic cell culture, which opened the door for creating more physiological 3D culture models of the liver. Here, we describe the method for the construction of hepatic tissue structures using a microfluidic device which has a 3D gel region with adjacent microchannels. Primary rat hepatocytes are seeded into a microchannel in a microfluidic device. The cells are then cultured in interstitial flow conditions, which leads to the construction of 3D tissue structures.

A new microdevice controlling the curvature of cell adhering plane is proposed. This microdevice is composed of a cell culture chamber with thin silicone membrane, a microslit and a vacuum chamber. Controlling the air pressure, the curvature of the cell adhering plane can be flexibly tuned in a spatiotemporal way. To prove the concept, vascular smooth muscle is cultured on the curved surface and their response to sudden curvature change is monitored. This microdevice would contribute to pursue fundamental question on how cells recognize the surrounding geometry, which is one of the core interests in the mechanobiology field.