Three-dimensional Microvessel Research Articles

The role of adhesive receptor patterns on cell transport in complex microvessels.

Cell transport is governed by the interaction of fluid dynamic forces and biochemical factors such as adhesion receptor expression and concentration. Although the effect of endothelial receptor density is well understood, it is not clear how the spacing and local spatial distribution of receptors affect cell adhesion in three-dimensional microvessels. To elucidate the effect of vessel shape on cell trajectory and the arrangement of endothelial receptors on cell adhesion, we employed a three-dimensional deformable cell model that incorporates microscale interactions between the cell and the endothelium. Computational cellular adhesion models are systematically altered to assess the influence of receptor spacing. We demonstrate that the patterns of receptors on the vessel walls are a key factor guiding cell movement. In straight microvessels, we show a relationship between cell velocity and the spatial distribution of adhesive endothelial receptors, with larger receptor patches producing lower translational velocities. The joint effect of the complex vessel topology seen in microvessel shapes such as curved and bifurcated vessels when compared to straight tubes is explored with results which showed the spatial distribution of receptors affecting cell trajectory. Our findings here represent demonstration of the previously undescribed relationship between receptor pattern and geometry that guides cellular movement in complex microenvironments.

OrganoPlate Micro-fluidic Microvessel Culture and Analysis

The endothelial cells from the microvasculature are key drivers and targets of inflammatory and thrombotic processes in microvascular diseases. The study of bioactive lipids in inflammatory processes has been largely based on two-dimensional endothelial cell cultures. Three-dimensional microvessels-on-a-chip provides an opportunity to monitor the inflammatory phenotype of human microvessels in a more physiological-relevant environment. This protocol describes the culture of endothelial cells as three-dimensional microvessels in the OrganoPlate. The microvessels are treated with tumor necrosis factor alpha to induce inflammation. The collection of samples from the microvessels is optimized for measuring bioactive lipids with liquid chromatography-mass spectrometry, providing a more informative metabolic readout as compared with functional assays.

Long-Term Cryopreservation Preserves Blood-Brain Barrier Phenotype of iPSC-Derived Brain Microvascular Endothelial Cells and Three-Dimensional Microvessels.

Brain microvascular endothelial cells derived from induced pluripotent stem cells (dhBMECs) are a scalable and reproducible resource for studies of the human blood-brain barrier, including mechanisms and strategies for drug delivery. Confluent monolayers of dhBMECs recapitulate key in vivo functions including tight junctions to limit paracellular permeability and efflux and nutrient transport to regulate transcellular permeability. Techniques for cryopreservation of dhBMECs have been reported; however, functional validation studies after long-term cryopreservation have not been extensively performed. Here, we characterize dhBMECs after 1 year of cryopreservation using selective purification on extracellular matrix-treated surfaces and ROCK inhibition. One-year cryopreserved dhBMECs maintain functionality of tight junctions, efflux pumps, and nutrient transporters with stable protein localization and gene expression. Cryopreservation is associated with a decrease in the yield of adherent cells and unique responses to cell stress, resulting in altered paracellular permeability of Lucifer yellow. Additionally, cryopreserved dhBMECs reliably form functional three-dimensional microvessels independent of cryopreservation length, with permeabilities lower than non-cryopreserved two-dimensional models. Long-term cryopreservation of dhBMECs offers key advantages including increased scalability, reduced batch-to-batch effects, the ability to conduct well-controlled follow up studies, and support of multisite collaboration from the same cell stock, all while maintaining phenotype for screening pharmaceutical agents.

Motion, deformation, and aggregation of multiple red blood cells in three-dimensional microvessel bifurcations

Vessel bifurcation is a place where many diseases start, such as the atherosclerosis, because the flow in this place is complex involving secondary flow and/or stagnation regions, and the cells are often blocked to aggregate together. A numerical study has been conducted to analyze three-dimensional motion, deformation, and aggregation of multiple red blood cells (RBCs) in a microvessel bifurcation. A smoothed dissipative particle dynamics model is used to simulate the fluids inside and outside of the RBCs. The RBC membrane is modeled as a triangular network, associated with a deformation potential energy and an aggregation potential energy to describe the RBC deformation and aggregation, respectively. The interaction between the fluid and the RBCs is modeled by the immersed boundary method. The numerical models are first validated by examining the rheology of multiple RBCs in a cylindrical tube. Then, we investigate the effect of number of RBCs, mechanical properties, and interaction strength on their motion, deformation, and aggregation. The simulation results showed that the leading RBC has more deformation, compared with subsequent RBCs. The larger the RBC number, the easier the RBCs aggregate. The RBC deformation has an obvious effect on the RBC aggregation, whereas the RBC aggregation has a slight effect on the RBC deformation. Both the RBC deformation and aggregation can cause the RBC centroid to deviate at the apex of bifurcation, and this determines which branch the RBCs move into.

Phenotypic analysis of endothelial cell function by using three-dimensional microvessel model

要約：創薬において，in vitro では細胞レベルの解析しか行えず，in vivo ではその複雑さから組織特異的な現象の詳細な解析が行えない．この乖離をつなげるために，近年，三次元的に組織を模倣したモデルの構築が盛んに行われている．ボトムアップ的に構築された三次元人工微小血管モデルを用いることで，血管新生・透過性といった，血管組織構造レベルでのフェノタイプがライブイメージング可能となる．このフェノタイプ解析は，in vitro とin vivo のギャップを埋めるような詳細な解析が行えるだけでなく，標的薬剤・遺伝子が持つ血管に対する未知の機能を発見することができ，今まで説明できなかった作用機序や副作用の原理を解明できる．創薬の薬剤探索が終了した際には，まず三次元人工微小血管モデルを用いてフェノタイプ解析を行い，疾患改善の有意性を示す必要性があると考える．

Chemotherapeutic Drug Delivery and Quantitative Analysis of Proliferation, Apoptosis, and Migration in a Tissue-Engineered Three-Dimensional Microvessel Model of the Tumor Microenvironment.

Numerous approaches have been employed to improve the efficacy of drug and gene delivery systems, but their strategic development is hindered by a lack of mechanistic understanding and assessment of drug transport and action. Optimizing the efficiency of a drug delivery system requires a detailed understanding of the pharmacokinetics, transendothelial transport, distribution at the tumor site, and uptake in target cells. Elucidating transport kinetics and rate-limiting steps in animal models can be extremely challenging, while in vitro platforms often fail to recapitulate the complexities of drug transport in vivo. To recapitulate the critical aspects of delivery of anticancer agents, we have developed a 3D tissue-engineered microvessel model of the tumor microenvironment. Our model consists of single MDA-MB-231 breast cancer cells embedded within a collagen matrix that surrounds a perfusable cylindrical microvessel lined with human endothelial cells. Here we compare transport and action of free doxorubicin and Doxil, a liposomal formulation of doxorubicin. We show that the mode of drug delivery influences uptake in the vessel endothelium and tumor cells. Through quantification of endothelial and tumor cell proliferation, apoptosis, and motility, we profile the kinetics of drug action with mechanisms of drug transport across the vessel lumen and into the surrounding matrix. Our model can be customized to mimic specific tumor microenvironments and disease states within a physiologically relevant microfluidic platform and provides a basis for characterizing and optimizing drug delivery systems.

Investigation on vascular cytotoxicity and extravascular transport of cationic polymer nanoparticles using perfusable 3D microvessel model

Vascular networks are the first sites exposed to cationic polymer nanoparticles (NPs) administered intravenously, and thus function as a barrier for NPs reaching the target organ. While cationic polymer NPs have been intensively studied as non-viral delivery systems, their biological effects in human microvessels have been poorly investigated due to a lack of appropriate in vitro systems. Here, we employed a three-dimensional microvessel on a chip, which accurately models in vivo conditions. An open and perfused microvessel surrounded by pericytes was shown to reproduce the important features of living vasculature, including barrier function and biomarkers. Using this microvessel chip, we observed contraction of the microvascular lumen induced by perfused polyethylenimine (PEI)/DNA NPs. We demonstrated that the oxidative stress present when microvessels were exposed to PEI NPs led to rearrangement of microtubules resulting in microvessel contraction. Furthermore, the transcytotic behavior of PEI NPs was analyzed in the microvessel by monitoring the escape of PEI NPs from the microvascular lumen into the perivascular region, which was not possible in two-dimensional culture systems. With our new understanding of the different behaviors of cationic polymer NPs depending on their transcytotic route, we suggest that caveolae-mediated transcytosis is a powerful route for efficient extravascular transport. Statement of SignificanceMicrovascular networks are not only biological system constituting largest surface area in the body and but also first site exposed to nanoparticle in vivo. While cationic polymer NPs have been intensively studied as non-viral delivery systems, its biological effects in human microvessel have been poorly investigated due to lack of appropriate in vitro systems. Here, we microengineered an open and perfused 3D pericyte incorporated microvessel model which possesses same morphological characteristic of in vivo. Using the microengineered model, this study represents the first report of transcytotic behavior of NPs in 3D microvessel, and its effect on extravasation efficiency. Our study lays the groundwork for the integration of innovative technologies to examine blood vessel-nanoparticle interaction, which a critical but ill-defined phenomenon.

A Feasible Method of Angiogenesis Assessment in Gastric Cancer Using 3D Microvessel Density.

Background Cancer stem cell (CSC) promotes angiogenesis which plays an important role in tumor occurrence, growth, and metastasis. Accurately, quantifying the tumor vasculature can help understanding CSC characteristics and improve cancer diagnosis, therapy planning, and evaluation. The objective of this study is to present a method for improved angiogenesis assessment. Methods We proposed a three-dimensional microvessel density (3D MVD) to evaluate tumor angiogenesis and tested it in animal models. Six male Balb/c nude mice were divided into normal group and tumor group. The mice in tumor group were orthotopically implanted human gastric cancer, cell line BGC-823. The phase-contrast images were collected at Shanghai Synchrotron Radiation Facility BL13W beamline, which has much higher soft-tissue contrast and spatial resolution than conventional X-ray. After volume reconstruction and vessel extraction, the 3D models of the angiogenesis were established for MVD calculation. Results The results showed that the proposed 3D MVD is positively correlated with the pathological changes of the microvessels. It took the advantage of high resolution of the phase-contrast imaging and added three-dimensional information to the existing MVD measure. Conclusions Our study presents a feasible approach for better understanding of tumor angiogenesis. It may provide doctors and scientists a better tool for cancer investigation and improving medical outcomes.

Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to 14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14 μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60 μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood-brain barrier in immature vessels.

Fusible core molding for the fabrication of branched, perfusable, three-dimensional microvessels for vascular tissue engineering.

A novel method for fabrication of branched, tubular, perfusable microvessels for use in vascular tissue engineering is reported. A tubular, elastomeric, biodegradable scaffold is first fabricated via a new, double fusible injection molding technique that uses a ternary alloy with a low melting temperature, Field's metal, and paraffin as sacrificial components. A cylindrical core metal of 500 μm or lower dia-meter with the target branching scaffold geometry is first constructed, then the metal structure is coated with paraffin and, finally, the metal-paraffin construct is embedded in polydimethylsiloxane (PDMS). The paraffin layer is then removed by heating and replaced by a biodegradable elastomeric pre-polymer that is subsequently UV-cured inside the PDMS. Next, the metal core is melted away and the PDMS is removed to attain the branched tubular elastomeric biodegradable scaffold. Finally, it is also demonstrated that human umbilical vein endothelial cells (HUVEC) were able to spread on the surface of the scaffold and form a confluent monolayer, confirming the potential of this new technique for making engineered blood vessels.

Effect of Hypoxia on Formation of Three-Dimensional Microvessel Networks by Endothelial Cells in vitro

Effect of Hypoxia on Formation of Three-Dimensional Microvessel Networks by Endothelial Cells in vitro

P-18 Effect of shear stress on three-dimensional microvessel network formation of endothelial cells

P-18 Effect of shear stress on three-dimensional microvessel network formation of endothelial cells

Initial bFGF Distribution Affects the Depth of Three-dimensional Microvessel Networks in Vitro

Initial bFGF Distribution Affects the Depth of Three-dimensional Microvessel Networks in Vitro

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model.

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.