Microfluidic organ-on-a-chip designs are used to mimic human tissues, including the vasculature. Here we present a novel microfluidic device that allows the interaction of endothelial cells (ECs) with pericytes and the extracellular matrix (ECM) in full bio-matrix encased 3D vessel structures (neovessels) that can be subjected to continuous, unidirectional flow and perfusion with circulating immune cells. We designed a in a polydimethylsiloxane (PDMS) device with a reservoir for a 3D fibrinogen gel with pericytes. Open channels were created for ECs to form a monolayer. Controlled, continuous, and unidirectional flow was introduced via a pump system while the design facilitated 3D confocal imaging. In this vessel-on-a-chip system, ECs interact with pericytes to create a human cell derived blood vessel which maintains a perfusable lumen for up to 7 days. Dextran diffusion verified endothelial barrier function while demonstrating the beneficial role of supporting pericytes. Increased permeability after thrombin stimulation, showed the capacity of the neovessels to show natural vascular response. Perfusion of neovessels with circulating THP-1 cells demonstrated this system as a valuable platform for assessing interaction between the endothelium and immune cells in response to TNFα. In conclusion: We created a novel vascular microfluidic device that facilitates the fabrication of an array of parallel soft-channel structures in ECM gel that develop into biologically functional neovessels without hard-scaffold support. This model provides a unique tool to conduct live in vitro imaging of the human vasculature during perfusion with circulating cells to mimic (disease) environments in a highly systematic but freely configurable manner.

BACKGROUND: Realistic reconstruction of the in vivo human atherosclerotic environment requires the coculture of different cell types arranged in atherosclerotic vessel–like structures with exposure to flow and circulating cells, presenting challenges for disease modeling. This study aimed to develop a 3-dimensional tubular microfluidic model with quadruple coculture of human aortic smooth muscle cells, human umbilical cord vein endothelial cells, and foam cells to recreate a complex human atherosclerotic vessel in vitro to study the effects of flow and circulating immune cells. METHODS: We developed a coculture protocol utilizing BFP (blue fluorescent protein)-labeled human aortic smooth muscle cells, GFP (green fluorescent protein)-labeled human umbilical cord vein endothelial cells, and THP-1 macrophage-derived, Dil-labeled oxidized LDL (low-density lipoprotein) foam cells within a fibrinogen/collagen I–based 3-dimensional ECM (extracellular matrix). Perfusion experiments were conducted for 24 hours on both atherosclerotic vessels and healthy vessels (BFP-labeled human aortic smooth muscle cells and GFP-labeled human umbilical cord vein endothelial cells without foam cells). Additionally, perfusion with circulating THP-1 monocytes was performed to observe cell extravasation and recruitment. RESULTS: The resulting vessels displayed early lesion morphology, with a layered composition including an endothelium and media, and foam cells accumulating in the subendothelial space. The layered wall composition of both atherosclerotic and healthy vessels remained stable under perfusion. Circulating THP-1 monocytes demonstrated cell extravasation into the atherosclerotic vessel wall and recruitment to the foam cell core. The qPCR (quantitative polymerase chain reaction) analysis indicated increased expression of atherosclerosis markers in the atherosclerotic vessels and adaptation of vascular smooth muscle cell migration in response to flow and the plaque microenvironment, compared with control vessels. CONCLUSIONS: The human 3-dimensional atherosclerosis model demonstrated stability under perfusion and allowed for the observation of immune cell behavior, providing a valuable tool for the atherosclerosis research field.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are pivotal regulators of extracellular matrix (ECM) composition and could, due to their dynamic activity, function as prognostic tools for fibrosis and cardiac function in left ventricular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction (HFpEF). We conducted a systematic review on experimental animal models of LVDD and HFpEF published in MEDLINE or Embase. Twenty-three studies were included with a total of 36 comparisons that reported established LVDD, quantification of cardiac fibrosis and cardiac MMP or TIMP expression or activity. LVDD/HFpEF models were divided based on underlying pathology: hemodynamic overload (17 comparisons), metabolic alteration (16 comparisons) or ageing (3 comparisons). Meta-analysis showed that echocardiographic parameters were not consistently altered in LVDD/HFpEF with invasive hemodynamic measurements better representing LVDD. Increased myocardial fibrotic area indicated comparable characteristics between hemodynamic and metabolic models. Regarding MMPs and TIMPs; MMP2 and MMP9 activity and protein and TIMP1 protein levels were mainly enhanced in hemodynamic models. In most cases only mRNA was assessed and there were no correlations between cardiac tissue and plasma levels. Female gender, a known risk factor for LVDD and HFpEF, was underrepresented. Novel studies should detail relevant model characteristics and focus on MMP and TIMP protein expression and activity to identify predictive circulating markers in cardiac ECM remodeling.

For the survival and integration of complex large-sized tissue-engineered (TE) organ constructs that exceed the maximal nutrients and oxygen diffusion distance required for cell survival, graft (pre)vascularization to ensure medium or blood supply is crucial. To achieve this, the morphology and functionality of the microcapillary bed should be mimicked by incorporating vascular cell populations, including endothelium and mural cells. Pericytes play a crucial role in microvascular function, blood vessel stability, angiogenesis, and blood pressure regulation. In addition, tissue-specific pericytes are important in maintaining specific functions in different organs, including vitamin A storage in the liver, renin production in the kidneys and maintenance of the blood–brain-barrier. Together with their multipotential differentiation capacity, this makes pericytes the preferred cell type for application in TE grafts. The use of a tissue-specific pericyte cell population that matches the TE organ may benefit organ function. In this review, we provide an overview of the literature for graft (pre)-vascularization strategies and highlight the possible advantages of using tissue-specific pericytes for specific TE organ grafts. Impact statement: The use of a tissue-specific pericyte cell population that matches the tissue-engineered (TE) organ may benefit organ function. In this review, we provide an overview of the literature for graft (pre)vascularization strategies and highlight the possible advantages of using tissue-specific pericytes for specific TE organ grafts.

Cell-based approaches using tissue engineering and regenerative medicine to replace damaged renal tissue with 3D constructs is a promising emerging therapy for kidney disease. Besides living cells, a template provided by a scaffold based on biomaterials and bioactive factors is needed for successful kidney engineering. Nature's own template for a scaffolding system is the extracellular matrix (ECM). Research has focused on mapping the mature renal ECM; however, the developing fetal ECM matches more the active environment required in 3D renal constructs. Here, we characterized the differences between the human fetal and mature renal ECM using spectrometry-based proteomics of decellularized tissue. We identified 99 different renal ECM proteins of which the majority forms an overlapping core, but also includes proteins enriched in either the fetal or mature ECM. Relative protein quantification showed a significant dominance of EMILIN1 in the fetal ECM. We functionally tested the role of EMILIN1 in the ECM using a novel methodology that permits the reliable anchorage of native cell-secreted ECM to glass coverslips. Depletion of EMILIN1 from the ECM layer using siRNA mediated knock-down technologies does not affect renal epithelial cell growth, but does promote migration. Lack of EMILIN1 in the ECM layer reduces the adhesion strength of renal epithelial cells, shown by a decrease in focal adhesion points and associated stress fibers. We showed in this study the importance of a human renal fetal and mature ECM catalogue for identifying promising ECM components that have high implementation potential in scaffolds for 3D renal constructs.

Introduction: Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human-induced pluripotent stem-cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of three-dimensional (3D) substrate properties under cyclic strain on phenotypic adaptation in hiPSC-derived VSMCs. Here, we aim to investigate the impact of intrinsic substrate properties, such as the hydrogel's elastic modulus and cross-linking density in a 3D static and dynamic environment, on the phenotypical adaptation of human mural cells derived from hiPSC-derived organoids (ODMCs), compared to aortic VSMCs. Methods and results: ODMCs were cultured in two-dimensional (2D) conditions with synthetic or contractile differentiation medium or in 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate Young's modulus and cross-linking density. Cells in 3D substrates were exposed to cyclic, unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity similar to primary human VSMCs. In static 3D culture, a substrate with a higher Young's modulus and cross-linking density promoted a contractile phenotype in ODMCs and VSMCs. Dynamic stimulation in the 3D substrate promoted a switch toward a contractile phenotype in both cell types. Conclusion: Our study demonstrates phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human VSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modeling and regenerative strategies.