Abstract3D‐scaffold based in vitro human tissue models accelerate disease studies and screening of pharmaceutics while improving the clinical translation of findings. Here is reported the use of human induced pluripotent stem cell (hiPSC)‐derived vascular organoid cells as a new cell source for the creation of an electrospun polycaprolactone‐bisurea (PCL‐BU) 3D‐scaffold‐based, perfused human macrovessel model. A separation protocol is developed to obtain monocultures of organoid‐derived endothelial cells (ODECs) and mural cells (ODMCs) from hiPSC vascular organoids. Shear stress responses of ODECs versus HUVECs and barrier function (by trans endothelial electrical resistance) are measured. PCL‐BU scaffolds are seeded with ODECs and ODMCs, and tissue organization and flow adaptation are evaluated in a perfused bioreactor system. ODECs and ODMCs harvested from vascular organoids can be cryopreserved and expanded without loss of cell purity and proliferative capacity. ODECs are shear stress responsive and establish a functional barrier that self‐restores after the thrombin challenge. Static bioreactor culture of ODECs/ODMCs seeded scaffolds results in a biomimetic vascular bi‐layer hierarchy, which is preserved under laminar flow similar to scaffolds seeded with primary vascular cells. HiPSC‐derived vascular organoids can be used as a source of functional, flow‐adaptive vascular cells for the creation of 3D‐scaffold based human macrovascular models.
Background: Atherosclerosis is a complex inflammatory vascular disease characterized by lipid and immune cells accumulation in the vessel wall, leading to lumen narrowing. Although several 3D in vitro microfluidic systems were previously described, a realistic reconstruction of the in vivo human atherosclerotic environment requires co-culture of different cell types arranged in atherosclerotic vessel-like structures with exposure to flow and circulating cells, creating challenges for disease modelling. In this study we developed a 3D tubular microfluidic model with quadruple coculture of human aortic smooth muscle cells (hAoSMCs), human umbilical cord vein endothelial cells (HUVECs) and foam cells to re-create a complex human atherosclerotic vessel in vitro to study the effect of flow and circulating immune cells. Methods & Results: Our new co-culture protocol with BFP-labelled hAoSMCs, GFP-labelled HUVECs and THP-1 macrophages-derived, Dil-labelled Oxidized Low-Density Lipoprotein (Dil-Ox-LDL) foam cells in a fibrinogen-collagen-I based 3D extracellular matrix (ECM) resulted in vessels with an early lesion morphology, showing a layered vessel-like composition with an endothelium and media, with foam cells accumulating in the sub-endothelial space. Perfusion for 24 hours of atherosclerotic and "healthy" vessels (BFP hAoSMCs and GFP HUVECs without foam cells) showed that the layered wall composition remained stable. Perfusion with circulating THP-1 monocytes demonstrated cell extravasation into the atherosclerotic vessel wall and recruitment of THP-1 cells to the foam cell core. QPCR analysis revealed increased expression of atherosclerosis markers in the atherosclerotic vessels and adaptation in VSMCs migration to flow and the plaque microenvironment, compared to control vessels. Conclusion: We present a 3D tubular microfluidic model of a complex early atherosclerotic human vessel that can be exposed to flow and circulating THP-1 monocytes to study hemodynamic changes and immune cell recruitment under live confocal imaging. This novel atherosclerosis-on-a-chip model offers a humanized platform for in-depth mechanistic in vitro studies and drug testing.
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