Embedded macrophages induce intravascular coagulation in 3D blood vessel-on-chip

Abstract

Macrophages are innate immune cells that prevent infections and help in wound healing and vascular inflammation. While these cells are natural helper cells, they also contribute to chronic diseases, e.g., by infiltrating the endothelial layer in early atherosclerosis and by promoting vascular inflammation. There is a crosstalk between inflammatory pathways and key players in thrombosis, such as platelets and endothelial cells - a phenomenon known as 'thromboinflammation'. The role of the embedded macrophages in thromboinflammation in the context of vascular disease is incompletely understood. Blood vessels-on-chips, which are microfluidic vascular cell culture models, have been used extensively to study aspects of vascular disease, like permeability, immune cell adhesion and thrombosis. Blood perfusion assays in blood vessel-on-chip models benefit from multiple unique aspects of the models, such as control of microvessel structure and well-defined flow patterns, as well as the ability to perform live imaging. However, due to their simplified nature, blood vessels-on-chip models have not yet been used to capture the complex cellular crosstalk that is important in thromboinflammation. Using induced pluripotent stem cell-derived endothelial cells and polarized THP-1 monocytes, we have developed and systematically set up a 3D blood vessel-on-chip with embedded (lipid-laden) macrophages, which is created using sequential cell seeding in viscous finger patterned collagen hydrogels. We have set up a human whole blood perfusion assay for these 3D blood vessels-on-chip. An increased deposition of fibrin in the blood vessel-on-chip models containing lipid-laden macrophages was observed. We anticipate the future use of this advanced vascular in vitro model in drug development for early atherosclerosis or aspects of other vascular diseases.

Keywords: Macrophage; Organ-on-chip; THP-1; Thromboinflammation; Vessel-on-chip; hiPSC-EC.

Fig. 1

Protocol for establishing a complex 3D VoC, including characterization of its key components. a: Schematic overview of the protocol. 3D lumens are created in squared 500 × 500 μm collagen-filled channels. These channels are subsequently seeded with (lipid-laden) macrophages and hiPSC-EC; creating a 3D hiPSC-EC derived perfusable 3D blood vessel-on-chip with embedded macrophages. b: Viscous finger patterning is used to create a 3D lumen inside a channel filled with collagen type-1. Lumens can be perfused with microbeads (arrows). Scale bars represent 250 μm. c: Endothelial cell markers such as von Willebrand factor and vascular endothelial-cadherin (VE-cadherin) are present in hiPSC-derived endothelial cells. Scale bar represents 20 μm. d: Polarized THP-1 macrophages. Scale bar represents 100 μm. e: Polarized THP-1 macrophages can be loaded with Ox-LDL (green). Scale bar represents 100 μm. f: Percentage of polarized, lipid-laden macrophages as analyzed by fluorescence microscopy at multiple timepoints (‘Day 1’, ‘Day 2’, ‘Day 6’) and in the continued presence (‘PMA’) or absence (‘nPMA’) of the polarizing phorbol myristate acetate (PMA). Day 6 represents a combination of ‘PMA’ and ‘nPMA’

Fig. 2

Embedded lipid-laden macrophages in 3D blood vessel-on-chip. a: Collagen lumen (dashed lines) loaded with lipid-laden macrophages (green Dil-Ox-LDL) seeded at 1·10⁵ cells/ml. b: After lipid-laden macrophages were incubated for 30 minutes, hiPSC-EC were seeded to the channel at 5·10⁶ cells/ml. Lipid laden-macrophages are visibly embedded underneath the hiPSC-EC suspension (green Dil-Ox-LDL). c: Example of endothelial cell sprouting. d: Number of sprouts observed in three representative figures of a vessel lumen, in conditions with only endothelial medium (‘0:1 RPMI:EGM-2’) or a mixture of endothelial and macrophage medium (‘1:1 RPMI:EGM-2’). e: Percentage of the vessel area covered with green, fluorescent signal when seeding macrophages at multiple concentrations. f: Effect of seeding densities of macrophages on monolayer quality (‘Monolayer score’) of hiPSC-EC after 24 hours of incubation, with a bad monolayer scored as a 1 and a very good monolayer as 5. g: 3D confocal images show a monolayer of hiPSC-EC in the channel after 5 days of co-culture. Ox-LDL is still visibly embedded in the endothelial layer after 5 days of incubation (Nuclei: white; Actin: Red; Ox-LDL: Green). h: Endothelial layer assessment, with i: healthy endothelial layer (control); ii: macrophage embedded endothelial layer (sample) and iii: TNF-α treated endothelial layer (inflamed). In i and ii a cortical actin distribution can be observed, whereas iii shows an actin redistribution towards stress fibers (Nuclei: white; Actin: Red; Ox-LDL: Green). All scale bars represent 100 μm

Fig. 3

Blood perfusion assay results in a higher deposition of fibrin in the channels containing embedded macrophages; a: Blood vessel channel with a monoculture of hiPSC-EC (‘Control’). b: Blood vessel channel with co-cultured macrophages with a high % area coverage of fibrin; c,d: fibrin clot size and % area of fibrin coverage in monoculture (‘Control’) conditions and co-culture (‘Embedded macrophages) conditions. All scale bars represent 150 μm