In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

Abstract

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease. 15. In addition to lung emboli, it is postulated that the pulmonary endothelium provides a prothrombotic environment in CTEPH that

High titers and low fucosylation of early human anti–SARS-CoV-2 IgG promote inflammation by alveolar macrophages

“…These macrophages were then detached with TrypLE Select (Gibco) for further treatment and stimulation. Pulmonary artery endothelial cells (PAEC) were obtained from resected pulmonary artery tissue, obtained from lobectomy surgery performed at Amsterdam UMC and isolated according to the previously published protocol (35). Briefly, the endothelial cell layer was carefully scraped onto fibronectincoated (5 μg/mL) culture dishes (Corning, #3295), and maintained in culture in endothelial cell medium (ECM, ScienCell, #1001) supplemented with 1% Penicillin/Streptomycin, 1% endothelial cell growth supplement (ECGS), 5% FBS, and 1% non-essential amino acids (NEAA, Biowest, #X055-100).…”

Anti-SARS-CoV-2 IgG from severely ill COVID-19 patients promotes macrophage hyper-inflammatory responses

“…In addition to excessive lung inflammation through a cytokine storm, severe COVID-19 is characterized by pulmonary edema, following disruption of the microvascular endothelium (18), and by coagulopathy, which in many patients is characterized by pulmonary thrombosis (19). To test whether the excessive macrophage activation by anti-Spike IgG may contribute to pulmonary edema and thrombosis, we applied in vitro models for endothelial barrier integrity (20) and in situ thrombosis (21) using primary human pulmonary artery endothelial cells (HPAEC), where thrombocytes can be added under flow conditions. While conditioned medium of macrophages that had been stimulated with only PolyIC induced a transient drop in endothelial barrier integrity, co-stimulation of macrophages with Spike protein and serum of severe COVID-19 patients induced long-lasting endothelial barrier disruption ( Figure 2A).…”

“…Pulmonary artery endothelial cells (PAEC) were obtained from resected pulmonary artery tissue, obtained from lobectomy surgery performed at Amsterdam UMC, location VU University Medical Center, The Netherlands and isolated according to the previously published protocol (21). Briefly, the endothelial cell layer was carefully scraped onto fibronectin-coated (5 µg/mL) culture dishes (Corning, #3295), and maintained in culture in endothelial cell medium (ECM, ScienCell, #1001) supplemented with 1% Penicillin/Streptomycin, 1% endothelial cell growth supplement (ECGS), 5% FBS, and 1% nonessential amino acids (NEAA, Biowest, #X055-100).…”

Platelet and myeloid cell phenotypes in a rat model of Fabry disease

“…A microfluidic adhesion assay was adapted from Manz et al 33 In brief, fibrinogen (341 576; MilleporeSigma) was immobilized by adding 20 µL of fibrinogen diluted in Hepes buffer to a final concentration of 50 µg/mL in each microfluidic channel of an Ibidi µ‐slide VI 0.4 chip (80601; Ibidi, Fitchburg, WI, USA). Microfluidic chips were then incubated overnight at 4°C inside of a 100 mm petri dish wrapped in parafilm to prevent evaporation.…”