Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium

Abstract

Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils' importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.

Keywords: endothelium; microfluidics; neutrophil; platelet; thromboinflammation.

Figure 1

Design of endothelialised microfluidic chip. (A) Top and (B) side views of the Polydimethylsiloxane (PDMS) chip. Each chip consists of an inlet well, straight channel and an outlet pin connected to the pump which perfuses the inlet fluid at pre-determined shear rates. An endothelial cell suspension is added to the well, perfused over the channel and allowed to create a monolayer on the surface.

Figure 2

Microchannel coated with tumour necrosis factor alpha (TNF-α) treated human umbilical vein endothelial cells (HUVECs). Confocal micrographs showing tile-scans of endothelialised devices stained for actin (red), nuclei (blue), and intercellular adhesion molecule 1 (ICAM-1) (green). Scale bars represent 100 µm.

Figure 3

Expression of surface adhesion proteins on human umbilical vein endothelial cells (HUVECs) in the endothelialised chips. (A) Confocal micrographs showing maximum intensity projections of z-stacks stained for nuclei (blue), intercellular adhesion molecule 1 (ICAM-1) (red), E-selectin (green) and (B) von Willebrand factor (vWF) (yellow) of non-treated (no tumour necrosis factor alpha (“No TNF-α”) and TNF-α treated (“TNF-α”) HUVECs-on chip. Scale bars represent 20 µm.

Figure 4

Effect of anti-coagulant on performance of the thromboinflammation chip. Representative images of fibrin (magenta), platelet (green), and neutrophil (red) adhesion on endothelialised chip (A) without or (B) with recalcification of citrated blood with 10 mM CaCl₂; (C) without or (D) with addition of protamine to heparinized blood. Scale bar represents 20 µm.

Figure 5

Effect of endothelial substrate on performance of the thromboinflammation chip. Representative images of fibrin (magenta), platelet (green), and neutrophil (red) adhesion on endothelialised chip on (A) fibronectin or (B) collagen substrate. Red arrows denote the contact of neutrophils with platelet aggregates. (C) Fibrin, (D) platelet surface coverage, and (E) neutrophils per ×40 field on fibronectin coated (black columns) versus collagen-coated (blue columns) endothelialised chips. Data is mean ± SD of six fields of view from n = 3 separate donors. * p < 0.05; N.S = non-significant by two-tailed, paired non-parametric t-test (Wilcoxon).

Figure 6

Effect of storage on performance of the thromboinflammation chip. Whole blood was perfused on endothelialised chips stored for one day versus seven days after fixation. (A). Fibrin (B) platelet surface coverage and (C) neutrophils per ×40 field on one-day-old (black columns) versus seven-day-old chips (blue columns) from HUVECs from the same cord (four cords in total). Data is mean ± SD of 12 fields of view from n = 4 separate donors. N.S = non-significant by two-tailed, non-parametric t-test (Wilcoxon).

Figure 7

Effect of shear rate on performance of the thromboinflammation chip. Whole blood was perfused on endothelialised chips for 4 min at low (100 s⁻¹) and high (700 s⁻¹) shear. Representative images of fibrin (magenta), platelet (green), and neutrophil (red) adhesion on endothelialised chip after perfusion of recalcified citrated blood, at (A) low (100 s⁻¹) versus (B) high (700 s⁻¹) shear. (C) Fibrin and (D) platelet surface coverage and (E) neutrophils per 40× field at 4 min after perfusion at 100 s⁻¹ (black columns) and 700 s⁻¹ shear (blue columns). Data is mean ± SD of nine fields of view from n = 3 separate donors. *** p < 0.001, N.S = non-significant by two-tailed, non-parametric t-test (Wilcoxon).

Figure 8

Effect of anti-inflammatory agents on thromboinflammation. (A). Representative images of fibrin (magenta), platelets (green), and neutrophils (red) on endothelialized chips. Endothelial cells were treated with tumour necrosis factor alpha (“TNF-α”) or without TNF-α (“No- TNF-α”). Whole blood was incubated with M1/70 (inhibitory antibody to Mac-1), G1 (inhibitory antibody to P-selectin) and isoquercetin. Scale bar represents 20 µm. (B). Fibrin, (C). platelet surface coverage and (D). neutrophils per x40 field in No TNF-α (black columns), TNF-α (dark blue columns), TNF-α in the presence of M1/0 (violet columns), TNF-α in the presence of G1 (magenta columns) and TNF-α in the presence of isoquercetin (light blue columns). Data represents mean ± SD of 3-9 fields of view per donor, n = 4–6. ** p < 0.01; *** p < 0.001; **** p < 0.0001, N.S = non-significant by one-way ANOVA with Dunnett’s post-hoc multiple comparison..