Tracking the dynamics of thrombus formation in a blood vessel-on-chip with visible-light optical coherence tomography

Abstract

Thrombus formation is a physiological response to damage in a blood vessel that relies on a complex interplay of platelets, coagulation factors, immune cells, and the vessel wall. The dynamics of thrombus formation are essential for a deeper understanding of many disease processes, like bleeding, wound healing, and thrombosis. However, monitoring thrombus formation is challenging due to the limited imaging options available to analyze flowing blood. In this work, we use a visible-light optical coherence tomography (vis-OCT) system to monitor the dynamic process of the formation of thrombi in a microfluidic blood vessel-on-chip (VoC) device. Inside the VoC, thrombi form in a channel lined with a monolayer of endothelial cells and perfused by human whole blood. We show that the correlation of the vis-OCT signal can be utilized as a marker for thrombus formation. By thresholding the correlation during thrombus formation, we track and quantify the growth of the thrombi over time. We validate our results with fluorescence microscopic imaging of fibrin and platelet markers at the end of the blood perfusion assay. In conclusion, we demonstrate that the correlation of the vis-OCT signal can be used to visualize both the spatial and temporal behavior of the thrombus formation in flowing human whole blood.

Fig. 1.

Overview of blood perfusion assay using OCT imaging. (a) The microfluidic chip is made from PDMS and contains 4 straight channels assessable separately. (b) The microfluidic chip consists of a $1$ cm long channel with $300\times 52$ $\mu$ m width and height. These channels are seeded with HUVECs and after 24 hours a monolayer has formed, then blood can be perfused through the channels. (c) Overview of blood perfusion set-up when imaging using OCT. (d) Brightfield image of the quiescent cell monolayer. (e) Fluorescent microscopy image of the monolayer distinguishing nuclei in blue and F-actin in green. (f) After the blood perfusion assay thrombus markers such as fibrin (in purple) and platelets (in green) are imaged using a fluorescent microscope. (g) Schematic illustration of the vis-OCT set-up used to monitor thrombus formation. L: lens, SC: supercontinuum source, F: low-pass filter, NDF: neutral density filter, BS: beam splitter, DC: dispersion compensation element, M: mirror, GS: galvanometer scanner, SL: scanning lens.

Fig. 2.

OCT intensity and correlation at selected times during the blood perfusion essay. (a) En-face and cross-sectional views of the VoC with flowing blood at the beginning of the blood perfusion assay, the channel boundary is visible in the cross-sectional views. (b) En-face view and cross-sections of the correlation map $C_t$ overlaid with the intensity in (a) derived with Eq. (1) from (a). En-face images in (a) and (b) were produced by averaging the values within the red box indicated in the cross-sections. (c) En-face intensity and (d) correlation as a function of time during the blood perfusion assay. Red arrows indicate the origin of a thrombus. The blue arrow indicates a region without clotting. A video of the entire perfusion assay can be found in Visualization 1.

Fig. 3.

Thrombus formation and validation with microscopy images. (a) OCT intensity of the VoC after flushing. (b) Last correlation map before flushing the VoC, exhibiting the presence of thrombi. The red arrow indicates a clotted region inside the channel. As pointed out by the blue arrow, thrombi do not occupy the entire height of the channel, allowing blood to flow in the upper part of the channel. (c) OCT images from the regions of interest I and II in (a) and (b), and respective comparison with images obtained in the microscope consisting of brightfield, fibrin and platelets markers, and the corresponding overlay.

Fig. 4.

Thrombus growth and evolution over time. (a) Color-coded location of clotted blood over time. The color represents the time at which a $C_t\ge 0.4$ was registered. The clotted area increases over time as the assay progresses. The purple arrows indicate the presumed origin of two thrombi. (b) Violin plot showing the distribution of thrombi over time within the red box in (a) (excluding the flow channel boundaries). As the assay progresses, bigger clots appear as red blood cell aggregates grow. (c) Total percentage area of the channel with thrombi as a function of time. A video of the thrombus growth can be found in Visualization 2.