Abstract
IntroductionStudying arterial thrombus formation by in vitro flow assays is a widely used approach. Incorporating human atherosclerotic plaque material as a thrombogenic surface in these assays represents a method to model the pathophysiological environment of thrombus formation upon plaque disruption. Up until now, achieving a homogeneous coating of plaque material and subsequent reproducible platelet adhesion has been challenging. Here, we characterized a novel method for coating of plaque material on glass coverslips for use in thrombosis microfluidic assays.MethodsA homogenate of human atherosclerotic plaques was coated on glass coverslips by conventional manual droplet coating or by spin coating. Prior to coating, a subset of coverslips was plasma treated. Water contact angle measurements were performed as an indicator for the hydrophilicity of the coverslips. Homogeneity of plaque coatings was determined using profilometric analysis and scanning electron microscopy. Thrombogenicity of the plaque material was assessed in real time by microscopic imaging while perfusing whole blood at a shear rate of 1500 s−1 over the plaque material.ResultsPlasma treatment of glass coverslips, prior to spin coating with plaque material, increased the hydrophilicity of the coverslip compared to no plasma treatment. The most homogeneous plaque coating and highest platelet adhesion was obtained upon plasma treatment followed by spin coating of the plaque material. Manual plaque coating on non-plasma treated coverslips yielded lowest coating homogeneity and platelet adhesion and activation.ConclusionSpin coating of atherosclerotic plaque material on plasma treated coverslips leads to a more homogenous coating and improved platelet adhesion to the plaque when compared to conventional droplet coating on non-plasma treated coverslips. These properties are beneficial in ensuring the quality and reproducibility of flow experiments.
Highlights
Studying arterial thrombus formation by in vitro flow assays is a widely used approach
To improve mimicking of pathophysiological processes of arterial thrombosis in in vitro flow assays, human atherosclerotic plaque tissue can be used as a thrombogenic surface
Homogenates were coated on the glass using four different methods, (1) the droplet method, (2) additional plasma treatment of the glass coverslip before droplet method, (3) plasma treatment of the glass coverslip combined with spin coating of the plaque homogenate and (4) spin coating of the plaque homogenate without plasma treatment
Summary
Studying arterial thrombus formation by in vitro flow assays is a widely used approach. Results—Plasma treatment of glass coverslips, prior to spin coating with plaque material, increased the hydrophilicity of the coverslip compared to no plasma treatment. Manual plaque coating on non-plasma treated coverslips yielded lowest coating homogeneity and platelet adhesion and activation. Conclusion—Spin coating of atherosclerotic plaque material on plasma treated coverslips leads to a more homogenous coating and improved platelet adhesion to the plaque when. Over the past few years there has been an increasing interest to study thrombus formation using in vitro flow assays. With this technique whole blood is perfused over fibrillar collagen through a channel at predefined shear rates.
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