Well plate microfluidic system for investigation of dynamic platelet behavior under variable shear loads

Abstract

The study of platelet behavior in real-time under controlled shear stress offers insights into the underlying mechanisms of many vascular diseases and enables evaluation of platelet-focused therapeutics. The two most common methods used to study platelet behavior at the vessel wall under uniform shear flow are parallel plate flow chambers and cone-plate viscometers. Typically, these methods are difficult to use, lack experimental flexibility, provide low data content, are low in throughput, and require large reagent volumes. Here, we report a well plate microfluidic (WPM)-based system that offers high throughput, low reagent consumption, and high experimental flexibility in an easy to use well plate format. The system consists of well plates with an integrated array of microfluidic channels, a pneumatic interface, an automated microscope, and software. This WPM system was used to investigate dynamic platelet behavior under shear stress. Multiple channel designs are presented and tested for shear loads with whole blood to determine their applicability to study thrombus formation. Normal physiological shear (0.1-20 dyn/cm(2) ) and pathological shear (20-200 dyn/cm(2) ) devices were used to study platelet behavior in vitro under various shear, matrix coating, and monolayer conditions. The high physiological relevance, low blood consumption, and increased throughput create a valuable technique available to vascular biology researchers. The approach also has extensibility to other research areas including inflammation, cancer biology, and developmental/stem cell research.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

“…[2][3][4] Several attempts at developing a high-throughput platelet function assay are currently being investigated. These include devices that measure global platelet reactivity in response to shear alone, 5 microfluidic devices with precoated adhesion or activation molecules, [6][7][8] assays that measure calcium flux in platelets by fluorescent imaging, 9 enzyme-linked immunosorbent assay-type assays to capture platelets on agonist-coated surfaces, 10 luminometric assays of platelet secretion in response to various platelet agonists, 11 and flow cytometric counting techniques with platelet immunostaining. 12 Most of these techniques require specialized laboratory instruments (flow cytometers, osmotic pumps, microscopes, imaging devices) or can be expensive to perform (single use precoated cartridges or capillaries, fluorescent antibodies), and not all are sensitive to mild platelet inhibition, 10 making them unsuitable for use in nonspecialized centers.…”

Functional assay of antiplatelet drugs based on margination of platelets in flowing blood

“…Recent years have also seen the rise in use of microfluidic flow cells as essential tools for studying platelet activation and adhesion. 19,20 Gutierrez et al used extracellular matrix (ECM) coated substrates to investigate the influence of elevated shear rates on platelet adhesion with respect to the GPIIb/IIIa receptor. 21 Similar assays have also been used by Maloney et al to investigate patientspecific responses to antiplatelet therapies.…”

Mechanobiology of Platelets: Techniques to Study the Role of Fluid Flow and Platelet Retraction Forces at the Micro- and Nano-Scale

“…A variety of environments can be introduced under these shear rates, using patterning techniques to create wound-like environments with well-defined areas of adhesive protein (Figure 3a) [77]. They have enabled studies on aggregate formation to be run with multiple experiments simultaneously (Figure 3b) [74,82] or with controlled concentration of agonists (Figure 3c) [73]. Of particular noteworthiness, a novel microfluidic device has been used to investigate aggregate formation after exposure to micro-gradients in the shear rate (Figure 3d) [24,75].…”

“…Finally, microfluidic devices have been used to run parallel assays under different conditions [74,77,82]. Due to the fact that they work with very low blood volumes they can be used to run parallel varying a parameter such as shear rate [77,84], chemicals including inhibitors (Figure 3e) [83], and matrix coatings [82].…”