High Content Evaluation of Shear Dependent Platelet Function in a Microfluidic Flow Assay
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High Content Evaluation of Shear Dependent Platelet Function in a Microfluidic Flow Assay RYAN R. HANSEN,1 ADAM R. WUFSUS,1 STEVEN T. BARTON,1 ABIMBOLA A. ONASOGA,1 REBECCA M. JOHNSON-PABEN,1 and KEITH B. NEEVES1,2 1
Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, CO 80401, USA; and 2Department of Pediatrics, University of Colorado, Aurora, CO 80045, USA (Received 21 June 2012; accepted 11 September 2012; published online 22 September 2012) Associate Editor Michael R. King oversaw the review of this article.
motivation for developing flow-based in vitro methods of measuring platelet function. Flow-based devices for measuring platelet function include annular chambers, parallel-plate chambers, or cone-and-plate viscometers.18 However, the high volume requirements (10–100 mL) and low throughput (one shear rate per experiment) of these devices make them unsuitable for drug screening and clinical applications. Microfluidic devices have emerged as a suitable alternative to these conventional flow assays because of their decreased volume requirement.22 A particularly desirable characteristic is the simultaneous analysis of platelet function over the range of physiologic shear stresses. Gutierrez et al.10 reported a multishear microfluidic device capable of spanning a 100-fold range of shear rates over surfaces coated with fibrinogen and type 1 collagen. Commercial microfluidic systems are now available and have been used to study shear dependent platelet function.6,21 Another advantage of microfluidic approaches is that channel geometries can be defined to mimic vascular features such as stenosed vessels and valves.14,25,30 The throughput of flow assays can be increased using micropatterning methods to define many focal ‘‘injury’’ sites within a single channel. For example, Okorie and Diamond19 used a microarray pin-tool to define hundreds of collagen or collagen-von Willebrand factor (VWF) spots within a parallel plate flow chamber. The advantage of this pin-tool approach is that the composition of each spot can be different. The disadvantage is that the spot size is limited to greater than 150 lm, and therefore, may not be compatible with microfluidic channels that have dimensions on the order of 10–100 lm. Microfluidic patterning has been used to define 50–250 lm lines of collagen and VWF across the entire width of microfluidic channels in
Abstract—The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 lL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50–920 s21). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 lm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater
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