Microfluidic Models of the Microvasculature for Red Blood Cell Metrology in Sickle Cell Disease

The microvasculature plays an important role in the pathology of sickle cell disease. As erythrocytes deliver oxygen while traversing the arterial and capillary networks, deoxygenated sickle hemoglobin begins to polymerize into fibers, causing changes in erythrocyte mechanical and adhesive properties that contribute to the obstruction of capillaries and postcapillary venules. Micropipette aspiration, atomic force microscopy, optical tweezers, and other related experimental techniques have been used to measure mechanical and adhesive properties of erythrocytes, but these approaches require static conditions that ignore the effects of shear stress. More recently, microfluidic devices have been introduced that are capable of providing realistic shear rates, but employ channel diameters that are much larger than those of human capillaries. We used photolithography and soft lithography techniques to fabricate microfluidic devices from (poly)dimethylsiloxane that recapitulate the internal dimensions of human capillaries. The physical constraints imposed by these channels allow us to measure the dynamics of deformation and “reformation” ‐ how quickly a red cell recovers its biconcave disc shape upon exit from a capillary channel. In our devices, the initial inlet channel bifurcates five times, resulting in 32 capillaries that are 4 μm wide and 1 mm long. The channels are 2.5 μm tall, which orients the erythrocytes flat and keeps them within the focal plane of a microscope objective lens. We used high speed phase contrast video microscopy to record and analyze the cells under physiologically representative flow conditions at1% hematocrit. We measured the differences in erythrocyte elongation (the ratio of the maximum and minimum Feret diameter) and circularity (4π*Area/Perimeter 2 ), as well as characterizing the time to return from the constrained shape in the capillaries to the more relaxed discoid shape, in sickle cell patients and healthy controls under both oxygenated and deoxygenated conditions. These measurements provide quantitative assessment of erythrocyte deformability and shape recovery that we will use to study vascular occlusion that occurs in sickle cell disease. Support or Funding Information All samples were drawn from volunteers with informed consent under protocol 03‐H‐0015 as reviewed and approved by the National Heart, Lung, and Blood Institute's Institutional Review Board. This research was supported by the Division of Intramural Research of the National Heart, Lung, and Blood Institute and the Material Measurement Laboratory of the National Institute of Standards and Technology.