## 2 Materials and Methods
### Microfluidic Microcirculation Mimetic  
**Microfluidic Microcirculation Mimetic:** We developed the MMM to mimic the advection of cells in the human capillaries, which often have constrictions smaller than blood cell diameters (Figure 1). To simulate VOC ex vivo, we induced hypoxic conditions that lead to VOC using sodium metabisulfite (SM), a reducing agent which promotes sickling [29]. We also mimicked treatment conditions by treating sRBC of patients with hydroxyurea (HU), an FDA-approved drug whose clinical benefit varies between patients and whose mechanism of action in the case of SCD is not completely understood [25,30]. The MMM differs from existing microfluidic devices and point-of-care technologies [31, 32, 33] that model blood vessels in the lungs and measure deformability in that it does not involve branches or channel networks but remains serial. We have maintained a serial model for two reasons. Firstly, from the \"viewpoint\" of the cell, constrictions occur one after the other, branches or no branches. Secondly, a serial model enables a very large number of constrictions (here, 187) within the field of view of the microscope objective, allowing real-time observation of physiologically or pathophysiologically relevant phenomena such as vaso-occlusion. Detailed descriptions of the MMM can be found in previous work where it has been used to study various cell types in health and diseases, including macrophages [16], neutrophils [11, 12], leukemia cells [13] and mesenchymal stromal cells [14]. Briefly, following conceptualization, AutoCAD software was used to design three variants of the MMM: one without constrictions (constant width of 15 \(\upmu\)m) and the others with 5 and 7 \(\upmu\)m as the smallest constriction widths. All variants have a constant height of 15 \(\upmu\)m. A polyester photomask of the design was printed commercially (Photo Data and J.D. Photo-Tools, JD Photo Data, Hitchin, UK, and more recently by Potomac Photonics, Halethorpe, MD, USA). Using the photomask, the master molds were fabricated at the Institute of Semiconductors and Microsystems of TU Dresden, Germany, and at Potomac Photonics, Halethorpe, Maryland, USA, following standard photolithographic techniques. These master molds can then be used hundreds of times to make the MMM chips when needed, using soft lithography [11, 12, 13]. For soft lithography (currently done in Creighton University, Omaha, NE, USA), PDMS (Sylgard 184, Dow Corning, Midland, TX, USA) is degassed by centrifugation (1200 rpm for 20 min), poured onto molds and baked for about 20 min at 100 \({}^{\circ}\)C. Inlet and outlet holes are punched (Harris Uni-core, 1.5 mm hole) on the cut and peeled PDMS chip. The chips are bonded to 22 mm glass cover slips (Nr 1, Marienfeld, Germany) using oxygen plasma (Plasma Cleaner PDC-32G, Harrick Plasma). The inlet of the MMM chip is connected to a programmable double-channel microfluidics syringe pump (NE-4002X, New Era, New York, NY, USA) using flexible polycarbonate tubing and 1 mL syringe containing about 1000 \(\upmu\)L of cell suspension, for each experiment (Figure 1). Cells are advected sequentially through the microchannel using volume flow rates (0.9, 9 and 99 \(\upmu\)L/h) that include the physiological blood flow range. For imaging, the device was mounted on a phase-contrast inverted microscope (Amscope, Ningbo, China) with 20\(\times\) and 40\(\times\) magnification objectives for measuring cell size and 4-5\(\times\) objective for measuring the transit time through the entire device. A CCD camera (DMx 21 BF04, Germany, Imaging Source, Charlotte, NC, USA) was used to record advection videos at a frame rate of 60 frames per second.