A. Fabrication of microfluidic devices and experimental procedure

A microfluidic device [VA (viscosity-aggregation)] for measuring blood viscosity and RBC aggregation was composed of two inlets (A and B), one outlet (A), and a straight channel (width = 2 mm, length = 12 mm, and depth = 100 μm). Inlet (A) is connected to the middle position of the straight channel. In addition, the other microfluidic device [FD (fluid divider)] was designed to have four ports, and two identical side channels (width = 3 mm, length = 12 mm, and depth = 100 μm) connected with a bridge channel. A silicon-master mold was fabricated using conventional micro-electromechanical-system fabrication techniques, including photolithography and deep-reactive-ion etching. To conduct soft lithography with a silicon-master mold, polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning, Midland, MI, USA) was mixed with a curing agent at a ratio of 10:1. The PDMS mixture was then poured into the silicon-master mold placed in a Petri dish. Air bubbles dissolved in PDMS were completely removed with a vacuum pump. After curing the PDMS in a convection oven at 70 °C for 1 h, the PDMS block was peeled off from the silicon-master mold. The PDMS block was cut with a razor blade. Two inlets (A, B) and one outlet (A) of the VA were punched with a biopsy punch (outer diameter = 0.75 mm). The right-upper port of the FD was punched with a biopsy punch (outer diameter = 0.75 mm). The three other ports of the FD were punched with a biopsy punch (outer diameter = 1 mm). After treating the PDMS block and a glass substrate with oxygen plasma (CUTE-MPR, Femto Science Co., Korea), the VA and FD were prepared by bonding the PDMS block to the glass substrate. The microfluidic device was then placed on an optical microscope (BX51, Olympus, Japan) equipped with a 4× objective lens (NA = 0.1). After 1% bovine serum albumin (BSA) solution (∼5 ml) was dropped into a reservoir, the peristaltic pump was operated at rotation speed of ω = 15 rpm for 10 min. After the BSA solution was completely removed from the in-vitro closed-loop, blood (∼10 ml) was dropped into the reservoir.

Figure ​Figure11 shows the schematics for the periodic and simultaneous measurement of blood viscosity and RBC aggregation using a microfluidic platform under in-vitro closed-loop circulation. As shown in Fig. 1(A), the experimental setup was composed of in-vitro closed-loop circulation and a microfluidic platform, which was intended to quantify the blood viscosity and RBC aggregation. As depicted in Fig. (1A-a), in-vitro closed-loop circulation was established by connecting several components [peristaltic pump (Techpre pump, Jeniwell, Seoul, Korea), air compliance unit (ACU), fluid divider (FD), and reservoir] with polyethylene tubes (L₅, L₆, L₇, L₈, and L₉) in series. Here, the dimensions for each tube were as shown in Table S1 (supplementary material). The blood was then circulated by operating the peristaltic pump under an in-vitro closed-loop. To remove the alternating component of the blood flow-rate generated by operating the peristaltic pump, the air cavity in the ACU is adjusted to 2.5 ml with a disposable syringe (3 ml, Kovax-Syringe, Korea Vaccine, Korea).^44,45 As shown in Fig. S1 (supplementary material), the blood flow-rate remains constant with time (i.e., Q_Blood = 40.85 ± 0.59 ml/h) under in-vitro closed-loop circulation. To stimulate RBC aggregation in blood, a specific concentration of dextran solution (i.e., C_dextran = 100 mg/ml) was periodically delivered into the reservoir by operating a syringe pump. Here, a polyethylene tube (L₄) is connected from the driving syringe to the reservoir. As shown in Fig. 1(A-b), a microfluidic platform consists of a microfluidic device (VA), pinch valve (PV) and a syringe pump (neMESYS, Centoni Gmbh, Germany) for supplying phosphate-buffered saline (PBS) solution (1×, pH 7.4, Gibco, Life Technologies, USA). For convenience, the upper and lower areas of the straight channel are named the upper channel and lower channel, respectively. The outlet of the FD was connected to inlet (B) of the VA with a polyethylene tube (L₂). To control the blood flow from the FD to the microfluidic device (i.e., open or close), the PV was installed in front of inlet (B) of the VA. 1× PBS (Phosphate-buffered saline) was precisely supplied into inlet (A) of the VA with the syringe pump.

Schematics for the periodic and simultaneous measurement of blood viscosity RBC aggregation using a microfluidic platform under in-vitro closed-loop circulation. (A) Schematics of the experimental setup including an in-vitro closed-loop circulation and a microfluidic platform. (a) In-vitro closed-loop circulation established by connecting several components (peristaltic pump, ACU, FD, and reservoir) with polyethylene tubes (L₅, L₆, L₇, L₈, and L₉) in series. After the reservoir was filled with blood (10 ml), a peristaltic pump was set to ω = 15 rpm. The air cavity in the ACU was adjusted to 2.5 ml. A specific concentration of dextran solution (i.e., C_dextran = 100 mg/ml) was delivered into the reservoir by operating the syringe pump. (b) A microfluidic platform including a microfluidic device (VA), PV, and syringe pump. The outlet of the FD was connected to the inlet (B) of the VA with a polyethylene tube (L₂). To control the blood flow from the FD to the VA, a PV was installed in front of the inlet (B). 1× PBS solution was supplied into inlet (A) with a syringe pump. (B) Flow-rate profile of two fluids with a syringe pump (i.e., Q_PBS and Q_Dextran) and PV operation (i.e., open and close) for each period. (C) Quantification of image intensity (⟨I⟩) and blood flow-rate (Q_μPIV). (a) Quantification of the image intensity (⟨I⟩) within the ROI (484 × 150 pixels) in the upper channel. (b) Quantification of blood velocity fields with a time-resolved micro-PIV technique. The blood flow-rate (Q_μPIV) was quantified by multiplying averaged blood velocity (⟨U⟩) by the cross-sectional area (A_c) (i.e., Q_μPIV = ⟨U⟩·A_c). (c) A simple fluidic circuit with discrete elements including fluidic resistances (R_P, R_B), and flow rates (Q_PBS, Q_Blood). The interface between two fluids was evaluated by converting the gray image into a binary image. (D) For a preliminary demonstration, blood (Hct = 50%) was prepared by adding normal RBCs to autologous plasma. Here, the period was set to T = 400 s. The inset shows microscopic images at a specific time (t) [(a) t = 10 s, (b) t = 100 s, (c) t = 150 s, (d) t = 200 s, (e) t = 300 s, and (f) t = 400 s]. During each period, at constant blood flow (i.e., 0 < t < 100 s), the blood viscosity was quantified with the MPFM. Afterwards, at stationary blood flow (i.e., 100 s < t < 400 s), the RBC aggregation was quantified by evaluating variations in image intensity with respect to time.

Figure 1(B) represents the flow-rate profile of PBS and dextran solutions (i.e., Q_PBS and Q_Dextran), and operation of PV (i.e., open, and close) for every period. To measure the RBC aggregation and blood viscosity sequentially, the blood flow-rate was synchronized with the PBS flow-rate. PBS solution was supplied as a reference fluid at a flow rate of Q_PBS = Q₀ from t = 0 to t = 0.25 T. Here, T denoted the period. The blood flow-rate was stopped or flowed by operating the PV (Supa clip, Pankyo, Korea). In other words, the PV was only opened from t = 0 to t = 0.25 T for every period. The PV was closed for the remaining duration of each period. To stimulate the RBC aggregation of blood circulating under in-vitro closed loop, a specific concentration of dextran solution was delivered at the flow rate of Q_Dextran = Q₀ from t = 0.5 T to t = 0.75 T. All the experiments were conducted at a constant temperature (25 °C).