Independent parallel production of tunable blood clot analogues in hourglass-profiled circular PDMS fluidic channels

Rapid removal of occluding blood clots, whether by intravenous thrombolysis or endovascular thrombectomy, can be life-saving. Yet recanalization is not always successful, and the determinants remain incompletely understood. Thrombi exhibit heterogeneous compositions, while clinical samples and animal models are costly, variable, and often lack reproducibility. To address these challenges, we present a blood clot analogue production system capable of generating tunable clot analogues in parallel under precisely regulated flow and calcium ion conditions. The platform integrates one or multiple pressure-controlled fluidic loops operating independently with a single pump and multiple hourglass-profiled circular devices fabricated from poly(dimethylsiloxane) (PDMS), a gas-permeable material that replicates vascular occlusion hemodynamics. Using this system, clot analogues measuring approximately 0.8 cm to over 2.5 cm in length were generated, displaying heterogeneous upstream-constriction-downstream architectures in which the red blood cell (RBC)-fibrin fraction could be tuned between 23% and 76%, closely mirroring clinical thrombi. The composition was modulated through recalcification under various CaCl₂ concentrations (1.8-16.5 mM) and shear rates (10⁴-10⁶ s^-1). At higher Ca²⁺ levels (≥6.6 mM), compact RBC-fibrin-dominated clots formed rapidly (<10 minutes), displaying laminar "Zahn line" structures. In contrast, lower Ca²⁺ conditions (1.8-2.3 mM) delayed occlusion (>40 minutes) and yielded diffuse neutrophil extracellular trap (NET)-platelet networks. The system operates stably for >1000 minutes with recirculating blood, ensuring reproducibility and cost-effectiveness while reducing reliance on animal thrombosis models in accordance with the 3R (replacement, reduction, and refinement) principles. Collectively, this platform provides a controllable, ethically responsible, and physiologically relevant model for thrombogenesis and thrombectomy research.