High-throughput biomimetic cycling of red blood cells: elucidating the morpho-mechanical determinants of fatigue and clearance.

During their 120-day circulatory lifespan, red blood cells (RBCs) undergo repeated mechanical deformation as they traverse microcapillaries and splenic inter-endothelial slits (IES). This cyclic mechanical loading gradually impairs RBC deformability, ultimately leading to their clearance by the spleen. However, current platforms for investigating RBC fatigue often couple mechanical loading with real-time observation, which obscures the cumulative impact of cyclic strain. To address this limitation, we developed an integrated microfluidic chip equipped with a dedicated "S"-shaped fatigue zone-each RBC experiences hundreds of extrusion events during a single continuous pass through this zone-followed by a physically decoupled observation zone. This design enables clear separation of fatigue induction from biomechanical evaluation. Our findings show that cyclic extrusion drives a progressive morphological transition in the RBC population from discocytes to echinocytes and spherocytes, along with reduced cell volume and surface area, increased membrane shear modulus, and elevated sphericity. Combined experiments and simulations reveal that the passage of spherocytes depends not only on their deformability but also critically on the relative size of the cells versus the channel dimensions. Furthermore, simulations of splenic filtration identify the sphericity index-not membrane stiffness-as the primary geometric factor governing RBC retention in IES. This work presents a high-throughput, label-free platform that disentangles RBC fatigue induction from post-fatigue analysis. It provides mechanistic insights into how repetitive mechanical stress regulates RBC aging and clearance, offering a valuable tool for advancing our understanding of RBC physiology in health and disease.