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The spleen is a hematopoietic organ that participates in cellular and humoral immunity. It also serves as a quality control mechanism for removing senescent and/or poorly deformable red blood cells (RBCs) from circulation. Pitting is a specialized process by which the spleen extracts particles, including malaria parasites, from within circulating RBCs during their passage through the interendothelial slits (IES) in the splenic cords. To study this physiological function in vitro, we have developed two microfluidic devices modeling the IES, according to the hypothesis that at a certain range of mechanical stress on the RBC, regulated through both slit size and blood flow, would force it undergo the pitting process without affecting the cell integrity. To prove its functionality in replicating pitting of malaria parasites, we have performed a characterization of  P. falciparum -infected RBCs ( P.f. -RBCs) after their passage through the devices, determining hemolysis and the proportion of once-infected RBCs (O-iRBCs), defined by the presence of a parasite antigen and absence of DAPI staining of parasite DNA using a flow cytometry-based approach. The passage of  P.f. -RBCs through the devices at the physiological flow rate did not affect cell integrity and resulted in an increase of the frequency of O-iRBCs. Both microfluidic device models were capable to replicate the pitting of  P.f. -RBCs ex vivo by means of mechanical constraints without cellular involvement, shedding new insights on the role of the spleen in the pathophysiology of malaria.

Pitting of malaria parasites in microfluidic devices mimicking spleen interendothelial slits