02546nas a2200253   4500000000100000008004100001260001500042100001600057700002200073700001900095700001400114700001600128700001400144700002000158700002600178700002100204700001700225700001300242245013800255856005300393300001600446490000800462520182200470       2023                            d  c2023-02-071 aYuhao Qiang1 aAbdoulaye Sissoko1 aZixiang L. Liu1 aTing Dong1 aFuyin Zheng1 aFang Kong1 aJohn M. Higgins1 aGeorge E. Karniadakis1 aPierre A. Buffet1 aSubra Suresh1 aMing Dao00aMicrofluidic study of retention and elimination of abnormal red blood cells by human spleen with implications for sickle cell disease  uhttps://www.pnas.org/doi/10.1073/pnas.2217607120  ae22176071200 v1203 aThe spleen clears altered red blood cells (RBCs) from circulation, contributing to the balance between RBC formation (erythropoiesis) and removal. The splenic RBC retention and elimination occur predominantly in open circulation where RBCs flow through macrophages and inter-endothelial slits (IESs). The mechanisms underlying and interconnecting these processes significantly impact clinical outcomes. In sickle cell disease (SCD), blockage of intrasplenic sickled RBCs is observed in infants splenectomized due to acute splenic sequestration crisis (ASSC). This life-threatening RBC pooling and organ swelling event is plausibly triggered or enhanced by intra-tissular hypoxia. We present an oxygen-mediated spleen-on-a-chip platform for in vitro investigations of the homeostatic balance in the spleen. To demonstrate and validate the benefits of this general microfluidic platform, we focus on SCD and study the effects of hypoxia on splenic RBC retention and elimination. We observe that RBC retention by IESs and RBC–macrophage adhesion are faster in blood samples from SCD patients than those from healthy subjects. This difference is markedly exacerbated under hypoxia. Moreover, the sickled RBCs under hypoxia show distinctly different phagocytosis processes from those non-sickled RBCs under hypoxia or normoxia. We find that reoxygenation significantly alleviates RBC retention at IESs, and leads to rapid unsickling and fragmentation of the ingested sickled RBCs inside macrophages. These results provide unique mechanistic insights into how the spleen maintains its homeostatic balance between splenic RBC retention and elimination, and shed light on how disruptions in this balance could lead to anemia, splenomegaly, and ASSC in SCD and possible clinical manifestations in other hematologic diseases.