Physical mechanisms of red blood cell splenic filtration

Introduction: The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 μm RBCs pass through 0.3 μm-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 μm-wide rigid slits at 37 °C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28% of RBCs in a 0.4 μm-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a 1 and 3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Materials and

Methods: The microfluidic device was made of polydimethylsiloxaneand consisted of a PDMS chip, molded from a silicon master, and covalently assembled to a PDMS-coated glass coverslip. The microfluidic device was placed on an inverted microscope (Olympus IX71) equipped with 20×/60×/100× objectives, temperature controller, and cameras. The device surfaces were coated with 1% BSA before injection of 1% Hct suspensions. The flow of the RBC suspension was pressure-controlled (MFCS-8C, Fluigent). Typically, RBCs were observed in brightfield microscopy and movies were acquired at a high frame rate using a high-speed camera (Fastcam Mini camera, Photron).

For dynamic simulations, we applied an in-house code with coupled finite element (FEM) and boundary integral methods to study the dynamics of RBCs passing through the slits and their transit time with full fluid–structure interaction considered. We simulated the full fluid–cell interaction under prescribed pressure drop. The boundary integral method was used to simulate the surrounding Stokes flows, while the FEM was used to model the RBC lipid bilayer and cytoskeleton as two interacting shells. The same mesh of lipid bilayer was used in finite element and boundary integral simulations to satisfy the velocity and stress jump conditions on the interface. For quasi-static simulations, we applied the commercial FEM package ABAQUS to study the retention of RBCs in the slits and the corresponding critical pressure drop without modeling the fluid flow explicitly because the dynamics, such as viscosity, does not play a role in the critical pressure, and the quasi-static approach is much more efficient.

Results, Conclusions, and Discussions: We found that the first splenic selection of RBC deformability is of geometrical origin. The strong dumbbell cell deformation with a very thin neck inside the slit and spherical caps at the entrance and exit of the slit that we evidenced indeed requires that the RBC surface area-to-volume ratio be higher than that of two equal spheres. This is observed, strikingly, on circulating RBCs. Even more striking is that the surface area-to-volume ratio of circulating RBCs is not more than 15% larger than that of two equal spheres. Thus, there is no additional benefit to having more excess surface area than that required to pass through the slits. Yet, a greater excess of surface area would decrease the membrane tension and limit the need for spectrin unfolding in the slits. This is not the case retained by nature, maybe because an excess of surface area has a high biological cost or is quickly lost by membrane vesiculation as RBCs pass through the slits. The second splenic selection of RBC deformability is of mechanical origin. We targeted the two identified pathways potentially able to dynamically modify RBC mechanical properties. The first pathway involves the mechanosensitive PIEZO1 channel known to participate in regulating RBC volume. Our experimental results strongly suggest that no PIEZO1 effect is involved in the RBC passage. This may be due to the too short transit times of RBCs through the slits that do not allow a significant calcium influx. Indeed, most RBC transit times were less than one hundred milliseconds under physiological conditions in agreement with those previously observed for mice . The second pathway involves the spectrin network, which underlies the RBC membrane and is known to unfold under an external mechanical stress. We used an in silico approach, as, to our knowledge, there is no direct method for visualizing spectrin unfolding in nonmodified living RBCs. It shows that local spectrin unfolding within the narrowest slits is crucial for the passage of RBCs with less surface area.

Acknowledgements (Optional): The project leading to this publication has received funding from France 2030, the French Government program managed by the French National Research Agency (ANR-16-CONV-0001 and ANR-20-CE17-0024). We thank I. Ozerov and F. Bedu from PLANETE microfabrication facility. We thank Pr. Pierre Buffet and his group for providing us spherocytic and irreversible sickle RBCs. Z.P. and H.L. acknowledge the supports from US NSF grants NSF CBET-1706436/1948347 and NSF DMS-1951526. E.H., A.V., and Z.P. are also supported by a joint NSF-ANR grant (NSF-PHY2210366 and ANR-22-CE95-0004-01). E.H. belongs to the French Consortium Approches Quantitatives du Vivant/Quantitative approaches to living systems.