Pitting of malaria parasites in microfluidic devices mimicking spleen interendothelial slits

Abstract

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.

Biophysical Tools and Concepts Enable Understanding of Asexual Blood Stage Malaria

“…Additionally, microfluidic devices have been used to mimic spleen filtration and the process of pitting with an array of rectangular pillars of PDMS with tuneable slit sizes down to few micrometres ( Figure 3A ) ( Rigat-Brugarolas, 2014 ). Elizalde-Torrent and colleagues showed that by regulating the slit size and the blood flow it is possible to remove the solid parasite from the cytoplasm of an iRBC without destructing the cell ( Elizalde-Torrent et al., 2021 ). Taken together, Pf- iRBCs become more rigid as the parasite progresses through the blood developmental cycle, increasing their vulnerability to spleen clearance as shown by the use of microfluidic devices.…”

Microfluidics Approach to the Mechanical Properties of Red Blood Cell Membrane and Their Effect on Blood Rheology

“…All these conformational changes strongly affect the cells' mechanical properties, modifying the rheological properties of blood [197,198]. As in the case of non-infectious diseases, in recent years, microfluidic devices have been developed to support Malaria diagnosis mostly directed to low-resource locations [199][200][201] or to find possible treatments [202][203][204].…”

Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Mouse Models and Organs-on-Chips

“…Importantly, this precludes the comprehensive understanding of the human parasite-immune cell interactions occurring within this niche. Additionally, scarce literature exists regarding the in vitro biomimicry of the Plasmodium -infected erythrocyte clearance performed at the splenic red pulp interendothelial slits using a microfluidics-based approach ( Rigat-Brugarolas et al., 2014 ) ( Picot et al., 2015 ) ( Guo et al., 2012 ) ( Elizalde-Torrent et al., 2021 ) (see Figure 3 ).…”

“…The development of minimal functional units of these organs-on-a-chip thus offer another unprecedented opportunities to study these niches. Minimal functional units of the spleen-on-a-chip have been reported ( Rigat-Brugarolas et al., 2014 ) ( Elizalde-Torrent et al., 2021 ) ( Picot et al., 2015 ); yet, in addition to rheological studies of infected blood they need now to incorporate ECM matrices and cells. In contrast, elegant OOC of the bone marrow showing sustain expansion of CD34 + cells, differentiation and egress of cells from the chip emulating vascular and bone marrow channels ( Chou et al., 2020 ) ( Glaser et al., 2022 ).…”