Cerebral malaria

Around 1% of all symptomatic cases of malaria result in severe disease, of which cerebral malaria is the most complicated manifestation, associated with BBB breakdown and brain swelling [33]. The pathogenesis of cerebral malaria is multifactorial and still not well understood. Histopathology studies consistently find sequestration of Plasmodium-infected erythrocytes in the brain microvasculature [34], and recent studies have shown accumulation of immune cells as well [35]. Nevertheless, histopathological analyses of human samples bring their own challenges in terms of the type of research that can be performed, costs, and ethical limitations. Researchers have resorted to studying rodent malaria as a proxy for human disease, but many key mechanisms differ between these two diseases, including its hallmark: infected red blood cell sequestration to the brain vascular endothelium [36].

In recent years, 3D models of both the brain microvasculature and the BBB have been developed to study cerebral malaria [17]. For instance, 3D grid-like perfusable microvasculature models created by soft lithography (see concept in Box 1) recapitulate multiple flow conditions in a single device. 3D microvessels are organ-on-a-chip models of the vasculature: miniature, microfluidic devices that, under controlled flow conditions, mimic the dynamic microenvironment, architecture, and physiological functions of human tissues. The interaction of P. falciparum with receptors in brain endothelium is exclusively found in human and great apes and not in rodent experimental cerebral malaria models. Therefore, 3D microvessels have been essential to elucidate several aspects of cerebral malaria, including the effects of differential flow on infected erythrocyte sequestration [37], the dynamics of endothelial inflammatory response mechanisms [38], the roles of BBB cell types in cerebral malaria pathogenesis [16,39], and reproduced the disruption of the angiopoietin-Tie2 axis, a well-known disrupted pathway in patients [39] (Fig 1).

Box 1. Microfabrication techniques

Microfabrication is the creation of microstructures and patterns using different techniques. This technology is widely applied in several fields, such as electronics, physics, pharmacology, and medicine [98]. In recent years, new developments have been made in the bioengineering field, namely in the recreation of MPS [3]. Soft lithography stands out as the most widely utilized technique for crafting MPS in host–parasite interaction research. Here, we describe this method alongside a broad spectrum of potential techniques [99] (Fig 2):

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Fig 2. Potential techniques for fabricating MPS in host–parasite research.

The radar plot provides a qualitative assessment, encompassing the following factors: (i) accessibility (considering equipment acquisition, maintenance, infrastructure requirements, and technical expertise), (ii) scalability and throughput (ease of production parallelization and scale-up), (iii) dimensionality range (design flexibility across the three spatial axes: X, Y, and Z), and (iv) resolution (the minimum achievable feature size).

https://doi.org/10.1371/journal.ppat.1013088.g002

• 3D Bioprinting: Additive manufacturing process, where the materials, known as bioinks, are deposited layer-by-layer to obtain the final 3D construct. Bioinks are composed of hydrogel polymers (natural, synthetic, or composites), cell laden, or fully acellular, and may contain additional bioactive components, such as fibers, minerals, particles, small peptides, growth factors, and extracellular matrix components. While new 3D bioprinting technologies keep regularly emerging [100], the extrusion-based is still the most popular, where the bioink is melted and/or reshaped as filaments via extrusion through a nozzle, deposited, and (if needed) hardened on a building platform. Hardening can be achieved by cooling or via crosslinking (e.g., aerosol). The main limitation of extrusion is size. Alternatively, higher resolutions are usually achieved with droplet-based or material jetting, where the bioink is ejected through a printing nozzle or inkjet head as a droplet or liquid jet, by a thermal or piezoelectric actuator, and sequentially getting cured using a specific wavelength. Other approaches use a laser scan or an image projection to crosslink photosensitive bioinks—a technology known as stereolithography. Depending on the 3D bioprinting technology, the smallest feature size can range between 5 µm and hundreds of micrometers. Two-photon polymerization (2PP) has emerged as a method to create highly precise and intricate microstructures down to a few hundreds of nanometers. 2PP uses two photons absorbed simultaneously by a photosensitive bioink (or other resins) to trigger polymerization at the focal point of the laser.
• Lithography-based techniques: Microfabrication of a master mold using a combinatorial workflow of additive, modifying, and subtractive unit processes such as deposition, patterning, and etching, respectively. Photosensitive chemicals, known as photoresists, are homogeneously spread with a defined thickness on a flat substrate, usually glass or silicon wafers, baked, and modified with ultraviolet (UV) or infra-red (IR) light exposure to generate patterns down to sub-micrometer resolution in mask-based (optical photolithography) or maskless (direct laser writing) systems. Scanning a focused beam of electrons (E-Beam Lithography) can be used for features down to just a few tens of nanometers. Final patterns are obtained after immersing the exposed substrate in a developer. Additionally, the substrate or added deposited films sensitive to (dry or wet) etching can be subtracted. Commonly, the master mold is used to generate inverted replicas made of the silicone elastomer PDMS by soft lithography, or made of thermoplastics, such as polystyrene (PS), polymethacrylate (PMMA), or cyclic olefin copolymer (COC), by thermal nanoimprinting lithography, as structured coatings on wafers, or by hot embossing of blocks or film sheets. Photo or UV-based is another variant of nanoimprinting lithography to solidify the resist coating by UV exposure, without requiring heat. As there is a limited number of molding and demolding cycles that can be made, it is often useful to convert the master mold or even the replicas in other types of materials. For instance, using PDMS as stamps to generate patterned matrices of high-performance biocompatible or native polymers.
• Electrical and vapor deposition: used to create thin films, coatings, or 3D scaffolds by depositing material onto a substrate. Applying an electric field is possible to deposit polymers, metals, ceramics, or composite materials onto a substrate or mold, forming films or scaffolds with specific physical properties such as porosity, morphology, and electrical conductivity. These methods include electrospinning, electroforming, electrochemical, and electrophoretic deposition. Other procedures rely on generation and deposition of material in the vapor form, typically under a controlled atmosphere. Such methods include Physical Vapor Deposition, Chemical Vapor Deposition, Atomic Layer Deposition, Plasma Spray Deposition, and Vapor Phase Polymerization. Bioactive coatings (e.g., hydroxyapatite, calcium phosphates), particles, fibers, or biomolecules are frequently added to enhance biocompatibility and tailor the mechanical, rheological, and biological biomimicking properties.
• Hydrogels and scaffold molding: gelation or liquid-to-solid modifying methods can be used to generate 3D structures inside prefabricated molds or containers. These include sol-gel transition, which involves the conversion of a solution into a gel via a temperature or pH shift (e.g., Poly(N-isopropylacrylamide) (PNIPAAm), chitosan); physical (via ionic or hydrogen bonding) or chemical (via covalent bonding) crosslinking (e.g., glutaraldehyde and calcium ions (for alginate)); and freeze-drying (lyophilization) where water is removed from a hydrogel via freezing and then sublimation, leaving behind a porous, sponge-like structure.
• Modifying laser-assisted techniques: Lasers can be used to create microstructures in different materials at a microscopic scale. As the laser energy is absorbed by the material, depending on the material properties and the laser parameters, it is possible to perform operations such as precise and localized melting, sintering, or photoablation (from engraving to a total cut).

A hallmark of severe malaria is microcirculatory obstruction, which occurs in high-density capillaries as narrow as 5 μm in diameter. By combining soft lithography with multiphoton laser photoablation (i.e., ultrafast pulses enabling precise material removal at the focal point without thermal damage of surrounding areas), researchers have been able to create capillary-sized vessels that are later lined by migrating human primary endothelial cells [6]. Plasmodium falciparum perfusion of arteriole or venule-sized vessels joined by capillaries of 5–10 μm in diameter showed how infection affects red blood cell speed, deformability and its interaction with the capillary vessel wall, which ultimately exacerbates sequestration and vascular occlusion [6].

Cerebral animal african trypanosomiasis

Animal African Trypanosomiasis (AAT) is primarily caused by Trypanosoma congolense, Trypanosoma vivax, and Trypanosoma brucei, leading to considerable direct and indirect economic losses [40]. Trypanosoma congolense-induced AAT can progress to acute cerebral trypanosomiasis, characterized by increased sequestration to the BBB and lethal neuro-immunopathology [8]. Whilst a mouse model of acute cerebral trypanosomiasis exists and offers views on multiple important factors, including infection outcomes, virulence, drug resistance, and disease tolerance, decoupling immune response-mediated mechanisms from those directly determined by parasite sequestration is challenging to achieve in vivo. Recently, a 3D microvessel model that mimics the bovine brain microvasculature has been developed [32]. It offers a cheaper and controlled alternative to study ligand–receptor interactions, the role of biomechanics, and the direct contributions of parasite-intrinsic factors to vascular pathology in the absence of the immune system. It has been useful to assess the contribution of flow velocity, parasite strain, and endothelial cell bed on parasite sequestration in a model of the natural host. The engineering system used to fabricate bovine microvessels is the same as the human, which highlights the versatility of soft lithography generated microenvironments and their applicability to multiple hosts, tissues, and diseases.

Granulomatous amebic encephalitis

Granulomatous amebic encephalitis (GAE) is a rare but highly fatal human disease that can be caused by free living ameba, such as Acanthamoeba spp., Sappinia pedata, or Balamuthia mandrillaris [41]. It is endemic worldwide, with ~23,000 cases being reported yearly, mostly in immunocompromised individuals. In these conditions, GAE is a severe infection of the CNS, with more than 90% mortality rate [42]. Current knowledge derives from case reports [43], in vitro systems, and mouse models of infection [44].

Acanthamoeba castellanii attachment to human BBB under flow has been studied using a monolayer of human brain microvasculature endothelial cells perfused with media containing A. castellanii at different flow rates. There were significant differences between binding rates in traditional static monolayer assays and under different flow conditions [45]. Authors also found that secreted amebicidal/parasite proteins may play a role in BBB breakdown during an A. castellanii infection. In an alternative system, neurospheroids were successfully used to assess amebicidal activity and neural cytotoxicity against B. mandrillaris. Spheroids are 3D cell aggregates that self-assemble in a non-adherent 3D environment and mimic certain aspects of tissue organization and function. They are simpler than organoids and do not require cellular differentiation, which makes them more scalable. These spheroid models were previously developed for cancer studies [46] and later adapted for the study of GAE. Spheroids were grown using human neuroblastoma SH-SY5Y cells and human lung carcinoma A549 cells, in specialized 24-well plates that allow waste drainage, preventing parasite overgrowth. This allowed researchers to overcome the difficulties associated with assessing human cell viability being in the absence of B. mandrillaris overgrowth [47]. We did not find additional MPS studies for GAE, which potentially reflects the small size of its research community. The high mortality and low incidence limit clinical data availability, making it harder to design MPS that accurately reflect human pathology. Still, this gap presents a critical opportunity to develop such advanced MPS model to uncover the GAE disease mechanisms, improve drug screening, and bridge the lack of clinical data.

Toxoplasmosis

Toxoplasma gondii, the causative agent of toxoplasmosis, infects around one-third of the human population, and despite being largely asymptomatic, it causes disease in immunocompromised individuals and unborn babies [48]. Toxoplasma gondii is highly promiscuous, infecting a broad range of cells [49], and therefore can cause assorted clinical outcomes. It has been recently shown that T. gondii interacts differently with different host cell types [50], but further studies are necessary to fully comprehend these host–parasite interaction mechanisms. The use of microenvironments may help in decoding infection mechanisms in distinct environments.

Toxoplasma gondii has two stages: tachyzoite, responsible for acute infection, and bradyzoite, a cyst form responsible for latent infection in humans [48]. Using a soft lithography, a recent model that allows the visualization of the crucial stages of T. gondii infection of vascular endothelial cells has been developed [18] (Fig 1). With this model, researchers were able to assess in real time the tachyzoite infection of vascular endothelial cells under flow conditions in co-culture with neurons. This system allowed to characterize for the first time a more complete timeline of the events surrounding tachyzoite infection, namely parasite movement and transmission between host cells.

Chagas disease

Chagas disease, caused by Trypanosoma cruzi infection, is a parasitic disease endemic to South America. Upon entering the chronic stage of the disease, 20%–30% of infected individuals develop heart complications (e.g., myocarditis and myocytolysis), commonly known as Chagas cardiomyopathy. Despite comprising the most common cause of death from Chagas disease, the mechanisms behind it remain poorly understood [52].

Recently, MPS have been applied to the study of Chagas cardiomyopathy [31,53]. Here, researchers have opted for 3D cell culture systems that require the seeding of the cells onto a pre-molded microstructure or substrate, allowing them to grow like they would on a natural substrate [54], and assembling into 3D structures, such as spheroids, rods, toroids, and honeycomb or other mesh-like architectures [55]. By seeding cardiomyocytes in agarose coated u-well plates, spheroids that resembled cardiac tissue were created, with spontaneous contractility, cell differentiation, and production of growth factors [31]. Upon T. cruzi infection, these spheroids were able to properly develop cardiac fibrosis, enabling further research on this pathology (Fig 1).

Animal African trypanosomiasis

In T. congolense-induced AAT, sequestration occurs beyond the brain, in tissues including the heart, brain, spleen, kidney, and adipose tissue [2]. During chronic disease, parasites can accumulate in the heart and coronary vasculature. In a recent study, researchers have been able to create a bovine cardiac 3D model, using a similar rationale as the model described for cerebral AAT [32] (Fig 1). In this study, these microvessels were seeded with bovine aortic endothelial cells, to mimic the cardiac vasculature. Different parasite strains were shown to adhere differently to different endothelial cell beds and cyclic AMP homeostasis was found to play a critical role in parasite sequestration [32], corroborating what was previously observed in parasite attachment to plastic substrate [56].

Malaria

Inside the mammalian host, Plasmodium spp. has two stages: the liver stage and the blood stage (Box 2). The liver stage is an obligatory stage of infection, as parasites need to infect hepatocytes to develop into merozoites, which are then released back into the blood [57]. A human liver platform comprised of hepatocytes and fibroblasts seeded on a micropatterned (see Lithography-Based Techniques section in Box 1) collagen model supports P. falciparum and P. vivax infection [58] (Fig 1). The same model allowed the characterization of the developing P. vivax liver stages, including the analysis of the hypnozoite transcriptome [25]. It also allowed the assessment of relapse frequencies between strains and primaquine clearance. In 2022, this model was used to culture patient-derived P. vivax parasites to obtain single-cell transcriptomes for the study of malaria relapse in humans [59].

Amebic liver abscess

Entamoeba histolytica is a protozoan parasite with tropism to the gut, and infection is mostly asymptomatic. However, in around 9% of cases, E. histolytica trophozoites may cross the intestinal epithelium and invade the liver, forming amebic liver abscesses (ALA) [60]. Every year, around 40,000–100,000 people die of amebic liver abscesses [61], making it the second leading cause of death among parasitic infections, after malaria.

Whilst animal models of ALA have been successfully used to study key aspects of disease, MPS can be useful to elucidate mechanistic insights that require more controlled microenvironments. In this light, a 3D liver model was developed, which helped to overcome the many constraints behind the use of human samples [62] (Fig 1). This model allowed the study the role of galectins in liver infection. In previous animal models, it was shown that amebic adhesion to host cells was dependent on galactose- or N-acetyl-galactosamine-inhibitable (Gal/GalNAc) lectin, but the use of the 3D model revealed the mechanistic details of this dependence. We did not find additional examples of MPS uses for ALA research, which likely reflects its small field.

Malaria

The spleen is important for clearance of infected red blood cells and the development of malaria immunity [63]. For some Plasmodium species, such as P. vivax, the spleen may also act as an infection reservoir, leading to parasite persistence [64].

The spleen is responsible for, among other functions, removing deformed or senescent erythrocytes and other particles, including Plasmodium spp., from blood circulation [65]. A process that has been recapitulated in soft lithography-generated microfluidic models [66], which mimicked the complex circulation of the spleen and allowed the study of the biomechanics of Plasmodium-spleen interactions. During the spleen filtering process, erythrocytes undergo a process known as “pitting”, after passage through interendothelial slits, all unwanted particles and damaged erythrocytes are removed, including parasites, without damaging the infected host cell. Splenic pitting of malaria parasites has been recreated in an acellular microfluidic device generated by soft lithography [27] in a reductionist model that achieves filtration without compromising cell integrity, showing that immune phagocytosis is not essential for pitting (Fig 1). This illustrates the importance of accurately mimicking tissue mechanical cues, even in the absence of cells and other biological elements.

Giardiasis

Giardia lamblia (i.e., intestinalis) is the most common cause of parasitic diarrhea in the world [68] and consequently a public health concern. G. lamblia attachment to the intestinal epithelium is a crucial step for infection and parasite survival in the host. Although many models have been created to study G. lamblia attachment to the gut epithelium, understanding of attachment mechanisms, such as adhesion force under flow, remains elusive [21,69].

Gut-on-a-chip models have primed the discovery of the molecular factors driving parasite attachment to the gut, using similar soft lithography techniques than those highlighted previously in liver and spleen studies. G. lamblia trophozoites have been cultured inside a perfusable microfluidic chips to evaluate attachment efficiency under different flow rates [69] (Fig 1). The microfluidic network was designed to test in parallel different shear stress levels to simulate the intestinal tract microenvironment. This allowed estimation of adhesion force and determination of EC50 of compounds of interest in a single assay. Although this model did not include host epithelial cells, a previous microfluidic model including an intestinal cell monolayer system was sufficient to assess the role of other factors, such as tonicity, in G. lamblia detachment [70].

Intestinal amebiasis

Intestinal amebiasis is caused by the protozoan parasite E. histolytica. It is mainly transmitted through the oral-fecal route and may present some mild to severe symptoms, such as diarrhea, and abdominal pain. In more severe cases, it can progress to peritonitis and be associated with anemia [71].

Polydimethylsiloxane (PDMS)-based microfluidic models can exploit the addition of two lateral vacuum chambers that generate strain and mechanical stretching to mimic intestinal peristalsis. A system like this has been used to assess the effects of mechanical cues on E. histolytica intestinal tissue invasion of colorectal adenocarcinoma cells (Caco-2) [19] (Fig 1). This is a recent model, but that has the potential to accelerate fundamental research on intestinal amebiasis and be adapted to other intestinal pathogens.

Cryptosporidiosis

Cryptosporidiosis is caused by Cryptosporidium spp., mainly C. parvum and C. hominis, transmitted orally through the ingestion of contaminated food and/or water [72]. It is a major cause of diarrheal illness and poses a significant threat to children in developing countries, being one of the leading causes of diarrhea deaths in children under 5 years of age [73].

In Wilke and colleagues [74], an air–liquid interface model was developed that allowed for the full development of C. parvum in vitro. With this model, researchers were able to mimic the intestinal microenvironment and assess the gene expression differences across the parasite life cycle. An organoid alternative, capable of supporting the full C. parvum development, was later developed [75]. Organoids are 3D miniaturized, self-organizing tissue cultures derived from stem cells that replicate key structural and functional features of real organs. This model involves the formation of mini-gut tubes through laser ablation of a collagen-Matrigel hydrogel and supports long-term C. parvum infection [75]. Indeed, one challenge of printed microenvironments is maintaining them long-term, both due to material breakdown and loss of intrinsic cellular characteristics. However, long-term maintenance is crucial to understand parasitic diseases with long infection or incubation times. An early attempt at long-term culturing of C. parvum used a 3D human intestinal model containing intestinal myofibroblasts based on a silk scaffold seeded with colorectal adenocarcinoma cells (Caco-2) and goblet cells (HT29-MTX) [20] (Fig 1).

Helminthiasis

Teladorsagia circumcincta is a nematode of significant animal health importance, causing parasitic gastroenteritis in sheep, but available treatments are becoming increasingly ineffective [76], reinforcing the need to develop novel methods to study this helminth and potentially find new drugs [76]. Due to their customization potential, MPS can be useful to gradually increase biological complexity. An example is the integration of naturally occurring gut flora to address host–microbe–parasite interaction. A 3D GI tract model on a collagen hydrogel that couples immune and intestinal cells in co-culture with Escherichia coli, a commensal gut bacterium, has been useful to assess the host immune response to T. circumcincta and has become a successful proof-of-concept of a complex host–microbe model to study gut–helminth interactions [77].

Malaria

Pregnant women living in malaria endemic regions are susceptible to placenta malaria, which is characterized by severe complications for the mother, such as anemia and hypertension, and for the babies, including stillbirth or low birth weight [81]. Placental malaria is caused by cytoadhesion of P. falciparum infected erythrocytes to placenta trophoblasts through binding to low-sulphated placental chondroitin sulphate A [82]. A recent three-line microfluidic model has been developed, including a channel seeded with human umbilical vein endothelial cells and a third channel including trophoblasts (BeWO cells), both separated by a middle channel containing collagen type I (Fig 1). The use of this model revealed that binding of infected erythrocytes causes a decrease in glucose transport across the placental barrier [30].

Congenital Chagas disease

Trypanosoma cruzi infection during pregnancy results in congenital transmission, as well as significant risk of premature and stillbirth. Although not all parasite strains can be vertically transmitted, the reasons behind these differences remain unknown [83]. In 2024, Apodaca and colleagues used BeWo-based human trophoblasts spheroids seeded on agarose molds to study placental T. cruzi infections [29] (Fig 1). Cells exhibited functions resembling in vivo conditions, such as proliferation and morphogenesis. Assessment of T. cruzi infection of this placental model revealed that infection distribution and progression is strain dependent [29]. Previous attempts to infect spheroids with T. cruzi had failed, suggesting reduced susceptibility of JEG-3 cells to T. cruzi infection when grown in 3D [84]. This highlights the importance of choosing adequate host cells that can support infection.

Sleeping sickness

The skin can act as a reservoir of T. brucei during sleeping sickness [85], so the mechanisms behind parasite survival in the skin are of high relevance. Yet, studying skin-residing trypanosomes in vivo is hard for many reasons including the low numbers of infected human patients, the large area of skin in affected animals which challenges trypanosome detection and harvesting, and the invasiveness of acquiring skin biopsies. A high-density skin model obtained through compression of multiple layers of normal primary human epidermal keratinocytes and dermal fibroblasts in collagen was used to study T. brucei skin-form parasites [22] (Fig 1). This skin model supports differentiation of natural skin layers and withstands the bite of the tsetse fly (Glossina morsitans morsitans). Importantly, this system allowed the characterization of the T. brucei population dynamics in the skin: after establishing a proliferative population, T. brucei skin-form parasites strongly reduce their replication frequency and metabolic demands, becoming a reversible quiescent population in vitro, which may explain chronicity and/or persistence observed in patients [22].

Schistosomiasis and strongyloidiasis

Strongyloides ratti and Schistosoma mansoni are nematodes of medical importance: S. ratti is often used as an alternative to study human strongyloidiasis and S. mansoni is a prominent intestinal parasite causing schistosomiasis [86]. Schistosomiasis has an estimated burden of 2.19 million disability-adjusted life-years and remains a main public health concern [86].

The challenges associated with studying helminth skin forms in vivo are similar to those discussed for T. brucei. Two models based on primary human epidermal keratinocytes and human dermal fibroblasts grown in a collagen matrix first grown in a liquid–liquid and subsequently in an air–liquid interface were used to study percutaneous S. ratti and S. mansoni invasion of epidermis [23] (Fig 1). Both parasitic worms invaded a human epidermis equivalent and a human full thickness skin equivalent with similar success rates to in vivo studies.

Onchocerciasis

Onchocerca volvulus is the causative agent for onchocerciasis, or river blindness. It is an obligate human parasite, endemic to Africa, with some foci in South America and the Middle East [87]. There are no animal models nor well-established long-term in vitro cell culture methods for adult forms [24], and most research relies on the study of similar filarial models, such as cattle Onchocerca spp. [88].

MPS may offer an alternative for parasites refractory to standard in vitro conditions. Two models have been generated to support cultivation of O. volvulus young adult forms, a skin tissue model, made of primary human epidermal keratinocytes and dermal fibroblasts isolated from juvenile foreskin onto a collagen matrix, and an adipose tissue model, made of human bone marrow derived mesenchymal stromal cells seeded onto a u-shaped 96-well plate to form a cell aggregate [24] (Fig 1). This study provides evidence that the microenvironment, specifically the direct contact with cells and ECM, is important for O. volvulus development. Further improvement on this model to support full development to mature adult forms is necessary, but larval forms can be harvested from this system for continued culture. An aspect that is particularly important when establishing new culture systems for previously intractable pathogens, such as O. volvulus, is to ensure that cultured parasite forms retain their transcriptomic and proteomic profiles, as well as their infectivity and virulence if transferred to an animal host.

Malaria

Adults living in malaria endemic areas develop immunity to severe disease. The P. falciparum parasite ligand that mediates binding to endothelial receptors presents a broad sequence diversity. This argued against the possibility of broadly reactive antibodies. Recently P. falciparum binding inhibition studies in a grid 3D microfluidic revealed the existence of monoclonal antibodies that could recognize and prevent the binding of different subsets of parasites with binding affinity to Endothelial Protein C Receptor, one of the main determinants of severe malaria [89]. This study opens the door to study therapeutic strategies to prevent severe disease.

The use of microfluidic models has also been applied for antimalarial research. A multi-organ-on-a-chip model, which combines multiple organ-on-chip models in a single device to recapitulate a system, has been developed for antimalarial efficacy studies [28]. A microfluidic pathway with three chambers was designed and fabricated using laser cutting on clear cast acrylic sheets in combination with PDMS sheets. Each chamber contains different cell types: primary human hepatocytes as a proxy for the liver, human umbilical vein endothelial cells as the vascular endothelium, and primary human splenocyte cells to mimic the spleen (Fig 1). This platform can be used for therapeutic assessment, both drug efficacy and cytotoxicity, where multiple microenvironments are tested simultaneously [28].

Leishmaniasis

Leishmaniasis is caused by Leishmania spp. parasites and has several distinct clinical manifestations. Leishmania spp. promastigotes enter the mammalian host via a phlebotomine sandfly bite and are phagocytized by mononuclear cells, such as macrophages. Inside these macrophages, parasites then transform into the amastigote form, where they proliferate [90]. In recent years, the development of new treatment alternatives has become a priority because of increased drug resistance and reduced treatment efficacy. To better study drug efficiency in the treatment of Leishmania infected macrophages, there is a need to create a more realistic simulation of drug distribution. In O’Keeffe and colleagues [91], authors assessed several physiologically relevant assays, namely a previously described perfusion-based cell culture model, where infected macrophages are cultured under constant flow [92] and 3D cell culture models, in which infected macrophages are cultured in prefabricated scaffolds. Authors also tested several cell lines and origins of macrophages, namely the use of iPSC-derived macrophages in place of traditional cell lines (human and rodent primary and immortalized cell lines). They used these models and different cell combinations to assess the efficiency of drug uptake by macrophages when perfused with media containing the drugs of interest. This not only served as a comparison study between 3D models and traditional 2D models but also showed that variations in cell types and model constructions directly impact drug activity research, raising awareness on the need of using cell types from multiple donors when assessing drug efficacy.