REVIEW published: 04 July 2022 doi: 10.3389/fcimb.2022.920204 Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Edited by: Juan F. Quintana, University of Glasgow, Mouse Models and Organs-on-Chips United Kingdom Iris Aparici Herraiz 1,2†, Hugo R. Caires 3†, Óscar Castillo-Fernández 1,2,4, Núria Sima 1,2, Reviewed by: Lourdes Méndez-Mora 5, Ruth M. Risueño 6, Jetsumon Sattabongkot 7, Sudhir Kumar, Wanlapa Roobsoong 7, Aurora Hernández-Machado 4,5,8, Carmen Fernandez-Becerra 1,2, Seattle Children’s Research Institute, Cristina C. Barrias 3,9,10 and Hernando A. del Portillo 1,2,11* United States Andreia Barroso, 1 Barcelona Institute for Global Health (ISGlobal), Hospital Clı´nic - Universitat de Barcelona, Barcelona, Spain, 2 Institut d’Investigació IQVIA (Brazil), Brazil en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain, 3 i3S - Instituto de Investigação e Inovação em Saúde, Universidade Glenn McConkey, do Porto, Porto, Portugal, 4 Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, Barcelona, Spain, University of Leeds, United Kingdom 5 Department of Condensed Matter Physics, University of Barcelona (UB), Barcelona, Spain, 6 Josep Carreras Leukaemia Research Shiroh Iwanaga, Institute (IJC), Barcelona, Spain, 7 Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, Osaka University, Japan 8 Centre de Recerca Matemàtica (CRM), Barcelona, Spain, 9 INEB – Instituto de Engenharia Biomédica, Universidade do Porto, *Correspondence: Porto, Portugal, 10 ICBAS – Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal, 11 Institució Hernando A. del Portillo Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain hernandoa.delportillo@isglobal.org † These authors have contributed Plasmodium vivax is the most widely distributed human malaria parasite representing equally to this work 36.3% of disease burden in the South-East Asia region and the most predominant Specialty section: species in the region of the Americas. Recent estimates indicate that 3.3 billion of people This article was submitted to are under risk of infection with circa 7 million clinical cases reported each year. This burden Parasite and Host, a section of the journal is certainly underestimated as the vast majority of chronic infections are asymptomatic. Frontiers in Cellular and For centuries, it has been widely accepted that the only source of cryptic parasites is the Infection Microbiology liver dormant stages known as hypnozoites. However, recent evidence indicates that Received: 14 April 2022 Accepted: 06 June 2022 niches outside the liver, in particular in the spleen and the bone marrow, can represent a Published: 04 July 2022 major source of cryptic chronic erythrocytic infections. The origin of such chronic Citation: infections is highly controversial as many key knowledge gaps remain unanswered. Yet, Aparici Herraiz I, Caires HR, as parasites in these niches seem to be sheltered from immune response and antimalarial Castillo-Fernández Ó, Sima N, Méndez-Mora L, Risueño RM, drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Sattabongkot J, Roobsoong W, Due to ethical and technical considerations, working with the liver, bone marrow and Hernández-Machado A, Fernandez-Becerra C, Barrias CC and spleen from natural infections is very difficult. Recent advances in the development of del Portillo HA (2022) Advancing Key humanized mouse models and organs-on-a-chip models, offer novel technological Gaps in the Knowledge of frontiers to study human diseases, vaccine validation and drug discovery. Here, we Plasmodium vivax Cryptic Infections Using Humanized Mouse review current data of these frontier technologies in malaria, highlighting major challenges Models and Organs-on-Chips. ahead to study P. vivax cryptic niches, which perpetuate transmission and burden. Front. Cell. Infect. Microbiol. 12:920204. doi: 10.3389/fcimb.2022.920204 Keywords: humanized mouse, organs-on-a-chip, Plasmodium vivax, models, Key gaps in the knowledge Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax BACKGROUND by reticulocyte-prone malaria parasites (del Portillo et al., 2004). Remarkably, recent studies of spleen rupture in Timika, Human malaria is a disease caused by five species of plasmodia of Indonesia, have unequivocally demonstrated that the largest which Plasmodium falciparum and Plasmodium vivax represent parasite biomass of P. vivax during chronic asymptomatic the vast majority of burden whereas Plasmodium ovale, infections is observed in the reticulocyte-rich spleen (Kho Plasmodium malaria and the recent zoonosis caused by et al., 2021a) (Kho et al., 2021b). Also, P. vivax parasites have Plasmodium knowlesi in South East Asia, are likely to been suspected to reside in the BM during chronic asymptomatic contribute to less than 5% of such burden (Battle et al., 2019). infections as originally observed in the late 19 th century In the particular case of P. vivax, its burden has dramatically (Bignami, 1894). Morphological and molecular evidence of the decreased from 11.9-22 million cases in 2013 to 7 million clinical parasite in this hemopoietic tissues have been recently observed cases in 2020 (WHO, 2020). This panorama would indicate that in patients (Baro et al., 2017; Brito et al., 2020). Noticeably, all elimination of P. vivax can be achieved with the present control patients presented defects in erythropoiesis and global tools available. However, in low-endemic countries that have transcriptional analysis corroborated these morphological been targeting vivax malaria for elimination, an increasing observation (Baro et al., 2017; Brito et al., 2020). All together, number of asymptomatic infections capable of transmitting to these data call for a new paradigm in P. vivax research which mosquitoes are being reported (Cheng et al., 2015). In fact, should now incorporate these cryptic infections into its life cycle during chronic infections, numerous epidemiological field (Figure 1). This will clearly require a renewed emphasis on studies support that >90% of infections are sub-microscopic understanding the origin and significance of these cryptic and asymptomatic (Angrisano and Robinson, 2022). Thus, the erythrocytic niches, if elimination of malaria is to be achieved. global burden of this species is certainly underestimated. For ethical and technical reasons, working with the human Moreover, experts agree that P. vivax will be the last human spleen have been mostly limited to post-mortem analysis (Imbert malaria parasite species to be eliminated due to its unique et al., 2009) and studies on bone marrow aspirates are limited as biology, including (i) the presence of hypnozoites, a latent it is considered a highly invasive clinical procedure. Here, we will (dormant) form of the parasite that develops in the liver review new enabling technologies on humanized mouse models (Krotoski, 1985) which can reactivate weeks or months after and organs-on-a-chip, promising approaches to unravel the the primary infection causing clinical relapses (White, 2011); (ii) mechanistic particularities of these largely unexplored cryptic the limitation of using primaquine and tafenoquine to kill infections of P. vivax. hypnozoites in pregnant women and G6PD-defficient individuals due to the risk of acute hemolytic anemia (Baird, 2019); (iii) the risk of primaquine treatment failure in patients HUMANIZED MOUSE MODELS with particular cytochrome P450 isozyme 2D6 (CYP2D6) polymorphisms (Suarez-Kurtz, 2021); (iv) the recent findings The interest in engrafting human immune systems to that invasion of merozoites into reticulocytes is not limited to the immunocompromised mice started with the discovery of the Duffy binding protein (Mé nard et al., 2010) and the detection of nude mouse in the laboratory of Dr. N.R. Grist (Flanagan, P. vivax in sub-Saharan Africa, where this blood group is nearly 1966). However, this early mouse strain was unable to sustain absent (Zimmerman, 2017); (v) the outdoor biting behaviour of the engraftment of human bone marrow cells, thus failing to vectors transmitting P. vivax, that results on low efficacy control establish their growth (Ganick et al., 1980) (Macchiarini et al., measures when impregnated bed nets are used. All together, 2005). A breakthrough came with the discovery of these unique aspects of the biology of P. vivax strongly support immunodeficient mice with the Prkdc-scid mutation, which in the generalized view that this species will be the last human humans is called Severe Combined Immunodeficiency (SCID) malaria parasite to be eliminated (Mueller et al., 2009). resulting in the C.B-17-Prkdcscid mouse (Pearson et al., 2008). The Cryptic stages can be defined as parasites difficult to detect severe combined immunodeficiency (SCID) is a loss-of-function with currently available tools which persist in different host mutation, which affects the protein kinase, DNA-activated tissues during chronic asymptomatic infections. For centuries, catalytic polypeptide (PRKDC), leading to an inefficient DNA it was amply accepted that the only source of cryptic stages in P. non-homologous end joining repair that is required for T cell and vivax were hypnozoites inside infected-hepatocytes (Krotoski, B cell receptor rearrangement, resulting in a lack of mature T and 1985). As this liver stage cannot be detected using currently B cells (Blunt et al., 1995) (Bosma et al., 1983) (Kirchgessner et al., available diagnostic methods, it constitutes a silent reservoir of 1995) (Mosier et al., 1988). Comparing SCID mice with nude mice the disease and a major threat for malaria elimination. However, there is a clear reduction of immune responses whereas the as recently reviewed (Fernandez-Becerra et al., 2022), new residual immune responses in nude mice are higher. Thus, evidence indicates that cryptic niches outside the liver, in SCID mice have been extensively used as recipients for human particular in the bone marrow (BM) and the spleen, represent cell and tissue xenografts in vivo. a major source of hypnozoites-unrelated recrudescence. Thus, Another genetic deficiency that leads into a reduction of an early studies on a clinical case of spontaneous spleen rupture adaptive immune response is the mutation in one of the revealed the presence of large numbers of intact parasites in the recombination-activating genes Rag1 and Rag2, responsible for red pulp (Machado Siqueira et al., 2012). This observation raised T cell and B cell receptor rearrangements (VDJ recombination), evidence of a long standing hypothesis of spleen cytoadherence thus leading to lack of B and T cells (Shinkai et al., 1992). Rag- Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax term studies and systemic reconstitution with human cells (Murphy et al., 1987). An important advancement in the development of more immunodeficient mouse strains was the production of murine strains that have a mutation or deletion on the interleukin (IL)-2 receptor common g chain (Il2rg or gc). The IL-2 receptor common gamma chain is an important cytokine receptor subunit for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 and is indispensable for high-affinity binding and signaling of these cytokines (Cao et al., 1995) (DiSanto et al., 1995) (Ohbo et al., 1996). This gene disruption results in critical cytokine signaling networks that are required for both adaptive and innate immune responses leading to the lack of NK cells characterized by the absence of IL-15 signaling (Kennedy et al., 2000) (Kerre et al., 2002) (Ranson et al., 2003) (Traggiai et al., 2004) (Walsh et al., 2017). However, there is still some remaining phagocytic activity which triggers-on the development of graft-versus-host disease (GVHD) after the engraftment of human cells (Greenblatt et al., 2012). The Cluster of Differentiation 47 (CD47) acts as a marker of self (Oldenborg et al., 2000), and its interaction with the inhibitory FIGURE 1 | Life cycle of Plasmodium vivax highlighting new cryptic receptor signal regulatory protein a (SIRPa) on macrophages erythrocytic stages. During a blood meal, malaria-infected mosquitos inject delivers a “do not eat-me” signal preventing phagocytosis of self- sporozoites which after reaching the bloodstream enter hepatocytes initiating the pre-erythrocytic cycle. Within the liver, P. vivax either differentiates (i) into a cells by macrophages. Therefore, if this interaction is not avoided dormant stage called a hypnozoite which, upon reactivation, causes clinical on the murine environment, mouse SIRPa receptors will not relapses, or (ii) into tissue schizonts, which after thousands of mitotic recognize human CD47 and human engrafted cells will be replications in membranous sacks known as merosomes, release merozoites phagocytized by murine macrophages. Fortunately, a into the bloodstream initiating the erythrocytic cycle. In this cycle, P. vivax merozoites predominantly, if not exclusively, invade reticulocytes starting asexual polymorphism in the mouse Sirpa gene found in the non-obese blood stage differentiation of rings, trophozoites, schizonts and egress to invade diabetic genetic background (NOD) resembles to the human new red blood cells. This cyclical developmental process takes about 48 h. In SIRPA gene, thus achieving phagocytic tolerance by the addition, P. vivax produces specific proteins to create caveola–vesicle interaction via CD47- SIRPA (Yamauchi et al., 2013). Phagocytic complexes that appear as profuse speckling in Giemsa-stained blood smears, tolerance can also be achieved by the transgenic expression of known as Schüfnner’s dots. Moreover, some P. vivax parasites can differentiate into mature gametocytes before a clinical infection and illness develops, thus mouse CD47 on human hematopoietic cells (Takenaka et al., 2007; having the advantage of continued transmission to the insect vector before the Legrand et al., 2011). On the other hand, phagocytic tolerance can appearance of clinical symptoms and subsequent treatment. Remarkably, be achieved temporarily by eliminFortunately, a polymorphism presence of parasites in the spleen and the bone marrow represents novel inating recipient phagocytic cells using clodronate-containing cryptic erythrocytic infections that need to be incorporated in the life cycle of this liposomes (Clo-lip), small hydrophilic molecule ingested by a species (boxed). Infections of these organs can be either directly from invasion of merozoites into the reticulocyte-rich bone marrow and spleen, or by infected- macrophage in a liposome-encapsulated form. It will be reticulocytes in peripheral blood. Circulating gametocytes are rounded shape accumulated within the cell as soon as the liposomes are digested and on uptake in the blood meal of anopheles mosquitoes begin the sexual with the help of its lysosomal phospholipases. At a certain cycle. This includes release of the male and female gametes, fertilisation, and intracellular clodronate concentration, the macrophage is formation of a motile ookinete that crosses the midgut epithelium. Differentiation eliminated by apoptosis. Alternatively, if phagocytic cells develop into a new replicative form known as the oocyst, release of sporozoites, migration, and invasion of the salivary glands ends this complex life cycle in in an environment without CD47, then they become tolerized to which the parasite undergoes more than ten stages of cellular differentiation and cells that lack CD47 (Wang et al., 2007). Natural killer cells also invades at least four types of cells within two different hosts. (Created with have limited activity in NOD mice because of a defect in the BioRender.com by Carmen Fernandez-Becerra). NKG2D receptor (Ogasawara et al., 2003). All these properties have made the NOD background preferred for the development of models that receive xenotransplantation of cells and tissues from knockout (Rag KO) strains of mice are also used as recipients for human origin. human cell xenografts. These immunodeficient mouse strains are The combination of IL2rg knockout with SCID or Rag KO very useful as human cell transplant recipients, but they have mutations leads to highly immunodeficient strains that have several limitations, including a small production of T and B cells neither mature T and B cells, nor NK cells, with severely in older SCID mice and an increased susceptibility of SCID mice debilitated monocyte/macrophage function. Reported strains to preconditioning by radiation due to inefficient DNA repair. that use IL2rg mutation are the NSG (Blunt et al., 1995; Moreover, there is still high levels of innate immune cells, which Kirchgessner et al., 1995), NOG (Blunt et al., 1995; Kirchgessner includes substantial NK cells that complicate their use in long- et al., 1995), NRG, and BRG (Shultz et al., 2007; Shultz et al., 2012; Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Akkina et al., 2016) models. Taking into account this mutation, bloodstream before homing to the liver initiating asexual these strains can support more efficient, long-term, stable, and differentiation to form liver-stage schizonts. Upon completion systemic engraftment with human cells and tissues. Additional of liver-stage development, thousands of merozoites are released mutations in cytokine genes, the transgenic expression of human into the blood circulation starting erythrocytic infections. cytokines by exogenous plasmid injection, recombinant protein Noticeably, in the case of P. vivax and P. ovale, a proportion of injection or the expression of additional transgene can help on the sporozoites enter a quiescent stage, the hypnozoite, after development of a specific cell type of interest. Yet, most strains hepatocyte invasion. Without including the 8-aminoquinoline support humanization of lymphoid and not myeloid hypnozoitocidal drug in the standard anti-malarial treatment, erythropoietic lineages, thus limiting their use for studies on the hypnozoite remains in the liver causing relapses of the asexual blood stages in human malaria. disease. These relapses accounted for over 70% of clinical cases Noticeably, NSGW41 or NBSGW strains were created to in endemic areas (Commons et al., 2020) causing significant overcome a lack of erythropoiesis and megakaryopoiesis in morbidity and sustaining the transmission cycle. Primaquine and humanized mouse models. These strains support long-term tafenoquine have been proven to effectively eradicate hypnozoite engraftment of human hematopoietic stem cells (HSCs) without (John et al., 2012; Commons et al., 2018; Llanos-Cuentas et al., previous pre-conditioning therapy due to the loss of endogenous Kit 2019) but the liabilities on the 14-days treatment regimen function in KitW-41J allele (Yurino et al., 2016). This KIT-deficient (primaquine) and the risk of hemolytic anemia in G6PD mice demonstrated improved erythropoiesis formation into NOD/ deficient patients have to be overcome. Therefore, there SCID/Il2rg-/- (NSG) background (Cosgun et al., 2014; McIntosh remains a major need of new drugs. et al., 2015; Rahmig et al., 2016). After reconstitution, significant The lack of in vivo models to study liver-stage has been an numbers of mature thrombocytes were present in the peripheral obstacle for studying the biology of hypnozoite formation and blood while human erythroblasts were seen in the bone marrow drug development. Indeed, as detailed below, this was only (BM). In addition, the morphology, composition, and enucleation possible through the recent development of humanized mouse ability of de novo generated human erythroblasts were similar with models engrafted with human hepatocytes. Interestingly, this those in human BM (Rahmig et al., 2016). After humanization of mouse liver repopulation with human hepatocytes required the unconditioned NSGW41 or NBSGW mice, no human red blood establishment of two premises: (i) an immuno-compromised cells (huRBCs) or lower numbers are detected in circulation, recipient mouse and (ii) the initiation of liver injury that depletes whereas the BM is highly repopulated with human erythroid mouse hepatocytes, thus creating a niche that allows human progenitor cells, suggesting that human HSC engraftment hepatocytes to repopulate the mouse liver. Currently, there are supports an increased erythroid lineage production. All three humanized mouse models that can be repopulated with differentiation stages of human erythroid precursors have been human hepatocytes and have been mostly used to study detected and increased numbers of huRBC progenitors are present Plasmodium infections: suggesting that human erythropoiesis and differentiation up to The first liver-humanized model described was the albumin- nucleated erythroid progenitor stages is supported by the murine urokinase-type plasminogen activator (Alb-uPA) transgenic microenvironment in these mouse strains (McIntosh et al., 2015; SCID mouse in which the urokinase transgene is linked to an Rahmig et al., 2016). The enucleation frequency in vivo is totally albumin promoter (Mercer et al., 2001). This resulting in the different between humanized mice and human BM due to the elevated level of plasma uPA, hypofibrinogenemia and formation of erythroblastic islands, which probably requires factors accelerated hepatocyte cell death causing sub-acute liver that are incompatible between both species (Dzierzak and failure. The transplantation of human hepatocyte into 7-12 Philipsen, 2013). Checking the expression of transcripts encoding days Alb-uPA mouse allow a high repopulation of human for adult-type a-, b-, g- and d-globin in NSGW41 model showed hepatocytes resulting in human/mouse chimeric liver with 60- that there is no block in human erythrocyte maturation and give 70% observable human hepatocytes. The Alb-uPA human liver paucity of huRBCs to either insufficient in vivo enucleation or chimeric SCID mouse model (Alb-uPA huHep mouse) has been SIRPa-independent phagocytosis (Rahmig et al., 2016). NSGW41 shown to support P. falciparum infection (Sacci et al., 2006). One and NBSGW strains are thus an interesting model to study P. vivax disadvantage of this humanized mouse model is the continuous blood stages in the bone marrow during infection since these are expression of uPA transgene that causes a progressive damage to able to sustain human eythrocytic precursors by the engraftment the liver parenchyma probably via activation of plasminogen, with HSCs and can be maintained for longer periods of time if which regulates the activity of matrix metalloproteinases that are clodronate liposomes are administered depleting the remaining critical for liver cell growth. Therefore, Alb-uPA model/uPA- murine macrophages. dependent models have a limited utility for many applications due to this disadvantage, as well as a few others such as very poor Humanized Mouse Models in Malaria breeding efficiency, renal disease, and a very narrow time Pre-Erythrocytic Infections window for transplantation before the mice submit to their The pre-erythrocytic cycle of Plasmodium spp. infection begins bleeding (Heckel et al., 1990). when salivary gland sporozoites enter the human body through After Alb-uPA huHep mouse model, the FRG KO mouse model the bite of female infected Anopheles spp. mosquitoes during with triple knock out of tyrosine catabolic enzyme fumaryl- blood meals. Sporozoite then cross the skin barrier to enter the acetoacetate hydrolase FAH, Rag2-/- and IL2rgnull was developed. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 4 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax The fumaryl-acetoacetate hydrolase (FAH) KO leads to the toxic Injection of human reticulocytes/erythrocytes into the FRG accumulation of fumaryl-acetoacetate, an intermediate of the huHep mouse resulted in successful transplantation of human tyrosine hormone metabolism (Azuma et al., 2007) causing liver blood generating FRG huHep-blood mouse model. The FRG KO injury. Like in Alb-uPA model, the FRG KO model can be huHep-blood mouse model has been shown to support P. vivax transplanted with human hepatocytes (changing its name to FRG liver-stage and transition to blood-stage enabling in vivo drug KO huHep) (Azuma et al., 2007). These FAH KO mice maintain efficacy testing (Mikolajczak et al., 2015). By injecting human their hepatocytes only in the presence of 2-(2-nitro-4- reticulocyte on day 9 and day 10 post sporozoite injection, blood- trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) and lose stages of P. vivax were observed as early as 4 hours after them when the drug is withdrawn (Grompe et al., 1995). This reticulocytes injection. Thus, the model can be used for model is also able to support complete development of P. falciparum studying the liver stage-to-blood stage transition of P. vivax liver-stage (LS) infections (Vaughan et al., 2012). Vaughan and (Mikolajczak et al., 2015). The model has also been used to test collaborators have observed that backcrossing the FRG KO huHep the efficacy of a pre-erythrocytic vaccine against P. vivax by model on the NOD background (FRG NOD mouse) results in the passive immunization of anti-PvCSP antibodies prior to transition of exo-erythrocytic merozoites stage to blood stage sporozoite injection, demonstrating that the vaccine could be infection (Vaughan et al., 2012). This model also supports of high benefit by reducing the hypnozoite reservoir and thereby successful transition of recombinant P. falciparum parasites from reducing the number of relapses (Schafer et al., 2021). various experimental genetic crosses (Vendrely et al., 2020). The The FRG huHep mouse model has been improved recently FAH KO mice also show some disadvantages including the (Schafer et al., 2020) by combining an immunomodulatory development of liver carcinomas because of their type I treatment in which Clodronate liposomes and Cyclophosphamide tyrosinemia and the continued or intermittent drug treatment were co-administered to deplete murine macrophages and after humanization to suppress the development of liver cancer neutrophils, respectively (Foquet et al., 2018). This help to increase (Bissig et al., 2010). FRG KO huHep mouse model can also support the lifespan of the infused human reticulocytes in the so called complete development of liver-stage of P. vivax an importantly FRGNKOhuHep/huRetic mouse model. Blood chimerism reached hypnozoite formation. (Mikolajczak et al., 2015) (Schafer 30% after the second infusion of reticulocytes. Liver stages et al., 2020). development in this mouse model resulted in the release of The third liver chimeric humanized mouse model expresses merozoites which were able to invade the infused human the herpes simplex virus thymidine kinase (HSVtk) transgene reticulocytes starting on day 9 onwards with the highest blood under the control of a mouse albumin enhancer/promoter in the parasitemia on day 10 post sporozoite injection. This procedure liver of NOG mice (TK-NOG) (Hasegawa et al., 2011). In this allows efficient and reproducible transition of P. vivax liver-stage to model, the HSVtk mRNA is selectively expressed causing severe blood-stage and specially gametocyte. The FRGNKOhuHep/huRetic parenchymal liver damage after ganciclovir (GCV) treatment model allows the study of vaccine candidates for the blockage of blood which allows repopulation of human hepatocytes (Hasegawa stages (Schafer et al., 2020). Moreover, in FRGNKOhuHep/huRetic et al., 2011). This TK-NOG huHep mouse model has model was detected a subset of exo-erythrocytic schizonts expressing demonstrated a normal systemic and metabolic function and the sexual stage marker Pvs16 as early as 2 days after the beginning of can be maintained without administration of exogenous drugs. blood stage infection indicating that exo-erythrocytic merozoites Noticeably, the humanized TK-NOG huHep mouse model might be pre-programmed to become gametocytes in the first cycle maintains their synthetic function for a prolonged period of of blood stage infection. However, the mature gametocyte marker time (over 8 months), as well as very high plasma human Pvs230 was not detected. Therefore, FRGNKOhuHep/huRetic model albumin levels. Moreover, additional administration of GCV allows the natural route of infection, liver-stage development and doses after engrafted with human hepatocytes enables the transition to blood-stage, providing a valuable system to test liver- depletion of residual mouse hepatocytes after human cell and blood-stage vaccines and drug candidates (see Figure 2). reconstitution (Hasegawa et al., 2011). This model enables the study of P. falciparum and P. ovale liver-stage. Therefore, TK- Perspectives and Challenges of NOG huHep mouse model may also serve as a suitable model for Humanized Mouse Models for Studying P. vivax liver-stage infection. P. vivax Malaria Advances on humanized mouse models are leading to a better Erythrocytic Infections understanding of malaria parasite biology, pathogenesis, and Once the sporozoites invade and establish themselves in the liver, immunology as well as allowing testing, discovery and validation parasites undergo asexual multiplication and develop into of new drugs and antigens for vaccination. Therefore, humanized schizont stages that finally generates exo-erythrocytic mouse models can be seen as the link between rodent models and merozoites. Upon released, thousands of merozoites enter the human infections to translate knowledge from both. In the case bloodstream and initiate erythrocytic cycle. In bloodstream, of Plasmodium vivax, this is of outmost importance as this some parasites undergo gametocytogenesis therefore species lacks a continuous in vitro culture system for blood developing sexual stages which can be transmitted to mosquito stages (Noulin et al., 2013) and, in addition to the liver, the bone vector during the blood meal. The in vivo study of Plasmodium marrow and spleen have recently shown to represent a large infection is more advanced in the FRG huHep mouse model. biomass of hidden parasites in natural infections. In that sense, Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 5 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax FIGURE 2 | Plasmodium falciparum and Plasmodium vivax life cycle together with the currently available immunodeficient mouse models to study the different parasite stages. Models available to study liver stages are Alb-uPA, FRG (N) huHep, TK-NOG and AFC8 model (this last one has not already been tested for Plasmodium infections). Models to study blood stages are FRG (N) huHep model with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection) administration; NSG, NOG and NRG models engrafted with human HSCs and treated with exogenous human cytokines or directly administered with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection); NSGW41 (or NBSGW) and MISTRG models engrafted with human HSC (this last one has not already been tested for Plasmodium infections). Models to study sexual stages are DRAG model engrafted with human HSC, FRG (N) huHep model with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection) administration, NSG, NOG and NRG models engrafted with exogenous huRBCs (for Pf infection) or huRetics (for Pv infection), NSG model engrafted with human HSC, treated with Clo-Lip and NIMP-R14 to deplete remaining murine neutrophils. *Clo-Lip treatment improves huRBCs engraftment/development. Human hepatocytes (huhep); human red blood cells (huRBC); human reticulocytes (huRetics); hematopoietic stem cells (HSC); Clodronate liposomes (Clo-Lip); Plasmodium falciparum (Pf); Plasmodium vivax (Pv); albumin-urokinase-type plasminogen activator (Alb-uPA); Knock-out (KO); FAH, Rag2-/- and IL2rgnull (FRG); thymidine kinase NOG (TK-NOG); NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NGS); NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG); NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG); C;129S4-Rag2tm1.1Flv Csf tm1(CSF1)FlvCsf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2gtm1.1Flv Tg(SIRPA)1Flv/J (MISTRG); NOD.Cg-KitW−41J Prkdcscid Il2rgtm1Wjl/ WaskJ (NSGW41); HLA-DR4 Rag-/- IL2rg-/- NOD (DRAG). (Created with Inkscape by Iris Aparici-Herraiz). the generation of FRGNKOhuHep/huRetic model represented a coagulated frog lymph (Harrison et al., 1907) and the use of Petri major breakthrough for the study of P. vivax liver stages and its dishes named after the inventor (Fischer, 1887). In 2D cultures, transition to blood stages since this model can sustain both cells are grown as a monolayer under controlled physicochemical stages, as well as, the possibility to study gametocytogenesis since parameters such as oxygen, pH and temperature, as well as sexual stage marker Pvs16 was early identified in the onset of the suitable growth medium, for trying to recapitulate as closely as infection (Schafer et al., 2020). However, next-generation possible the cellular microenvironment of the cells. 2D cell humanized mouse models for vivax malaria research will culture systems have advanced significantly our knowledge on require humanization of the liver, bone marrow and spleen for cell biology and for many simple applications they will continue studies on cryptic pre-erythrocytic and erythrocytic infections. providing new insights into cell biology. However, as cells grow, Unfortunately, access to human fetal tissues and broad their morphology changes by flattening and distorting as well as availability to the research community will limit their use. by forming a simple monolayer where they present forced, Therefore, novel enabling technologies reducing the use of artificial polarization. Moreover, cells are subjected to excessive animal experimentation and widely available, are also needed nutrition, molecular gradients cannot be reproduced and the to advance research knowledge on cryptic pre-erythrocytic and characteristics of the extracellular matrix (ECM), including its erythrocytic infections. architecture and stiffness, are also altered (Pampaloni et al., 2007). The simplicity of these traditional 2D culture systems, usually consisting of a single-cell type, makes them robust to ORGANS-ON-A-CHIP perform high-throughput experiments; yet, they provide little information about complex systems such as tissues or organs, The development and implementation of two-dimensional (2D) where cell-cell and cell-matrix communication is essential and cultures in cell biology has revolutionized our knowledge since where cellular in vitro mimicry of the micro-geometry of native the 19th century description of frog embryos in hanging drops of in vivo environments is needed. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Microfluidics Models removing cells’ metabolic waste - maintaining a stable Back in the 70’s of last century, the revolution that supposed the microenvironment - for optimal cell growth and function. use of the micro-fabrication methods from the industrial Moreover, microfluidics allows cells to experience stable microelectronics applied to other materials, such as glass and molecule gradients and shear stress mimicking the in vivo polymers, lead to the development of miniaturized electro- blood physics in microcapillaries. Interestingly, this approach mechanical components with sensors and acturators called has the inherent flexibility of allowing multiple human cell types Micro-ElectroMecanical Systems (MEMS). Noticeably, when cultured in distinct compartments to be connected for inter- MEMS were used to handle fluids, the term microfluidics was tissue modelling obviating the inter-species discrepancies of introduced (Whitesides, 2006). The main advantage of these animal models (Maschmeyer et al., 2015) (Picollet-D'hahan devices relies on the capability to handle small fluid volumes, for et al., 2021). Nevertheless, the first-generation microfluidics instance a microchannel of hundreds of microns can hold fluid approach frequently cultured cells as a 2D monolayer. In this volumes at the nano liter range (100 x 100 x 100 mm = 1nL). This sense, these cell-on-a-chip approaches failed to recapitulate the capability of generating controlled environments with low complex 3D cell–cell and cell-ECM interactions observed at the volume increased the processing velocity (Whitesides, 2006). in vivo microenvironments. Afterwards, microfluidics research centered on the development Accumulating evidence clearly highlights the importance of of analytical devices called Lab-on-a-Chip (LOC) and micro- tridimensionality for in vitro cell culture. Indeed, cells in vivo are Total analysis (µTAS) in order to develop point of care (POC) often surrounded by ECM, which is pivotal to maintain the in devices (Dittrich et al., 2006) (Yager et al., 2006). On these POC vivo-like cell behavior including cell polarity, proliferation, devices, researches were looking to develop fast, reliable and low migration and gene expression (Malinen et al., 2014) cost diagnostics capable to process and analyze proteins, (Fontoura et al., 2020). Thus, the successful development of enzymes or cells (Yager et al., 2006). Organ-on-a-chip models requires the culture of multiple cell Nowadays, it has been demonstrated that cell viability can be types in a highly controlled 3D microenvironment. This maintained in these micro systems with the appropriated ECM highlights the importance of developing fully customizable 3D coatings, culture media and flow conditions. Then, as for the matrices that resemble the native environment of each cell type. conventional in vitro models these cultured cells can be induced In this sense, hydrogels emerge as an important tool to take to express and maintain specific tissue functions in a controllable microfluidic models to the organ level. environment, with the capacity of replicating tissue/organ microstructures. These devices, now known as “organs-on- Hydrogels for Tailored Organs-on-a-Chip chips” (OOC) (Huh et al., 2011) (Hammel et al., 2021) Hydrogels are polymeric materials of cross-linked hydrophilic (Moraes et al., 2012) (Bhatia and Ingber, 2014) are essentially, macromolecules with the ability to retain high amounts of micro structured microreactors containing microchannels water. Importantly, several types of natural and synthetic created on polymeric materials or glass that contain and hydrogels are available possessing a wide range of compartmentalize cultured cells. The greater promise of these relevant biochemical and biophysical features including microsystems lies in the accuracy to recreate physical and cytocompatibility, biodegradability, and viscoelastic properties biochemical microenvironments of specific and key (Neves et al., 2020). Moreover, the easy visualization of cells compartments of living organs that are crucial for organ- embedded within the hydrogel, often transparent, is a plus for the level functions. real-time monitoring of cell behavior, essential for any organ-on- a-chip approach. Notably, hydrogels need to be carefully selected Platform Fabrication and their inherent properties fine-tuned depending on the Soft lithography with PDMS is the most used technique for the intended use. Natural hydrogels including Matrigel, collagen Organ on a Chip platforms fabrication (Huh et al., 2011). This and alginate typically display milder gelling conditions elastomeric material is cheap, transparent, gas permeable and compared to synthetic ones as polyacrylamide or polyethylene biocompatible. However, PDMS absorbs hydrophobic molecules glycol (Hu et al., 2022). Indeed, most of 3D cultures have relied that could affect some analytical processes (Toepke and Beebe, on the use of natural hydrogels (Santos Rosalem et al., 2020). 2006) that would be important for the final purpose of the OOC Basement membrane-derived matrigel is possibly the most model. Therefore, this possibility should be taken in widely used basement membrane hydrogel in 3D organoid consideration, and, if needed be addressed by surface culture experiments (Rossi et al., 2018). Matrigel is composed modification (Herland and Yoon, 2020) or by using alternative by a plethora of different rat basement membrane components materials (Campbell et al., 2021). including laminin-1, collagen IV, entactin, and heparin sulphate proteoglycans, as well as numerous growth factors (Blondel and Adding Dimensionality Lutolf, 2019). Nevertheless, several drawbacks of this hydrogel Over the last decades, the superiority of microfluidic-based cell include (i) unquantifiable lot-to-lot variability which undermines culture platform has been demonstrated in comparison with inter-laboratory reproducibility, (ii) presence of ill-defined static 2D culture in regard to the growth of cells and specific amounts of growth factors making this approach unsuitable for physiological functions (Jang et al., 2015), (Terrell et al., 2020) understanding cell signaling and (iii) their high susceptibility to (Chou et al., 2020). Indeed, microfluidic-based culture platforms protease-mediated degradation (impacting the matrix stability) allows a continuous supply of nutrients and oxygen while which may often preclude long-term cell culture (Blondel and Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Lutolf, 2019). On the other hand, alginate-based hydrogels are composed by more than 150 different proteins, including the non-adhesive biomaterials, as cells cannot establish specific most abundant, fibronectins, elastin, and fibrillar collagens attachment points with the polymer itself (Bidarra and Barrias, (Naba et al., 2014; Arteel and Naba, 2020). 2019) (Neves et al., 2020). Interestingly, this feature is rather useful in artificial microenvironment design as these materials Organ Infection by P. vivax can act as blank-slates. Importantly, natural alginate can be During Plasmodium vivax infection the female Anopheles chemically modified to promote specific biological responses in inoculated sporozoites, will reach the liver via blood circulation a highly tunable fashion through the incorporation of moieties and invade the target hepatocytes either through Kupffer cells or that specifically modulate cell-material interactions (Bidarra via sinusoid endothelial cells (Pradel and Frevert, 2001; Tavares et al., 2011) (Caires et al., 2015). Indeed, depending on the et al., 2013; Venugopal et al., 2020). However, others claim that a intended need alginate has been shown to acquire key biological direct invasion of hepatocytes by sporozoites is also possible features as cell adhesiveness, guided differentiation and matrix (Frevert et al., 1993; Ejigiri and Sinnis, 2009). Within invaded proteolytic degradation through chemical modification with hepatocytes these sporozoites rapidly proliferate and generate instructive peptides. These designer hydrogels have been thousands of merozoites giving rise to schizonts. This parasitic shown to offer unprecedent control over the ECM viscoelastic intrahepatic cycle develops over 7 days until the rupture the liver and biochemical properties and cell fate (Bidarra et al., 2010; schizonts from infected/dying hepatocytes (Venugopal et al., Bidarra et al., 2011; Fonseca et al., 2011; Maia et al., 2014). 2020). Importantly, this rupture mediates the release of Collectively, this next generation of bioengineered organ-on-a- merozoites into blood circulation, where they preferentially chip hold the promise to bridge even further the gap invade young reticulocytes to start the intraerythrocytic life between standard 2D cell culture and the more complex, cycle. In contrast to this route, some P. vivax sporozoites can expensive and ethically controversial animal experimentation undergo a non-proliferative, metabolically quiescent stage in malaria research. known as hypnozoites (Gural et al., 2018b; Sylvester et al., Since the study of P. vivax infection encompasses the 2021). Most importantly, these hypothetical liver-dwelling interrogation of their distinct stages over multiple organs, in hypnozoites may be reactivated months or years and are a the next section we will explore the minimal functional units of major cause of clinical relapse in P. vivax (Markus, 2018; liver, bone marrow and spleen. Most importantly, we will then Taylor et al., 2019). Unfortunately, the current inability to review the available in vitro models that attempt to recapitulate perform long-term in vitro culture of P. vivax severely P. vivax life cycle in each of these organs. Finally, we will address hampers the controlled interrogation of this parasitic the outstanding questions and challenges for the next generation dormancy and re-activation processes. of bioengineered 3D OOC models in P. vivax research. 2D Attempts to Replicate the Liver Stage The Liver Niche So far, most of the key experimental insights on malaria research The liver is a major organ that performs more than 500 was provided either by in vivo rodent models or by the in vitro physiological functions including compound detoxification, 2D monoculture (Voorberg-van der Wel et al., 2020a). decomposition of red blood cells and production of hormones Interestingly, most of these studies use hepatoma-derived cell (Calitz et al., 2018). The portal hepatic lobules are the minimal line model systems such as HepG2, Huh7 and HC-04 functional units of liver, which includes three classical hexagonal (Hollingdale et al., 1985; Kaushansky et al., 2015; Ribeiro et al., hepatic lobules, and are considered the structural unit of the 2016; Tweedell et al., 2019). While this provides a constant and liver. These structures are composed mostly by parenchymal cells reproducible source of host cells, these largely differ from hepatocytes (up to 60%) while the remaining includes non- primary hepatocytes in key biological features for Plasmodium parenchymal cells as hepatic stem cells, connective tissue cells, infection (Manzoni et al., 2017), including the lack of important hepatic stellate cells, monocytic Kupffer cells and endothelial hepatic receptors and functions, lower metabolic activity and cells (Miyajima et al., 2014). The lobular acinus can be further high dependence on glucose uptake (Castell et al., 2006; Meireles divided into three main zones of decreasing oxygen tension due et al., 2017; Tripathi et al., 2020). Additionally, the high to increasing distance towards the radial hepatic arterioles proliferative capacity of these cell lines precludes their (Panday et al., 2022). Most notably the liver zonation seems to application in the study of human P. vivax and hypnozoite display distinct hepatocyte metabolic profiles and functions. formation, which present prolonged development times (more Nevertheless, their role in host-pathogen interaction than 7 days). Interestingly, most of these bottleneck´s issues that remains unknown. hampered the in vitro study of P. vivax liver stage seem to be partially overcome by the use of primary human hepatocyte Biophysical Features of Native ECM (PHH) cells. Nevertheless, upon 2D culture, the PHH The native ECM composes up to 10% of the liver volume, progressively de-differentiate, losing their hepatic in vivo providing structural support, and has a Young’s Elastic phenotype (Du et al., 2006). Indeed, several strategies have Modulus that ranges between 300 and 600 Pa (Wu et al., 2018; been explored to counter PHH hepatic dedifferentiation Passi and Zahler, 2021) to provide structural support. Most through limited biomimicry of hepatic microenvironment. notably, it has been shown that a healthy liver ECM is Some of these attempts include (i) media supplementation Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 8 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax with molecules that inhibit TGF-b, Wnt, Notch and BMP de- of P. vivax including the formation and reactivation of differentiation hepatocyte signaling pathways (Xiang et al., 2019; hypnozoites in vitro (March et al., 2015; Gural et al., 2018b). Lucifora et al., 2020), (ii) coating of the 2D substrate with In a similar multiwell-based approach, it was also highlighted hepatic-like ECM components as collagens (Itsara et al., 2018; that substrate stiffness can also play a role in maintaining PHH Roth et al., 2018), (iii) co-culture with non-parenchymal cells phenotype (Maher et al., 2020). Indeed, these authors obtained such as fibroblasts (March et al., 2013; Gural et al., 2018b), or (iv) similar results for P. vivax infection by simply exploring the use the use of multiple bioengineering approaches to implement the of soft PDMS molds to emboss hepatocyte-confining proper 3D tissue architecture (Chua et al., 2019; Mellin and microfeatures into standard culture polystyrene microplates. Boddey, 2020). Although further improvements in terms of tissue engineering complexity (i.e. with integrated dimensionality, heterotypic co- Available 3D Models in Malaria (Focus on P. vivax) culture, improved infection rate, among others) are required to Pioneering work by Dembele et al. has shown that it was possible resemble the in vivo situation, this type of systems provided the to extend the lifespan/viability of sporozoite-infected simian first steps towards the future in vitro study of the liver hypnozoite hepatocytes in culture, via co-culture with human hepatoma dormancy/activation of the human P. vivax. cells HepaRG (to compensate infected hepatocyte cell loss) in a sandwich culture system (Dembele et al., 2014). Most notably, the use of collagen I coating allowed hepatocytes anchoring to The Bone Marrow Niche the bottom of the culture dish while matrigel was placed on top The bone marrow is located in the trabecular cavities of the long of the cultured cells to impact ECM dimensionality and cell bones, pelvis, sternum, etc. and is composed by multiple cell types polarization. Interestingly, the authors have demonstrated that surrounded by an heterogeneous ECM within an intricate this approach led to a higher in vitro infectivity of P. falciparum microvascular network (Crane et al., 2017). Most importantly, (40-50 times) and that P. cynomolgi sporozoites infection (simian the BM microenvironment is responsible for the de novo surrogate of P. vivax) of hepatocytes developed into both large generation of 5 × 1011 blood components per day, including dividing, but also, in small nondividing forms of the parasite platelets, immune cells and red blood cells (Nombela-Arrieta during 40 days of culture (Dembele et al., 2014). Collectively, the and Manz, 2017). This is achieved throughout distinct BM- authors reported the completion of the full liver cycle of the niches in a highly regulated process known as hematopoiesis parasite with development of schizonts, hypnozoites formation (Nombela-Arrieta and Manz, 2017). For this process several cell up to 15 days with subsequent functional re-activation in culture. types including mesenchymal stem/stromal cells (MSCs), Most recently, Voorberg-van der Wel et al. also demonstrated osteoblasts, endothelial cells and others articulate in exquisite that P. cynomolgi-infected primary rhesus hepatocyte cultured in niches - local microenvironments that maintain and regulate collagen-coated substrates could be routinely maintained for 3 stem cell fate - to simultaneously support hematopoietic stem weeks (Voorberg-van der Wel et al., 2020b). This feature was also cell asymmetric division, progenitor cell proliferation and lineage achieved by Roth et al. with PHH-infected with P. vivax using the commitment into the required blood components (Méndez-Ferrer same commercially available system (Roth et al., 2018). et al., 2020) (Crane et al., 2017). Interestingly, it was shown that, under such conditions, it was possible to explore malaria hypnozoite reactivation in vitro using Biophysical Features of Native ECM a dual fluorescent P. cynomolgi reporter line (Voorberg-van der The BM can be functionally divided into 3 distinct regions: Wel et al., 2020b). endosteal, central and perivascular niches (Nombela-Arrieta and Tissue dimensionality is a key factor for imposing the right Manz, 2017). Structurally, the endosteal niche is located in the cell polarity and functionality. In this sense, Arez et al. have vicinity of BM cortical bone and is composed by osteoblasts and employed a stirred tank culture system to generate spheroids of osteoclasts and MSCs surrounded by a 35 to 40 kPa stiff ECM of human hepatic cell lines with a stable hepatic phenotype up to collagen type I and IV, osteopontin and fibronectin (Nilsson four weeks. Interestingly, P. berghei invasion and development et al., 2005; Engler et al., 2006). The central niche is mostly were recapitulated in these hepatic spheroids, yielding functional inhabited by adipocytes, macrophages and fibroblasts embedded blood-infective merozoites (Arez et al., 2019). Most importantly, in a softer 0.3 kPa ECM containing laminin, fibronectin, heparin, Chua et al. have demonstrated that the formation of spheroids of hyaluronic acid (Nilsson et al., 1998; Omatsu et al., 2010; Shin simian and PHH in the soft macroporous 3D Cellusponge et al., 2014). This is followed by the perivascular niche with a platform, which resemble the native liver ECM, supported the reported ECM stiffness of 2-10 kPa composed of collagen IV, complete liver stage life cycle of both P. cynomolgi and P. vivax fibronectin, and laminin, where endothelial, stromal cells and parasites in vitro for up to 30 days (Chua et al., 2019). MSCs are in close contact with the arterial and sinusoidal blood The importance of heterotypic cell-cell interaction was vessels (Siler et al., 2000; Nelson and Roy, 2016). As expected, the illustrated by showing that a combination of PHHs with relative proximity of the niches with the vascular network creates supportive fibroblasts in a 2D multiwell micropatterned co- particular oxygen gradients, decreasing oxygen tension from the culture (MPCC) format was able to stabilize in vivo-like perivascular to central and endosteal niches. This feature hepatocyte-specific functions and metabolism up to 4-6 weeks combined with niche-specific cytokine gradients are (Gural et al., 2018b). Most interestingly, it was demonstrated that instrumental for the regulation of HSC function and this MPCC platform was also able to support the full liver cycle quiescence (Spencer et al., 2014). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Bioengineered Organ Models arteriolar surrounded by (ii) the lymph follicles where B cells Not surprisingly, even with the recent improvements of 3D divide and maturate (Bowdler, 2002). These regions are followed culture, we still fail to provide an adequate in vitro biomimicry by a marginal zone composed by antigen presenting cells of the complex nature of the bone marrow microenvironment (dendritic cells and macrophages) which are in close proximity (Raic et al., 2019). Several studies have faced difficulties to to the red pulp. The red pulp represents up to 75% of the splenic integrate the tissue-tissue interactions, biochemical gradients tissue and is formed by reticular cells and immune cells and mechanical forces acting in the distinct BM-niches (Santos (granulocytes and monocytes/macrophages) (Bowdler, 2002). Rosalem et al., 2020). Even so, the existing attempts to closely Importantly, the circulatory network in this region contains mimic BM niches are best provided by Organ-on-a-chip open spaces with a complex reticular mesh known as splenic approaches. These microfluidic-based approaches can be cords. Before exiting this compartment the cells and RBCs pass broadly divided by the cell type (single (Sung and Shuler, 2009; through 1–2 µm open interendothelial slits (IES), before re- Sung et al., 2010; Torisawa et al., 2016) vs co-culture (Carrion entering into venous sinuses circulation (Bowdler, 2002). et al., 2010; Khin et al., 2014; Zheng et al., 2016)) and type of Importantly, during this stage the stiffer dysfunctional or tridimensional ECM (Hydrogels (Thon et al., 2014; Torisawa infected RBCs are unable to squeeze through being cleared from et al., 2014; Bruce et al., 2015; Aleman et al., 2019; McAleer et al., circulation by resident macrophages (del Portillo et al., 2012). 2019; Chou et al., 2020; Herland et al., 2020; Glaser et al., 2022) vs Scaffolds (Houshmand et al., 2017; Marturano-Kruik et al., Biophysical Features of Native ECM 2018; Sieber et al., 2018) vs Cell-generated ECM (Zhang et al., Apart from identifying the splenic region by the residing 2014; Zhang et al., 2015; Wuchter et al., 2016)) used for the immune populations in these niches, each of these biomimicry of BM compartments. compartments can also be distinguished by the type of Representing some of these examples, Chou et al. have reported reticular network organization and matrisome (Lokmic et al., a vascularized human BM-on-a-chip (BM chip) that supports the 2008). Interestingly, despite several studies reported the macro- differentiation and maturation of multiple blood cell lineages, stiffness of a healthy spleen in the order of 15-20 KPa, the ECM including reticulocytes, over 4 weeks (Chou et al., 2020). stiffness attributed to each splenic niche remains largely Interestingly, the authors demonstrate that this 3D fibrin/ unknown (Giuffrè et al., 2019) (Veiga et al., 2017). collagen hydrogel-based microfluidic culture system had vastly improved CD34+ cell maintenance and function over the standard Organ Function During P. vivax Infection 2D and 3D bulk approaches, modulating more accurately distinct Not surprisingly, during the intra-erythrocytic life cycle of P. aspects of hematopoiesis and BM pathophysiology. More recently, vivax, the spleen constitutes the main organ involved both in Glaser et al. has also demonstrated that it is possible to study immune recognition and elimination of parasitized reticulocytes niche-specific functions on BM pathophysiology using a 3D (del Portillo et al., 2012). Interestingly, recent data strongly fibrin-based microfluidic co-culture system. For this, the authors suggests that besides facing destruction the P. vivax parasites have simultaneously used distinct chambers in the same chip that can also be actively accumulating in this organ as part of their recapitulated either the endosteal and perivascular niches (Glaser asexual blood stage lifecycle (Kho et al., 2021a). This would et al., 2022). Most interestingly, this system had fully perfusable constitute an important cryptic niche/reservoir of the parasite. vascular networks that allowed the maintenance of CD34+ HSCs Indeed, several reports pinpoint the ability of P. vivax in but also their proliferation and differentiation along myeloid and modulating VIR proteins for enhanced parasitic cytoadhesion erythroid lineages with the release and the egress of neutrophils to human spleen fibroblasts (Fernandez-Becerra et al., 2020). (CD66b+) through the microvascular network. Nevertheless, the questions remain on whether P. vivax The described BM-on- a-chip systems have a wide range of cytoadhesive mechanisms can be used to home to these splenic applicability. Yet, none of these systems has been applied to the cryptic niches, what is the nature of these niches and how do they malaria research so far. Instead, the few reported studies in enable parasite survival in such harsh lymphoid environment. P. vivax research remain limited to address fundamental BM- related questions in 2D culture systems (Panichakul et al., 2007; Available/Reported Models in Malaria Fernandez-Becerra et al., 2013; Martin-Jaular et al., 2013; Noulin Unfortunately, our current understanding of P. vivax-infected et al., 2014). RBCs interaction with the spleen derives mostly from post- mortem spleen sections (Machado Siqueira et al., 2012). (Kho The Spleen Niche et al., 2021a) (Kho et al., 2021b) (Imbert et al., 2009). One of the The spleen is the largest secondary lymphoid organ which is few examples that attempted to dissect the human parasitic primarily responsible for blood immune-surveillance and infection in the human spleen was provided by Buffets’ group. erythrocyte recycling (Bowdler, 2002) (Mebius and Kraal, 2005). Indeed, these authors have developed a technically challenging ex The spleen’s architecture is composed by two minimal functional vivo perfused model of the human spleen that was able to units, the white pulp and red pulp regions, which are connected by maintain clearing functions of P. falciparum-infected RBCs for a marginal zone. The white pulp encompasses nearly 25% of the two hours experiments (Safeukui et al., 2008). Given the splenic tissue and is composed by lymphoid tissue (Kashimura, structural complexity of the splenic white pulp, no 2020). Here, two regions can be distinguished: (i) the periarteriolar bioengineered in vitro 3D models were reported so far. lymphoid sheaths which have mostly T cells lining the central Importantly, this precludes the comprehensive understanding Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 10 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax of the human parasite-immune cell interactions occurring within reside making studies of these organs from natural infections, this niche. Additionally, scarce literature exists regarding the in extremely challenging or not feasible. Previous development of a vitro biomimicry of the Plasmodium-infected erythrocyte microscale human liver platform had demonstrated the feasibility of clearance performed at the splenic red pulp interendothelial mimicking this organ in 2D to study human malaria liver infections slits using a microfluidics-based approach (Rigat-Brugarolas (March et al., 2013). Later, the development of functional units of a et al., 2014) (Picot et al., 2015) (Guo et al., 2012) (Elizalde- liver-on-a-chip emulating the endothelial barrier of a liver sinusoid Torrent et al., 2021) (see Figure 3). physically separated form primary human hepatocytes, showed the FIGURE 3 | Organs-on-a-chip for cryptic infections in malaria research. Microfluidic system connecting the minimal functional units of a liver, bone marrow and spleen-on-a-chip on a PDMS support with controlled perfusion rate. Different molecular-design hydrogels can be used for tissue dimensionality, i.e., matrigel, alginate, fibrin/collagen. The small circles show the presence of malaria parasites and extracellular vesicles; the big circles show 3D cultures with illustrative examples of cell types present in each specific organ (Created with BioRender.com by Nuria Sima). Key Challenges of OOC in potential of using OOC of this organ in studies of physiology and Malaria Research drug-testing (Gural et al., 2018a). Understanding the interactions of malaria parasites during In addition to latent liver infections, P. vivax evolved cryptic different life stages with the vascular endothelium under flow erythrocytic infections in the spleen where the largest parasite conditions is essential for unveiling pathophysiological biomass is accumulated and in the bone marrow where sexual mechanisms in malaria (Bernabeu et al., 2021). Initial stages developed before reaching circulation (Fernandez-Becerra examples of microfluidic devices containing endothelial cells et al., 2022). The development of minimal functional units of cultured under flow conditions used commercial channel these organs-on-a-chip thus offer another unprecedented arrays. These approaches consisted on channel surface opportunities to study these niches. Minimal functional units functionalization with collagen for allowing adherence and of the spleen-on-a-chip have been reported (Rigat-Brugarolas endothelization of these device structures with HUVEC cells et al., 2014) (Elizalde-Torrent et al., 2021) (Picot et al., 2015); yet, (Introini et al., 2018). Recently, a vascular structure formed by in addition to rheological studies of infected blood they need now endothelium pipes was also developed by culturing HUVEC cells to incorporate ECM matrices and cells. In contrast, elegant OOC on a microfluidic device that emulate arteriole, capillary and of the bone marrow showing sustain expansion of CD34+ cells, venule vessels geometry. Interestingly, the authors could monitor differentiation and egress of cells from the chip emulating the spatial location and travel dynamics and the interaction of vascular and bone marrow channels (Chou et al., 2020) (Glaser the infected red blood cells at different stages (Arakawa et al., et al., 2022). It is therefore legitimate to speculate that human 2020). These elegant studies demonstrated the proof-of-principle bone marrow and spleen OOC models will soon be applied to that OOC offer the potential of a transformative technology for advance our knowledge of these cryptic erythrocytic infections studying malaria and other diseases. In fact, OOC are and to screen for novel drugs as parasites in those niches seem to revolutionizing studies on physiopathology, drug development, be shelter from antimalarial drugs (Lee et al., 2018). and POC diagnostics in several different human diseases As a bona fide aspect, OOC will also offer the opportunity to (Ingber, 2022). study the role of extracellular vesicles (EVs), nanovesicles of In the case of Plasmodium vivax, OOC offers an unprecedented endocytic origin, in the formation/activation of such niches at a opportunity to study cryptic niches of infection, which perpetuate space and velocity mimicking functional units of these organs in transmission and challenges malaria elimination. Noticeably, the liver, 3D. Of note, P. vivax has a tropism for reticulocytes, young red bone marrow and spleen are the organs where these hidden parasites cells residing in the bone marrow and the spleen, and which in Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 11 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax BOX | Advances and challenges of humanized mouse models and organs-on-a-chip for malaria research. Humanized Mice Organs on a Chip Advances Technical Limitations Advances Technical Limitations Generation of – Incomplete mouse immunodeficiency hinders Microfluidics – Bubbles, fluidic connections, manipulation, tubbing and immunocompromised engraftment pumping systems must be robust and easy to use mice to engraft human cells of human cells – Interconnection between different organ models to emulate and tissues – Access to human fetal tissues tissue-tissue interaction. – Minimal group size Models for liver stages Alb-uPA – Collateral effects due to the continuous expression PDMS platform – Absorption of hydrophobic molecules could be an issue for of uPA transgene: Liver parenchyma damage, poor free vesicle circulation breeding efficiency, renal disease, narrow time window for transplantation FRG (N) huHep – Liver carcinomas Hydrogels – Ensure the homogeneous diffusion of oxygen, medium, TK-NOG Matrigel* blood, extracellular vesicles. Models for blood stages – Incompatibility between human and mice factors Alginate – Emulate the mechanical properties of ECM of organs. leads to inefficient differentiation ofthe HSC Fibrin/ – Allow internal vascularization and biochemical gradient NSG, NOG, NRG – Dependence on human cytokines administration collagen formation. – Low capacity for human erythroid cells – Lot-to-lot variability development – *Unsuitable for understanding cell signaling NSGW41 – Human erythroid lineage engraftment in the bone Organs-on-a- – Dependence on primary cells from human donors. marrow; yet, few or no red blood cells are observed chip BM- – Donor-to-d onor variability in circulation BM-on-a-chip – Emulate the mechanical properties of circulating blood. FRG(N) huHep – Periodical infusion of huRBCs or huRetics required Splenon-on-a- – Tissue complexity TK-NOG chip – Low P. vivax infection rate Alb-uPA, albumin-urokinase-type plasminogen activator; KO, Knock-out; FRG, FAH, Rag2-/- IL2rgnull; TK-NOG, thymidine kinase NOG; NGS, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; NOG, NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac; NRG, NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ; NSGW41, NOD.Cg-KitW−41J Prkdcscid Il2rgtm1Wjl/WaskJ; hHSCs, human Hematopoietic Stem Cells; huRBCs, human Red Blood Cells; huRetics, human reticulocytes; PDMS, Polydimethylsiloxane; MPCC, come micropatterned cocultures; BM, bone marrow. their maturation to erythrocytes release EVs (Harding et al., AUTHOR CONTRIBUTIONS 1983; Pan and Johnstone, 1983). Noticeably, circulating EVs from natural vivax infections have recently shown to contain Wrote the manuscript: IAH, HC, HP. Contributed to the final parasite proteins and signal spleen fibroblast to increase surface manuscript editing: OC-F, NS, LM-M, RR, JS, WR, AH-M, CF-B, expression of ICAM-1, thus facilitating cytoadherence of P. CB. Figures have been idealised and made by CF-B (Figure 1), vivax-infected reticulocytes directly obtained from patients IA-H (Figure 2), and NS (Figure 3). All authors reviewed the (Toda et al., 2020). EVs thus should contain precious insights manuscript and approved the submitted version. into the formation of such niches. FUNDING CONCLUDING REMARKS IA-H is a predoctoral fellow supported by the Ministerio de Economia y Competitividad (FPI BES-2017081657). Work on Wrongly considered benign, vivax malaria has been a neglected humanized mouse models is funded by the Ministerio de disease. To further complicate matters, in spite of more than a Ciencia e Innovació n (PID2019-111795RB-I00) and on Organs- century of research investigations, this species still lacks a continuous on-a-Chip by “la Caixa” Foundation (LCF/PR/HR21/52410021). in vitro culture system for blood stages, which has severely hampered “This research is part of the ISGlobal “Centro de Excelencia Severo research of it. It is now clear that (i) P. vivax can cause severe disease Ochoa 2019-2023” Program (CEX2018-000806-S) and of the (Baird, 2007), that (ii) chronic infections are associated with a higher “Molecular Mechanisms of Malaria Program” partially funded risk of death than those caused by P. falciparum (Chen et al., 2016) by Fundació n Ramó n Areces. ISGlobal and IGTP are members of and that (iii) P. vivax is a resilient species towards malaria elimination the CERCA programme, Generalitat de Catalunya. (Fernandez-Becerra et al., 2022). Most of this resilience relies on the fact that the largest parasite biomass resides in cryptic niches of the spleen, bone marrow and the liver. Many gaps in the knowledge of these niches as well as technical challenges to study them remain to ACKNOWLEDGMENTS be investigated and solved (Box). Yet, the use of humanized mouse models and organs-on-a-chip technologies reviewed here offer a We thank Drs. Carola Schafer and Stefan H. Kappe for helpful technological breakthrough to study these niches, which ultimately discussions on humanized mouse models in malaria research. might give new clues to develop the continuous in vitro culture L.M.-M is funded by AGAUR (Generalitat de Catalunya) under system of P. vivax. Doctorat Industrial program, grant number (2018 DI 068). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 12 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax REFERENCES Bowdler, A. J. (2002). The Complete Spleen (Totowa: Humana Press). Brito, M. A. M., Baro, B., Raiol, T. C., Ayllon-Hermida, A., Safe, I. P., Deroost, K., Akkina, R., Allam, A., Balazs, A. B., Blankson, J. N., Burnett, J. C., Casares, S., et al. et al. (2020). Morphological and Transcriptional Changes in Human Bone (2016). Improvements and Limitations of Humanized Mouse Models for HIV Marrow During Natural Plasmodium Vivax Malaria Infections. J. Infect. Dis Research: NIH/NIAID "Meet the Experts" 2015 Workshop Summary. AIDS 225(7), 1274–1283. doi: 10.1093/infdis/jiaa177 Res. Hum. Retroviruses 32 (2), 109–119. doi: 10.1089/AID.2015.0258 Bruce, A., Evans, R., Mezan, R., Shi, L., Moses, B. S., Martin, K. H., et al. (2015). Aleman, J., George, S. K., Herberg, S., Devarasetty, M., Porada, C. D., Skardal, A., Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow et al. (2019). Deconstructed Microfluidic Bone Marrow On-A-Chip to Study Microenvironment for Study of Acute Lymphoblastic Leukemia. PloS One 10 Normal and Malignant Hemopoietic Cell-Niche Interactions. Small 15 (43), (10), e0140506. doi: 10.1371/journal.pone.0140506 e1902971. doi: 10.1002/smll.201902971 Caires, H. R., Gomez-Lazaro, M., Oliveira, C. M., Gomes, D., Mateus, D. D., Angrisano, F., and Robinson, L. J. (2022). Plasmodium Vivax - How Hidden Oliveira, C., et al. (2015). Finding and Tracing Human MSC in 3D Reservoirs Hinder Global Malaria Elimination. Parasitol. Int. 87, 102526. Microenvironments With the Photoconvertible Protein Dendra2. Sci. Rep. 5, doi: 10.1016/j.parint.2021.102526 10079. doi: 10.1038/srep10079 Arakawa, C., Gunnarsson, C., Howard, C., Bernabeu, M., Phong, K., Yang, E., et al. Calitz, C., Hamman, J. H., Fey, S. J., Wrzesinski, K., and Gouws, C. (2018). Recent (2020). Biophysical and Biomolecular Interactions of Malaria-Infected Advances in Three-Dimensional Cell Culturing to Assess Liver Function and Erythrocytes in Engineered Human Capillaries. Sci. Adv. 6 (3), eaay7243. Dysfunction: From a Drug Biotransformation and Toxicity Perspective. doi: 10.1126/sciadv.aay7243 Toxicol. Mech. Methods 28 (5), 369–385. doi: 10.1080/15376516.2017.1422580 Arez, F., Rebelo, S. P., Fontinha, D., Simao, D., Martins, T. R., Machado, M., et al. Campbell, S. B., Wu, Q., Yazbeck, J., Liu, C., Okhovatian, S., and Radisic, M. (2019). Flexible 3d Cell-Based Platforms for the Discovery and Profiling of (2021). Beyond Polydimethylsiloxane: Alternative Materials for Fabrication of Novel Drugs Targeting Plasmodium Hepatic Infection. ACS Infect. Dis. 5 (11), Organ-On-a-Chip Devices and Microphysiological Systems. ACS Biomater. 1831–1842. doi: 10.1021/acsinfecdis.9b00144 Sci. Eng. 7 (7), 2880–2899. doi: 10.1021/acsbiomaterials.0c00640 Arteel, G. E., and Naba, A. (2020). The Liver Matrisome - Looking Beyond Cao, X., Shores, E. W., Hu-Li, J., Anver, M. R., Kelsall, B. L., Russell, S. M., et al. Collagens. JHEP Rep. 2 (4), 100115. doi: 10.1016/j.jhepr.2020.100115 (1995). Defective Lymphoid Development in Mice Lacking Expression of the Azuma, H., Paulk, N., Ranade, A., Dorrell, C., Al-Dhalimy, M., Ellis, E., et al. Common Cytokine Receptor Gamma Chain. Immunity 2 (3), 223–238. (2007). Robust Expansion of Human Hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- doi: 10.1016/1074-7613(95)90047-0 Mice. Nat. Biotechnol. 25 (8), 903–910. doi: 10.1038/nbt1326 Carrion, B., Huang, C. P., Ghajar, C. M., Kachgal, S., Kniazeva, E., Jeon, N. L., et al. Baird, J. K. (2007). Neglect of Plasmodium Vivax Malaria. Trends Parasitol. 23 (2010). Recreating the Perivascular Niche Ex Vivo Using a Microfluidic (11), 533–539. doi: 10.1016/j.pt.2007.08.011 Approach. Biotechnol. Bioeng. 107 (6), 1020–1028. doi: 10.1002/bit.22891 Baird, J. K. (2019). 8-Aminoquinoline Therapy for Latent Malaria. Clin. Microbiol. Castell, J. V., Jover, R., Martinez-Jimenez, C. P., and Gomez-Lechon, M. J. (2006). Rev. 32 (4), e00011–00019. doi: 10.1128/CMR.00011-19 Hepatocyte Cell Lines: Their Use, Scope and Limitations in Drug Metabolism Baro, B., Deroost, K., Raiol, T., Brito, M., Almeida, A. C., de Menezes-Neto, A., Studies. Expert Opin. Drug Metab. Toxicol. 2 (2), 183–212. doi: 10.1517/ et al. (2017). Plasmodium Vivax Gametocytes in the Bone Marrow of an Acute 17425255.2.2.183 Malaria Patient and Changes in the Erythroid miRNA Profile. PloS Negl. Trop. Chen, I., Clarke, S. E., Gosling, R., Hamainza, B., Killeen, G., Magill, A., et al. (2016). Dis. 11 (4), e0005365. doi: 10.1371/journal.pntd.0005365 "Asymptomatic" Malaria: A Chronic and Debilitating Infection That Should Be Battle, K. E., Lucas, T. C. D., Nguyen, M., Howes, R. E., Nandi, A. K., Twohig, K. Treated. PloS Med. 13 (1), e1001942. doi: 10.1371/journal.pmed.1001942 A., et al. (2019). Mapping the Global Endemicity and Clinical Burden of Cheng, Q., Cunningham, J., and Gatton, M. L. (2015). Systematic Review of Sub- Plasmodium Vivax 2000-17: A Spatial and Temporal Modelling Study. Lancet Microscopic P. Vivax Infections: Prevalence and Determining Factors. PloS 394 (10195), 332–343. doi: 10.1016/S0140-6736(19)31096-7 Negl. Trop. Dis. 9 (1), e3413. doi: 10.1371/journal.pntd.0003413 Bernabeu, M., Howard, C., Zheng, Y., and Smith, J. D. (2021). Bioengineered 3d Chou, D. B., Frismantas, V., Milton, Y., David, R., Pop-Damkov, P., Ferguson, D., Microvessels for Investigating Plasmodium Falciparum Pathogenesis. Trends et al. (2020). On-Chip Recapitulation of Clinical Bone Marrow Toxicities and Parasitol. 37 (5), 401–413. doi: 10.1016/j.pt.2020.12.008 Patient-Specific Pathophysiology. Nat. BioMed. Eng. 4 (4), 394–406. Bhatia, S. N., and Ingber, D. E. (2014). Microfluidic Organs-on-Chips. Nat. doi: 10.1038/s41551-019-0495-z Biotechnol. 32 (8), 760–772. doi: 10.1038/nbt.2989 Chua, A. C. Y., Ananthanarayanan, A., Ong, J. J. Y., Wong, J. Y., Yip, A., Singh, N. Bidarra, S. J., and Barrias, C. C. (2019). 3d Culture of Mesenchymal Stem Cells in H., et al. (2019). Hepatic Spheroids Used as an In Vitro Model to Study Malaria Alginate Hydrogels. Methods Mol. Biol. 2002, 165–180. doi: 10.1007/ Relapse. Biomaterials 216, 119221. doi: 10.1016/j.biomaterials.2019.05.032 7651_2018_185 Commons, R. J., Simpson, J. A., Thriemer, K., Humphreys, G. S., Abreha, T., Bidarra, S. J., Barrias, C. C., Barbosa, M. A., Soares, R., and Granja, P. L. (2010). Alemu, S. G., et al. (2018). The Effect of Chloroquine Dose and Primaquine on Immobilization of Human Mesenchymal Stem Cells Within RGD-Grafted Plasmodium Vivax Recurrence: A WorldWide Antimalarial Resistance Alginate Microspheres and Assessment of Their Angiogenic Potential. Network Systematic Review and Individual Patient Pooled Meta-Analysis. Biomacromolecules 11 (8), 1956–1964. doi: 10.1021/bm100264a Lancet Infect. Dis. 18 (9), 1025–1034. doi: 10.1016/S1473-3099(18)30348-7 Bidarra, S. J., Barrias, C. C., Fonseca, K. B., Barbosa, M. A., Soares, R. A., and Commons, R. J., Simpson, J. A., Watson, J., White, N. J., and Price, R. N. (2020). Granja, P. L. (2011). Injectable in Situ Crosslinkable RGD-Modified Alginate Estimating the Proportion of Plasmodium Vivax Recurrences Caused by Matrix for Endothelial Cells Delivery. Biomaterials 32 (31), 7897–7904. Relapse: A Systematic Review and Meta-Analysis. Am. J. Trop. Med. Hyg. doi: 10.1016/j.biomaterials.2011.07.013 103 (3), 1094–1099. doi: 10.4269/ajtmh.20-0186 Bignami, M. (1894). On Summer-Autumnal Fever. New Sydenham Soc. 1–232. Cosgun, K. N., Rahmig, S., Mende, N., Reinke, S., Hauber, I., Schafer, C., et al. Bissig, K. D., Wieland, S. F., Tran, P., Isogawa, M., Le, T. T., Chisari, F. V., et al. (2014). Kit Regulates HSC Engraftment Across the Human-Mouse Species (2010). Human Liver Chimeric Mice Provide a Model for Hepatitis B and C Barrier. Cell Stem Cell 15 (2), 227–238. doi: 10.1016/j.stem.2014.06.001 Virus Infection and Treatment. J. Clin. Invest. 120 (3), 924–930. doi: 10.1172/ Crane, G. M., Jeffery, E., and Morrison, S. J. (2017). Adult Haematopoietic Stem JCI40094 Cell Niches. Nat. Rev. Immunol. 17 (9), 573–590. doi: 10.1038/nri.2017.53 Blondel, D., and Lutolf, M. P. (2019). Bioinspired Hydrogels for 3D Organoid del Portillo, H. A., Ferrer, M., Brugat, T., Martin-Jaular, L., Langhorne, J., and Culture. Chimia (Aarau) 73 (1), 81–85. doi: 10.2533/chimia.2019.81 Lacerda, M. V. (2012). The Role of the Spleen in Malaria. Cell Microbiol. 14 (3), Blunt, T., Finnie, N. J., Taccioli, G. E., Smith, G. C., Demengeot, J., Gottlieb, T. M., 343–355. doi: 10.1111/j.1462-5822.2011.01741.x et al. (1995). Defective DNA-Dependent Protein Kinase Activity is Linked to V del Portillo, H. A., Lanzer, M., Rodriguez-Malaga, S., Zavala, F., and Fernandez- (D)J Recombination and DNA Repair Defects Associated With the Murine Becerra, C. (2004). Variant Genes and the Spleen in Plasmodium Vivax Scid Mutation. Cell 80 (5), 813–823. doi: 10.1016/0092-8674(95)90360-7 Malaria. Int. J. Parasitol. 34 (13-14), 1547–1554. doi: 10.1016/ Bosma, G. C., Custer, R. P., and Bosma, M. J. (1983). A Severe Combined j.ijpara.2004.10.012 Immunodeficiency Mutation in the Mouse. Nature 301 (5900), 527–530. Dembele, L., Franetich, J. F., Lorthiois, A., Gego, A., Zeeman, A. M., Kocken, C. H., doi: 10.1038/301527a0 et al. (2014). Persistence and Activation of Malaria Hypnozoites in Long-Term Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 13 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Primary Hepatocyte Cultures. Nat. Med. 20 (3), 307–312. doi: 10.1038/ Glaser, D. E., Curtis, M. B., Sariano, P. A., Rollins, Z. A., Shergill, B. S., Anand, A., nm.3461 et al. (2022). Organ-On-a-Chip Model of Vascularized Human Bone Marrow DiSanto, J. P., Müller, W., Guy-Grand, D., Fischer, A., and Rajewsky, K. (1995). Niches. Biomaterials 280, 121245. doi: 10.1016/j.biomaterials.2021.121245 Lymphoid Development in Mice With a Targeted Deletion of the Interleukin 2 Greenblatt, M. B., Vrbanac, V., Vbranac, V., Tivey, T., Tsang, K., Tager, A. M., Receptor Gamma Chain. Proc. Natl. Acad. Sci. U.S.A. 92 (2), 377–381. et al. (2012). Graft Versus Host Disease in the Bone Marrow, Liver and doi: 10.1073/pnas.92.2.377 Thymus Humanized Mouse Model. PloS One 7 (9), e44664. doi: 10.1371/ Dittrich, P. S., Tachikawa, K., and Manz, A. (2006). Micro Total Analysis Systems. journal.pone.0044664 Latest Advancements and Trends. Anal. Chem. 78 (12), 3887–3908. Grompe, M., Lindstedt, S., al-Dhalimy, M., Kennaway, N. G., Papaconstantinou, J., doi: 10.1021/ac0605602 Torres-Ramos, C. A., et al. (1995). Pharmacological Correction of Neonatal Du, Y., Chia, S. M., Han, R., Chang, S., Tang, H., and Yu, H. (2006). 3D Hepatocyte Lethal Hepatic Dysfunction in a Murine Model of Hereditary Tyrosinaemia Monolayer on Hybrid RGD/galactose Substratum. Biomaterials 27 (33), 5669– Type I. Nat. Genet. 10 (4), 453–460. doi: 10.1038/ng0895-453 5680. doi: 10.1016/j.biomaterials.2006.07.018 Guo, Q., Reiling, S. J., Rohrbach, P., and Ma, H. (2012). Microfluidic Dzierzak, E., and Philipsen, S. (2013). Erythropoiesis: Development and Biomechanical Assay for Red Blood Cells Parasitized by Plasmodium Differentiation. Cold Spring Harb. Perspect. Med. 3 (4), a011601. Falciparum. Lab. Chip 12 (6), 1143–1150. doi: 10.1039/c2lc20857a doi: 10.1101/cshperspect.a011601 Gural, N., Mancio-Silva, L., He, J., and Bhatia, S. N. (2018a). Engineered Livers for Ejigiri, I., and Sinnis, P. (2009). Plasmodium Sporozoite-Host Interactions From Infectious Diseases. Cell Mol. Gastroenterol. Hepatol. 5 (2), 131–144. the Dermis to the Hepatocyte. Curr. Opin. Microbiol. 12 (4), 401–407. doi: 10.1016/j.jcmgh.2017.11.005 doi: 10.1016/j.mib.2009.06.006 Gural, N., Mancio-Silva, L., Miller, A. B., Galstian, A., Butty, V. L., Levine, S. S., Elizalde-Torrent, A., Trejo-Soto, C., Mé ndez-Mora, L., Nicolau, M., Ezama, O., et al. (2018b). In Vitro Culture, Drug Sensitivity, and Transcriptome of Gualdró n-Ló pez, M., et al. (2021). Pitting of Malaria Parasites in Microfluidic Plasmodium Vivax Hypnozoites. Cell Host Microbe 23 (3), 395–406.e394. Devices Mimicking Spleen Interendothelial Slits. Sci. Rep. 11 (1), 22099. doi: 10.1016/j.chom.2018.01.002 doi: 10.1038/s41598-021-01568-w Hammel, J. H., Cook, S. R., Belanger, M. C., Munson, J. M., and Pompano, R. R. (2021). Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E. (2006). Matrix Elasticity Modeling Immunity In Vitro: Slices, Chips, and Engineered Tissues. Annu. Rev. Directs Stem Cell Lineage Specification. Cell 126 (4), 677–689. doi: 10.1016/ BioMed. Eng. 23, 461–491. doi: 10.1146/annurev-bioeng-082420-124920 j.cell.2006.06.044 Harding, C., Heuser, J., and Stahl, P. (1983). Receptor-Mediated Endocytosis of Fernandez-Becerra, C., Aparici-Herraiz, I., and del Portillo, H. A. (2022). Cryptic Transferrin and Recycling of the Transferrin Receptor in Rat Reticulocytes. J. Erythrocytic Infections in Plasmodium Vivax, Another Challenge to its Cell Biol. 97 (2), 329–339. doi: 10.1083/jcb.97.2.329 Elimination. Parasitol. Int. 87, 102527. doi: 10.1016/j.parint.2021.102527 Harrison, R. G., Greenman, M. J., Mall, F. P., and Jackson, C. M. (1907). Fernandez-Becerra, C., Bernabeu, M., Castellanos, A., Correa, B. R., Obadia, T., Observations of the Living Developing Nerve Fiber. Anatomical. Rec. 1 (5), Ramirez, M., et al. (2020). Plasmodium Vivax Spleen-Dependent Genes 116–128. doi: 10.1002/ar.1090010503 Encode Antigens Associated With Cytoadhesion and Clinical Protection. Hasegawa, M., Kawai, K., Mitsui, T., Taniguchi, K., Monnai, M., Wakui, M., et al. Proc. Natl. Acad. Sci. U.S.A. 117 (23), 13056–13065. doi: 10.1073/ (2011). The Reconstituted 'Humanized Liver' in TK-NOG Mice is Mature and pnas.1920596117 Functional. Biochem. Biophys. Res. Commun. 405 (3), 405–410. doi: 10.1016/ Fernandez-Becerra, C., Lelievre, J., Ferrer, M., Anton, N., Thomson, R., Peligero, j.bbrc.2011.01.042 C., et al. (2013). Red Blood Cells Derived From Peripheral Blood and Bone Heckel, J. L., Sandgren, E. P., Degen, J. L., Palmiter, R. D., and Brinster, R. L. Marrow CD34(+) Human Haematopoietic Stem Cells are Permissive to (1990). Neonatal Bleeding in Transgenic Mice Expressing Urokinase-Type Plasmodium Parasites Infection. Mem. Inst. Oswaldo Cruz. 108 (6), 801–803. Plasminogen Activator. Cell 62 (3), 447–456. doi: 10.1016/0092-8674(90) doi: 10.1590/0074-0276108062013019 90010-c Fischer, G. (1887). Centralblatt Für Bakteriologie Und Parasitenkunde. https:// Herland, A., Maoz, B. M., Das, D., Somayaji, M. R., Prantil-Baun, R., Novak, R., www.biodiversitylibrary.org/item/210666 et al. (2020). Quantitative Prediction of Human Pharmacokinetic Responses to Flanagan, S. P. (1966). 'Nude', a New Hairless Gene With Pleiotropic Effects in the Drugs via Fluidically Coupled Vascularized Organ Chips. Nat. BioMed. Eng. 4 Mouse. Genet. Res. 8 (3), 295–309. doi: 10.1017/s0016672300010168 (4), 421–436. doi: 10.1038/s41551-019-0498-9 Fonseca, K. B., Bidarra, S. J., Oliveira, M. J., Granja, P. L., and Barrias, C. C. (2011). Herland, A., and Yoon, M. H. (2020). Macromolecular Bioelectronics. Macromol. Molecularly Designed Alginate Hydrogels Susceptible to Local Proteolysis as Biosci. 20 (11), e2000329. doi: 10.1002/mabi.202000329 Three-Dimensional Cellular Microenvironments. Acta Biomater. 7 (4), 1674– Hollingdale, M. R., Collins, W. E., Campbell, C. C., and Schwartz, A. L. (1985). In 1682. doi: 10.1016/j.actbio.2010.12.029 Vitro Culture of Two Populations (Dividing and Nondividing) of Fontoura, J. C., Viezzer, C., Dos Santos, F. G., Ligabue, R. A., Weinlich, R., Puga, R. Exoerythrocytic Parasites of Plasmodium Vivax. Am. J. Trop. Med. Hyg. 34 D., et al. (2020). Comparison of 2D and 3D Cell Culture Models for Cell (2), 216–222. doi: 10.4269/ajtmh.1985.34.216 Growth, Gene Expression and Drug Resistance. Mater. Sci. Eng. C. Mater. Biol. Houshmand, M., Soleimani, M., Atashi, A., Saglio, G., Abdollahi, M., and Appl. 107, 110264. doi: 10.1016/j.msec.2019.110264 Nikougoftar Zarif, M. (2017). Mimicking the Acute Myeloid Leukemia Foquet, L., Schafer, C., Minkah, N. K., Alanine, D. G. W., Flannery, E. L., Steel, R. Niche for Molecular Study and Drug Screening. Tissue Eng. Part C. Methods W. J., et al. (2018). Plasmodium Falciparum Liver Stage Infection and 23 (2), 72–85. doi: 10.1089/ten.TEC.2016.0404 Transition to Stable Blood Stage Infection in Liver-Humanized and Blood- Huh, D., Hamilton, G. A., and Ingber, D. E. (2011). From 3D Cell Culture to Humanized FRGN KO Mice Enables Testing of Blood Stage Inhibitory Organs-on-Chips. Trends Cell Biol. 21 (12), 745–754. doi: 10.1016/ Antibodies (Reticulocyte-Binding Protein Homolog 5) In Vivo. Front. j.tcb.2011.09.005 Immunol. 9. doi: 10.3389/fimmu.2018.00524 Hu, X., Xia, Z., and Cai, K. (2022). Recent Advances in 3D Hydrogel Culture Frevert, U., Sinnis, P., Cerami, C., Shreffler, W., Takacs, B., and Nussenzweig, V. Systems for Mesenchymal Stem Cell-Based Therapy and Cell Behavior (1993). Malaria Circumsporozoite Protein Binds to Heparan Sulfate Regulation. J. Mater. Chem. B. 10 (10), 1486–1507. doi: 10.1039/d1tb02537f Proteoglycans Associated With the Surface Membrane of Hepatocytes. J. Imbert, P., Rapp, C., and Buffet, P. A. (2009). Pathological Rupture of the Spleen in Exp. Med. 177 (5), 1287–1298. doi: 10.1084/jem.177.5.1287 Malaria: Analysis of 55 Cases, (1958-2008). Travel Med. Infect. Dis. 7 (3), 147– Ganick, D. J., Sarnwick, R. D., Shahidi, N. T., and Manning, D. D. (1980). Inability 159. doi: 10.1016/j.tmaid.2009.01.002 of Intravenously Injected Monocellular Suspensions of Human Bone Marrow Ingber, D. E. (2022). Human Organs-on-Chips for Disease Modelling, Drug to Establish in the Nude Mouse. Int. Arch. Allergy Appl. Immunol. 62 (3), 330– Development and Personalized Medicine. Nat. Rev. Genet, 1–25. 333. doi: 10.1159/000232530 doi: 10.1038/s41576-022-00466-9 Giuffrè, M., Macor, D., Masutti, F., Abazia, C., Tinè, F., Patti, R., et al. (2019). Introini, V., Carciati, A., Tomaiuolo, G., Cicuta, P., and Guido, S. (2018). Evaluation of Spleen Stiffness in Healthy Volunteers Using Point Shear Wave Endothelial Glycocalyx Regulates Cytoadherence in Plasmodium Falciparum Elastography. Ann. Hepatol. 18 (5), 736–741. doi: 10.1016/j.aohep.2019.03.004 Malaria. J. R. Soc. Interface 15 (149), 20180773. doi: 10.1098/rsif.2018.0773 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 14 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Itsara, L. S., Zhou, Y., Do, J., Dungel, S., Fishbaugher, M. E., Betz, W. W., et al. Machado Siqueira, A., Lopes Magalhaes, B. M., Cardoso Melo, G., Ferrer, M., (2018). PfCap380 as a Marker for Plasmodium Falciparum Oocyst Castillo, P., Martin-Jaular, L., et al. (2012). Spleen Rupture in a Case of Development In Vivo and In Vitro. Malar. J. 17 (1), 135. doi: 10.1186/ Untreated Plasmodium Vivax Infection. PloS Negl. Trop. Dis. 6 (12), e1934. s12936-018-2277-6 doi: 10.1371/journal.pntd.0001934 Jang, M., Neuzil, P., Volk, T., Manz, A., and Kleber, A. (2015). On-Chip Three- Maher, S. P., Conway, A. J., Roth, A., Adapa, S. R., Cualing, P., Andolina, C., et al. Dimensional Cell Culture in Phaseguides Improves Hepatocyte Functions In (2020). An Adaptable Soft-Mold Embossing Process for Fabricating Optically- Vitro. Biomicrofluidics 9 (3), 034113. doi: 10.1063/1.4922863 Accessible, Microfeature-Based Culture Systems and Application Toward Liver John, G. K., Douglas, N. M., von Seidlein, L., Nosten, F., Baird, J. K., White, N. J., Stage Antimalarial Compound Testing. Lab. Chip 20 (6), 1124–1139. et al. (2012). Primaquine Radical Cure of Plasmodium Vivax: A Critical Review doi: 10.1039/c9lc00921c of the Literature. Malar. J. 11, 280. doi: 10.1186/1475-2875-11-280 Maia, F. R., Barbosa, M., Gomes, D. B., Vale, N., Gomes, P., Granja, P. L., et al. Kashimura, M. (2020). The Human Spleen as the Center of the Blood Defense (2014). Hydrogel Depots for Local Co-Delivery of Osteoinductive Peptides and System. Int. J. Hematol. 112 (2), 147–158. doi: 10.1007/s12185-020-02912-y Mesenchymal Stem Cells. J. Control Release 189, 158–168. doi: 10.1016/ Kaushansky, A., Douglass, A. N., Arang, N., Vigdorovich, V., Dambrauskas, N., j.jconrel.2014.06.030 Kain, H. S., et al. (2015). Malaria Parasites Target the Hepatocyte Receptor Malinen, M. M., Kanninen, L. K., Corlu, A., Isoniemi, H. M., Lou, Y. R., EphA2 for Successful Host Infection. Science 350 (6264), 1089–1092. Yliperttula, M. L., et al. (2014). Differentiation of Liver Progenitor Cell Line doi: 10.1126/science.aad3318 to Functional Organotypic Cultures in 3D Nanofibrillar Cellulose and Kennedy, M. K., Glaccum, M., Brown, S. N., Butz, E. A., Viney, J. L., Embers, M., Hyaluronan-Gelatin Hydrogels. Biomaterials 35 (19), 5110–5121. et al. (2000). Reversible Defects in Natural Killer and Memory CD8 T Cell doi: 10.1016/j.biomaterials.2014.03.020 Lineages in Interleukin 15-Deficient Mice. J. Exp. Med. 191 (5), 771–780. Manzoni, G., Marinach, C., Topcu, S., Briquet, S., Grand, M., Tolle, M., et al. doi: 10.1084/jem.191.5.771 (2017). Plasmodium P36 Determines Host Cell Receptor Usage During Kerre, T. C., De Smet, G., De Smedt, M., Zippelius, A., Pittet, M. J., Langerak, A. Sporozoite Invasion. Elife 6, e25903. doi: 10.7554/eLife.25903 W., et al. (2002). Adapted NOD/SCID Model Supports Development of March, S., Ng, S., Velmurugan, S., Galstian, A., Shan, J., Logan, D. J., et al. (2013). Phenotypically and Functionally Mature T Cells From Human Umbilical A Microscale Human Liver Platform That Supports the Hepatic Stages of Cord Blood CD34(+) Cells. Blood 99 (5), 1620–1626. doi: 10.1182/ Plasmodium Falciparum and Vivax. Cell Host Microbe 14 (1), 104–115. blood.v99.5.1620 doi: 10.1016/j.chom.2013.06.005 Khin, Z. P., Ribeiro, M. L., Jacobson, T., Hazlehurst, L., Perez, L., Baz, R., et al. March, S., Ramanan, V., Trehan, K., Ng, S., Galstian, A., Gural, N., et al. (2015). (2014). A Preclinical Assay for Chemosensitivity in Multiple Myeloma. Cancer Micropatterned Coculture of Primary Human Hepatocytes and Supportive Res. 74 (1), 56–67. doi: 10.1158/0008-5472.CAN-13-2397 Cells for the Study of Hepatotropic Pathogens. Nat. Protoc. 10 (12), 2027–2053. Kho, S., Qotrunnada, L., Leonardo, L., Andries, B., Wardani, P. A. I., Fricot, A., doi: 10.1038/nprot.2015.128 et al. (2021a). Evaluation of Splenic Accumulation and Colocalization of Markus, M. B. (2018). Biological Concepts in Recurrent Plasmodium Vivax Immature Reticulocytes and Plasmodium Vivax in Asymptomatic Malaria: A Malaria. Parasitology 145 (13), 1765–1771. doi: 10.1017/S003118201 Prospective Human Splenectomy Study. PloS Med. 18 (5), e1003632. 800032X doi: 10.1371/journal.pmed.1003632 Martin-Jaular, L., Elizalde-Torrent, A., Thomson-Luque, R., Ferrer, M., Segovia, J. Kho, S., Qotrunnada, L., Leonardo, L., Andries, B., Wardani, P. A. I., Fricot, A., C., Herreros-Aviles, E., et al. (2013). Reticulocyte-Prone Malaria Parasites et al. (2021b). Hidden Biomass of Intact Malaria Parasites in the Human Predominantly Invade CD71hi Immature Cells: Implications for the Spleen. N. Engl. J. Med. 384 (21), 2067–2069. doi: 10.1056/NEJMc2023884 Development of an In Vitro Culture for Plasmodium Vivax. Malar. J. 12, Kirchgessner, C. U., Patil, C. K., Evans, J. W., Cuomo, C. A., Fried, L. M., Carter, 434. doi: 10.1186/1475-2875-12-434 T., et al. (1995). DNA-Dependent Kinase (P350) as a Candidate Gene for the Marturano-Kruik, A., Nava, M. M., Yeager, K., Chramiec, A., Hao, L., Robinson, Murine SCID Defect. Science 267 (5201), 1178–1183. doi: 10.1126/ S., et al. (2018). Human Bone Perivascular Niche-on-a-Chip for Studying science.7855601 Metastatic Colonization. Proc. Natl. Acad. Sci. U.S.A. 115 (6), 1256–1261. Krotoski, W. A. (1985). Discovery of the Hypnozoite and a New Theory of doi: 10.1073/pnas.1714282115 Malarial Relapse. Trans. R. Soc. Trop. Med. Hyg. 79 (1), 1–11. doi: 10.1016/ Maschmeyer, I., Lorenz, A. K., Schimek, K., Hasenberg, T., Ramme, A. P., Hübner, 0035-9203(85)90221-4 J., et al. (2015). A Four-Organ-Chip for Interconnected Long-Term Co-Culture Lee, R. S., Waters, A. P., and Brewer, J. M. (2018). A Cryptic Cycle in of Human Intestine, Liver, Skin and Kidney Equivalents. Lab. Chip 15 (12), Haematopoietic Niches Promotes Initiation of Malaria Transmission and 2688–2699. doi: 10.1039/c5lc00392j Evasion of Chemotherapy. Nat. Commun. 9 (1), 1689. doi: 10.1038/s41467- McAleer, C. W., Long, C. J., Elbrecht, D., Sasserath, T., Bridges, L. R., Rumsey, J. 018-04108-9 W., et al. (2019). Multi-Organ System for the Evaluation of Efficacy and Off- Legrand, N., Huntington, N. D., Nagasawa, M., Bakker, A. Q., Schotte, R., Strick- Target Toxicity of Anticancer Therapeutics. Sci. Transl. Med. 11 (497), Marchand, H., et al. (2011). Functional CD47/signal Regulatory Protein Alpha eaav1386. doi: 10.1126/scitranslmed.aav1386 (SIRP(alpha)) Interaction is Required for Optimal Human T- and Natural McIntosh, B. E., Brown, M. E., Duffin, B. M., Maufort, J. P., Vereide, D. T., Slukvin, Killer- (NK) Cell Homeostasis In Vivo. Proc. Natl. Acad. Sci. U.S.A. 108 (32), I. I., et al. (2015). Nonirradiated NOD,B6.SCID Il2rgamma-/- Kit(W41/W41) 13224–13229. doi: 10.1073/pnas.1101398108 (NBSGW) Mice Support Multilineage Engraftment of Human Hematopoietic Llanos-Cuentas, A., Lacerda, M. V. G., Hien, T. T., Velez, I. D., Namaik-Larp, C., Cells. Stem Cell Rep. 4 (2), 171–180. doi: 10.1016/j.stemcr.2014.12.005 Chu, C. S., et al. (2019). Tafenoquine Versus Primaquine to Prevent Relapse of Mebius, R. E., and Kraal, G. (2005). Structure and Function of the Spleen. Nat. Rev. Plasmodium Vivax Malaria. N. Engl. J. Med. 380 (3), 229–241. doi: 10.1056/ Immunol. 5 (8), 606–616. doi: 10.1038/nri1669 NEJMoa1802537 Meireles, P., Sales-Dias, J., Andrade, C. M., Mello-Vieira, J., Mancio-Silva, L., Lokmic, Z., Lämmermann, T., Sixt, M., Cardell, S., Hallmann, R., and Sorokin, L. Simas, J. P., et al. (2017). GLUT1-Mediated Glucose Uptake Plays a Crucial (2008). The Extracellular Matrix of the Spleen as a Potential Organizer of Role During Plasmodium Hepatic Infection. Cell Microbiol. 19 (2), e12646. Immune Cell Compartments. Semin. Immunol. 20 (1), 4–13. doi: 10.1016/ doi: 10.1111/cmi.12646 j.smim.2007.12.009 Mellin, R., and Boddey, J. A. (2020). Organoids for Liver Stage Malaria Research. Lucifora, J., Michelet, M., Rivoire, M., Protzer, U., Durantel, D., and Zoulim, F. Trends Parasitol. 36 (2), 158–169. doi: 10.1016/j.pt.2019.12.003 (2020). Two-Dimensional-Cultures of Primary Human Hepatocytes Allow Mé nard, D., Barnadas, C., Bouchier, C., Henry-Halldin, C., Gray, L. R., Efficient HBV Infection: Old Tricks Still Work! J. Hepatol. 73 (2), 449–451. Ratsimbasoa, A., et al. (2010). Plasmodium Vivax Clinical Malaria is doi: 10.1016/j.jhep.2020.03.042 Commonly Observed in Duffy-Negative Malagasy People. Proc. Natl. Acad. Macchiarini, F., Manz, M. G., Palucka, A. K., and Shultz, L. D. (2005). Humanized Sci. U.S.A. 107 (13), 5967–5971. doi: 10.1073/pnas.0912496107 Mice: Are We There Yet? J. Exp. Med. 202 (10), 1307–1311. doi: 10.1084/ Mé ndez-Ferrer, S., Bonnet, D., Steensma, D. P., Hasserjian, R. P., Ghobrial, I. M., jem.20051547 Gribben, J. G., et al. (2020). Bone Marrow Niches in Haematological Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 15 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Malignancies. Nat. Rev. Cancer 20 (5), 285–298. doi: 10.1038/s41568-020- Hematopoietic Stem and Progenitor Cell Niche. Immunity 33 (3), 387–399. 0245-2 doi: 10.1016/j.immuni.2010.08.017 Mercer, D. F., Schiller, D. E., Elliott, J. F., Douglas, D. N., Hao, C., Rinfret, A., et al. Pampaloni, F., Reynaud, E. G., and Stelzer, E. H. (2007). The Third Dimension (2001). Hepatitis C Virus Replication in Mice With Chimeric Human Livers. Bridges the Gap Between Cell Culture and Live Tissue. Nat. Rev. Mol. Cell Biol. Nat. Med. 7 (8), 927–933. doi: 10.1038/90968 8 (10), 839–845. doi: 10.1038/nrm2236 Mikolajczak, S. A., Vaughan, A. M., Kangwanrangsan, N., Roobsoong, W., Panday, R., Monckton, C. P., and Khetani, S. R. (2022). The Role of Liver Zonation Fishbaugher, M., Yimamnuaychok, N., et al. (2015). Plasmodium Vivax in Physiology, Regeneration, and Disease. Semin. Liver Dis. 42 (1), 1–16. Liver Stage Development and Hypnozoite Persistence in Human Liver- doi: 10.1055/s-0041-1742279 Chimeric Mice. Cell Host Microbe 17 (4), 526–535. doi: 10.1016/ Panichakul, T., Sattabongkot, J., Chotivanich, K., Sirichaisinthop, J., Cui, L., and j.chom.2015.02.011 Udomsangpetch, R. (2007). Production of Erythropoietic Cells In Vitro for Miyajima, A., Tanaka, M., and Itoh, T. (2014). Stem/progenitor Cells in Liver Continuous Culture of Plasmodium Vivax. Int. J. Parasitol. 37 (14), 1551– Development, Homeostasis, Regeneration, and Reprogramming. Cell Stem Cell 1557. doi: 10.1016/j.ijpara.2007.05.009 14 (5), 561–574. doi: 10.1016/j.stem.2014.04.010 Pan, B. T., and Johnstone, R. M. (1983). Fate of the Transferrin Receptor During Moraes, C., Mehta, G., Lesher-Perez, S. C., and Takayama, S. (2012). Organs-On- Maturation of Sheep Reticulocytes In Vitro: Selective Externalization of the a-Chip: A Focus on Compartmentalized Microdevices. Ann. BioMed. Eng. 40 Receptor. Cell 33 (3), 967–978. doi: 10.1016/0092-8674(83)90040-5 (6), 1211–1227. doi: 10.1007/s10439-011-0455-6 Passi, M., and Zahler, S. (2021). Mechano-Signaling Aspects of Hepatocellular Mosier, D. E., Gulizia, R. J., Baird, S. M., and Wilson, D. B. (1988). Transfer of a Carcinoma. J. Cancer 12 (21), 6411–6421. doi: 10.7150/jca.60102 Functional Human Immune System to Mice With Severe Combined Pearson, T., Greiner, D. L., and Shultz, L. D. (2008). Humanized SCID Mouse Immunodeficiency. Nature 335 (6187), 256–259. doi: 10.1038/335256a0 Models for Biomedical Research. Curr. Top. Microbiol. Immunol. 324, 25–51. Mueller, I., Galinski, M. R., Baird, J. K., Carlton, J. M., Kochar, D. K., Alonso, P. L., doi: 10.1007/978-3-540-75647-7_2 et al. (2009). Key Gaps in the Knowledge of Plasmodium Vivax, a Neglected Picollet-D'hahan, N., Zuchowska, A., Lemeunier, I., and Le Gac, S. (2021). Human Malaria Parasite. Lancet Infect. Dis. 9 (9), 555–566. doi: 10.1016/ Multiorgan-On-a-Chip: A Systemic Approach To Model and Decipher S1473-3099(09)70177-X Inter-Organ Communication. Trends Biotechnol. 39 (8), 788–810. Murphy, W. J., Kumar, V., and Bennett, M. (1987). Rejection of Bone Marrow doi: 10.1016/j.tibtech.2020.11.014 Allografts by Mice With Severe Combined Immune Deficiency (SCID). Picot, J., Ndour, P. A., Lefevre, S. D., El Nemer, W., Tawfik, H., Galimand, J., et al. Evidence That Natural Killer Cells can Mediate the Specificity of Marrow (2015). A Biomimetic Microfluidic Chip to Study the Circulation and Graft Rejection. J. Exp. Med. 165 (4), 1212–1217. doi: 10.1084/jem.165.4.1212 Mechanical Retention of Red Blood Cells in the Spleen. Am. J. Hematol. 90 Naba, A., Clauser, K. R., Whittaker, C. A., Carr, S. A., Tanabe, K. K., and Hynes, R. (4), 339–345. doi: 10.1002/ajh.23941 O. (2014). Extracellular Matrix Signatures of Human Primary Metastatic Pradel, G., and Frevert, U. (2001). Malaria Sporozoites Actively Enter and Pass Colon Cancers and Their Metastases to Liver. BMC Cancer 14, 518. Through Rat Kupffer Cells Prior to Hepatocyte Invasion. Hepatology 33 (5), doi: 10.1186/1471-2407-14-518 1154–1165. doi: 10.1053/jhep.2001.24237 Nelson, M. R., and Roy, K. (2016). Bone-Marrow Mimicking Biomaterial Niches Rahmig, S., Kronstein-Wiedemann, R., Fohgrub, J., Kronstein, N., for Studying Hematopoietic Stem and Progenitor Cells. J. Mater. Chem. B. 4 Nevmerzhitskaya, A., Bornhauser, M., et al. (2016). Improved Human (20), 3490–3503. doi: 10.1039/c5tb02644j Erythropoiesis and Platelet Formation in Humanized NSGW41 Mice. Stem Neves, S. C., Moroni, L., Barrias, C. C., and Granja, P. L. (2020). Leveling Up Cell Rep. 7 (4), 591–601. doi: 10.1016/j.stemcr.2016.08.005 Hydrogels: Hybrid Systems in Tissue Engineering. Trends Biotechnol. 38 (3), Raic, A., Naolou, T., Mohra, A., Chatterjee, C., and Lee-Thedieck, C. (2019). 3D 292–315. doi: 10.1016/j.tibtech.2019.09.004 Models of the Bone Marrow in Health and Disease: Yesterday, Today and Nilsson, S. K., Debatis, M. E., Dooner, M. S., Madri, J. A., Quesenberry, P. J., and Tomorrow. MRS Commun. 9 (1), 37–52. doi: 10.1557/mrc.2018.203 Becker, P. S. (1998). Immunofluorescence Characterization of Key Ranson, T., Vosshenrich, C. A., Corcuff, E., Richard, O., Müller, W., and Di Santo, Extracellular Matrix Proteins in Murine Bone Marrow in Situ. J. Histochem. J. P. (2003). IL-15 is an Essential Mediator of Peripheral NK-Cell Homeostasis. Cytochem. 46 (3), 371–377. doi: 10.1177/002215549804600311 Blood 101 (12), 4887–4893. doi: 10.1182/blood-2002-11-3392 Nilsson, S. K., Johnston, H. M., Whitty, G. A., Williams, B., Webb, R. J., Denhardt, Ribeiro, C. J., Espadinha, M., Machado, M., Gut, J., Goncalves, L. M., Rosenthal, P. J., D. T., et al. (2005). Osteopontin, a Key Component of the Hematopoietic Stem et al. (2016). Novel Squaramides With In Vitro Liver Stage Antiplasmodial Activity. Cell Niche and Regulator of Primitive Hematopoietic Progenitor Cells. Blood Bioorg. Med. Chem. 24 (8), 1786–1792. doi: 10.1016/j.bmc.2016.03.005 106 (4), 1232–1239. doi: 10.1182/blood-2004-11-4422 Rigat-Brugarolas, L. G., Elizalde-Torrent, A., Bernabeu, M., De Niz, M., Martin- Nombela-Arrieta, C., and Manz, M. G. (2017). Quantification and Three- Jaular, L., Fernandez-Becerra, C., et al. (2014). A Functional Microengineered Dimensional Microanatomical Organization of the Bone Marrow. Blood Model of the Human Splenon-on-a-Chip. Lab. Chip 14 (10), 1715–1724. Adv. 1 (6), 407–416. doi: 10.1182/bloodadvances.2016003194 doi: 10.1039/c3lc51449h Noulin, F., Borlon, C., Van Den Abbeele, J., D'Alessandro, U., and Erhart, A. Rossi, G., Manfrin, A., and Lutolf, M. P. (2018). Progress and Potential in (2013). 1912-2012: A Century of Research on Plasmodium Vivax In Vitro Organoid Research. Nat. Rev. Genet. 19 (11), 671–687. doi: 10.1038/s41576- Culture. Trends Parasitol. 29 (6), 286–294. doi: 10.1016/j.pt.2013.03.012 018-0051-9 Noulin, F., Manesia, J. K., Rosanas-Urgell, A., Erhart, A., Borlon, C., Van Den Roth, A., Maher, S. P., Conway, A. J., Ubalee, R., Chaumeau, V., Andolina, C., et al. Abbeele, J., et al. (2014). Hematopoietic Stem/Progenitor Cell Sources to (2018). Author Correction: A Comprehensive Model for Assessment of Liver Generate Reticulocytes for Plasmodium Vivax Culture. PloS One 9 (11), Stage Therapies Targeting Plasmodium Vivax and Plasmodium Falciparum. e112496. doi: 10.1371/journal.pone.0112496 Nat. Commun. 9 (1), 1837. doi: 10.1038/s41467-018-04817-1 Ogasawara, K., Hamerman, J. A., Hsin, H., Chikuma, S., Bour-Jordan, H., Chen, Sacci, J. B.Jr., Alam, U., Douglas, D., Lewis, J., Tyrrell, D. L., Azad, A. F., et al. T., et al. (2003). Impairment of NK Cell Function by NKG2D Modulation in (2006). Plasmodium Falciparum Infection and Exoerythrocytic Development NOD Mice. Immunity 18 (1), 41–51. doi: 10.1016/s1074-7613(02)00505-8 in Mice With Chimeric Human Livers. Int. J. Parasitol. 36 (3), 353–360. Ohbo, K., Suda, T., Hashiyama, M., Mantani, A., Ikebe, M., Miyakawa, K., et al. doi: 10.1016/j.ijpara.2005.10.014 (1996). Modulation of Hematopoiesis in Mice With a Truncated Mutant of the Safeukui, I., Correas, J. M., Brousse, V., Hirt, D., Deplaine, G., Mulé , S., et al. Interleukin-2 Receptor Gamma Chain. Blood 87 (3), 956–967. doi: 10.1182/ (2008). Retention of Plasmodium Falciparum Ring-Infected Erythrocytes in blood.V87.3.956.bloodjournal873956 the Slow, Open Microcirculation of the Human Spleen. Blood 112 (6), 2520– Oldenborg, P. A., Zheleznyak, A., Fang, Y. F., Lagenaur, C. F., Gresham, H. D., and 2528. doi: 10.1182/blood-2008-03-146779 Lindberg, F. P. (2000). Role of CD47 as a Marker of Self on Red Blood Cells. Santos Rosalem, G., Gonzales Torres, L. A., de Las Casas, E. B., Mathias, F. A. S., Science 288 (5473), 2051–2054. doi: 10.1126/science.288.5473.2051 Ruiz, J. C., and Carvalho, M. G. R. (2020). Microfluidics and Organ-on-a-Chip Omatsu, Y., Sugiyama, T., Kohara, H., Kondoh, G., Fujii, N., Kohno, K., et al. Technologies: A Systematic Review of the Methods Used to Mimic Bone (2010). The Essential Functions of Adipo-Osteogenic Progenitors as the Marrow. PloS One 15 (12), e0243840. doi: 10.1371/journal.pone.0243840 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 16 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax Schafer, C., Dambrauskas, N., Reynolds, L. M., Trakhimets, O., Raappana, A., Plasmodium Vivax Patients Signal Spleen Fibroblasts via NF-kB Flannery, E. L., et al. (2021). Partial Protection Against P. Vivax Infection Facilitating Parasite Cytoadherence. Nat. Commun. 11 (1):2761. Diminishes Hypnozoite Burden and Blood-Stage Relapses. Cell Host Microbe doi: 10.1038/s41467-020-16337-y 29 (5), 752–756.e754. doi: 10.1016/j.chom.2021.03.011 Toepke, M. W., and Beebe, D. J. (2006). PDMS Absorption of Small Molecules and Schafer, C., Roobsoong, W., Kangwanrangsan, N., Bardelli, M., Rawlinson, T. A., Consequences in Microfluidic Applications. Lab. Chip 6 (12), 1484–1486. Dambrauskas, N., et al. (2020). A Humanized Mouse Model for Plasmodium doi: 10.1039/b612140c Vivax to Test Interventions That Block Liver Stage to Blood Stage Transition Torisawa, Y. S., Mammoto, T., Jiang, E., Jiang, A., Mammoto, A., Watters, A. L., and Blood Stage Infection. iScience 23 (8), 101381. doi: 10.1016/ et al. (2016). Modeling Hematopoiesis and Responses to Radiation j.isci.2020.101381 Countermeasures in a Bone Marrow-On-a-Chip. Tissue Eng. Part C. Shin, J. W., Buxboim, A., Spinler, K. R., Swift, J., Christian, D. A., Hunter, C. A., Methods 22 (5), 509–515. doi: 10.1089/ten.TEC.2015.0507 et al. (2014). Contractile Forces Sustain and Polarize Hematopoiesis From Torisawa, Y. S., Spina, C. S., Mammoto, T., Mammoto, A., Weaver, J. C., Tat, T., Stem and Progenitor Cells. Cell Stem Cell 14 (1), 81–93. doi: 10.1016/ et al. (2014). Bone Marrow-on-a-Chip Replicates Hematopoietic Niche j.stem.2013.10.009 Physiology In Vitro. Nat. Methods 11 (6), 663–669. doi: 10.1038/nmeth.2938 Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., et al. Traggiai, E., Chicha, L., Mazzucchelli, L., Bronz, L., Piffaretti, J. C., Lanzavecchia, (1992). RAG-2-Deficient Mice Lack Mature Lymphocytes Owing to Inability to A., et al. (2004). Development of a Human Adaptive Immune System in Cord Initiate V(D)J Rearrangement. Cell 68 (5), 855–867. doi: 10.1016/0092-8674 Blood Cell-Transplanted Mice. Science 304 (5667), 104–107. doi: 10.1126/ (92)90029-c science.1093933 Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V., and Greiner, D. L. (2012). Tripathi, J., Segeritz, C. P., Griffiths, G., Bushell, W., Vallier, L., Skarnes, W. C., Humanized Mice for Immune System Investigation: Progress, Promise and et al. (2020). A Novel Chemically Differentiated Mouse Embryonic Stem Cell- Challenges. Nat. Rev. Immunol. 12 (11), 786–798. doi: 10.1038/nri3311 Based Model to Study Liver Stages of Plasmodium Berghei. Stem Cell Rep. 14 Shultz, L. D., Ishikawa, F., and Greiner, D. L. (2007). Humanized Mice in (6), 1123–1134. doi: 10.1016/j.stemcr.2020.04.010 Translational Biomedical Research. Nat. Rev. Immunol. 7 (2), 118–130. Tweedell, R. E., Tao, D., Hamerly, T., Robinson, T. M., Larsen, S., Gronning, A. doi: 10.1038/nri2017 G. B., et al. (2019). The Selection of a Hepatocyte Cell Line Susceptible to Sieber, S., Wirth, L., Cavak, N., Koenigsmark, M., Marx, U., Lauster, R., et al. Plasmodium Falciparum Sporozoite Invasion That Is Associated With (2018). Bone Marrow-on-a-Chip: Long-Term Culture of Human Expression of Glypican-3. Front. Microbiol. 10. doi: 10.3389/ Haematopoietic Stem Cells in a Three-Dimensional Microfluidic fmicb.2019.00127 Environment. J. Tissue Eng. Regener. Med. 12 (2), 479–489. doi: 10.1002/ Vaughan, A. M., Mikolajczak, S. A., Wilson, E. M., Grompe, M., Kaushansky, A., term.2507 Camargo, N., et al. (2012). Complete Plasmodium Falciparum Liver-Stage Siler, U., Seiffert, M., Puch, S., Richards, A., Torok-Storb, B., Muller, C. A., et al. Development in Liver-Chimeric Mice. J. Clin. Invest. 122 (10), 3618–3628. (2000). Characterization and Functional Analysis of Laminin Isoforms in doi: 10.1172/JCI62684 Human Bone Marrow. Blood 96 (13), 4194–4203. doi: 10.1182/ Veiga, Z. S. T., Villela-Nogueira, C. A., Fernandes, F. F., Cavalcanti, M. G., blood.V96.13.4194 Figueiredo, F. A., Pereira, J. L., et al. (2017). Transient Elastography Evaluation Spencer, J. A., Ferraro, F., Roussakis, E., Klein, A., Wu, J., Runnels, J. M., et al. (2014). of Hepatic and Spleen Stiffness in Patients With Hepatosplenic Direct Measurement of Local Oxygen Concentration in the Bone Marrow of Live Schistosomiasis. Eur. J. Gastroenterol. Hepatol. 29 (6), 730–735. doi: 10.1097/ Animals. Nature 508 (7495), 269–273. doi: 10.1038/nature13034 MEG.0000000000000853 Suarez-Kurtz, G. (2021). Impact of CYP2D6 Genetic Variation on Radical Cure of Vendrely, K. M., Kumar, S., Li, X., and Vaughan, A. M. (2020). Humanized Mice Plasmodium Vivax Malaria. Clin. Pharmacol. Ther. 110 (3), 595–598. and the Rebirth of Malaria Genetic Crosses. Trends Parasitol. 36 (10), 850–863. doi: 10.1002/cpt.2313 doi: 10.1016/j.pt.2020.07.009 Sung, J. H., Kam, C., and Shuler, M. L. (2010). A Microfluidic Device for a Venugopal, K., Hentzschel, F., Valkiunas, G., and Marti, M. (2020). Plasmodium Pharmacokinetic-Pharmacodynamic (PK-PD) Model on a Chip. Lab. Chip 10 Asexual Growth and Sexual Development in the Haematopoietic Niche of the (4), 446–455. doi: 10.1039/b917763a Host. Nat. Rev. Microbiol. 18 (3), 177–189. doi: 10.1038/s41579-019-0306-2 Sung, J. H., and Shuler, M. L. (2009). A Micro Cell Culture Analog (microCCA) Voorberg-van der Wel, A., Kocken, C. H. M., and Zeeman, A. M. (2020a). With 3-D Hydrogel Culture of Multiple Cell Lines to Assess Metabolism- Modeling Relapsing Malaria: Emerging Technologies to Study Parasite-Host Dependent Cytotoxicity of Anti-Cancer Drugs. Lab. Chip 9 (10), 1385–1394. Interactions in the Liver. Front. Cell Infect. Microbiol. 10. doi: 10.3389/ doi: 10.1039/b901377f fcimb.2020.606033 Sylvester, K., Maher, S. P., Posfai, D., Tran, M. K., Crawford, M. C., Vantaux, A., Voorberg-van der Wel, A. M., Zeeman, A. M., Nieuwenhuis, I. G., van der Werff, et al. (2021). Characterization of the Tubovesicular Network in Plasmodium N. M., Klooster, E. J., Klop, O., et al. (2020b). A Dual Fluorescent Plasmodium Vivax Liver Stage Hypnozoites and Schizonts. Front. Cell Infect. Microbiol. 11. Cynomolgi Reporter Line Reveals In Vitro Malaria Hypnozoite Reactivation. doi: 10.3389/fcimb.2021.687019 Commun. Biol. 3, 7. doi: 10.1038/s42003-019-0737-3 Takenaka, K., Prasolava, T. K., Wang, J. C., Mortin-Toth, S. M., Khalouei, S., Gan, Walsh, N. C., Kenney, L. L., Jangalwe, S., Aryee, K. E., Greiner, D. L., Brehm, M. A., O. I., et al. (2007). Polymorphism in Sirpa Modulates Engraftment of Human et al. (2017). Humanized Mouse Models of Clinical Disease. Annu. Rev. Pathol. Hematopoietic Stem Cells. Nat. Immunol. 8 (12), 1313–1323. doi: 10.1038/ 12, 187–215. doi: 10.1146/annurev-pathol-052016-100332 ni1527 Wang, H., Madariaga, M. L., Wang, S., Van Rooijen, N., Oldenborg, P. A., and Tavares, J., Formaglio, P., Thiberge, S., Mordelet, E., Van Rooijen, N., Medvinsky, Yang, Y. G. (2007). Lack of CD47 on Nonhematopoietic Cells Induces Split A., et al. (2013). Role of Host Cell Traversal by the Malaria Sporozoite During Macrophage Tolerance to CD47null Cells. Proc. Natl. Acad. Sci. U.S.A. 104 Liver Infection. J. Exp. Med. 210 (5), 905–915. doi: 10.1084/jem.20121130 (34), 13744–13749. doi: 10.1073/pnas.0702881104 Taylor, A. R., Watson, J. A., Chu, C. S., Puaprasert, K., Duanguppama, J., Day, White, N. J. (2011). Determinants of Relapse Periodicity in Plasmodium Vivax N. P. J., et al. (2019). Resolving the Cause of Recurrent Plasmodium Vivax Malaria. Malar. J. 10, 297. doi: 10.1186/1475-2875-10-297 Malaria Probabilistically. Nat. Commun. 10 (1), 5595. doi: 10.1038/s41467- Whitesides, G. M. (2006). The Origins and the Future of Microfluidics. Nature 442 019-13412-x (7101), 368–373. doi: 10.1038/nature05058 Terrell, J. A., Jones, C. G., Kabandana, G. K. M., and Chen, C. (2020). From Cells- World Health Organization (2020). World Malaria Report: 20 Years of Global on-a-Chip to Organs-on-a-Chip: Scaffolding Materials for 3D Cell Culture in Progress and Challenges. World Health Organization. Available at: https:// Microfluidics. J. Mater. Chem. B. 8 (31), 6667–6685. doi: 10.1039/d0tb00718h apps.who.int/iris/handle/10665/337660. Thon, J. N., Mazutis, L., Wu, S., Sylman, J. L., Ehrlicher, A., Machlus, K. R., et al. Wuchter, P., Saffrich, R., Giselbrecht, S., Nies, C., Lorig, H., Kolb, S., et al. (2016). Microcavity (2014). Platelet Bioreactor-on-a-Chip. Blood 124 (12), 1857–1867. Arrays as an In Vitro Model System of the Bone Marrow Niche for Hematopoietic Stem doi: 10.1182/blood-2014-05-574913 Cells. Cell Tissue Res. 364 (3), 573–584. doi: 10.1007/s00441-015-2348-8 Toda, H., Diaz-Varela, M., Segui-Barber, J., Roobsoong, W., Baro, B., Garcia- Wu, S., Zheng, Q., Xing, X., Dong, Y., Wang, Y., You, Y., et al. (2018). Matrix Silva, S., et al. (2020). Plasma-Derived Extracellular Vesicles From Stiffness-Upregulated LOXL2 Promotes Fibronectin Production, MMP9 and Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 17 July 2022 | Volume 12 | Article 920204 Aparici-Herraiz et al. Disruptive Technologies in P. vivax CXCL12 Expression and BMDCs Recruitment to Assist Pre-Metastatic Niche Angiogenesis. Adv. Healthc. Mater. 5 (9), 1014–1024. doi: 10.1002/ Formation. J. Exp. Clin. Cancer Res. 37 (1), 99. doi: 10.1186/s13046-018-0761-z adhm.201501007 Xiang, C., Du, Y., Meng, G., Soon Yi, L., Sun, S., Song, N., et al. (2019). Long-Term Zimmerman, P. A. (2017). Infection in Duffy-Negative People in Africa. Am. J. Functional Maintenance of Primary Human Hepatocytes In Vitro. Science 364 Trop. Med. Hyg. 97 (3), 636–638. doi: 10.4269/ajtmh.17-0461 (6438), 399–402. doi: 10.1126/science.aau7307 Yager, P., Edwards, T., Fu, E., Helton, K., Nelson, K., Tam, M. R., et al. (2006). Microfluidic Diagnostic Technologies for Global Public Health. Nature 442 Conflict of Interest: The authors declare that the research was conducted in the (7101), 412–418. doi: 10.1038/nature05064 absence of any commercial or financial relationships that could be construed as a Yamauchi, T., Takenaka, K., Urata, S., Shima, T., Kikushige, Y., Tokuyama, T., potential conflict of interest. et al. (2013). Polymorphic Sirpa is the Genetic Determinant for NOD-Based Mouse Lines to Achieve Efficient Human Cell Engraftment. Blood 121 (8), Publisher’s Note: All claims expressed in this article are solely those of the authors 1316–1325. doi: 10.1182/blood-2012-06-440354 and do not necessarily represent those of their affiliated organizations, or those of Yurino, A., Takenaka, K., Yamauchi, T., Nunomura, T., Uehara, Y., Jinnouchi, F., the publisher, the editors and the reviewers. Any product that may be evaluated in et al. (2016). Enhanced Reconstitution of Human Erythropoiesis and this article, or claim that may be made by its manufacturer, is not guaranteed or Thrombopoiesis in an Immunodeficient Mouse Model With Kit(Wv) endorsed by the publisher. Mutations. Stem Cell Rep. 7 (3), 425–438. doi: 10.1016/j.stemcr.2016.07.002 Zhang, W., Gu, Y., Sun, Q., Siegel, D. S., Tolias, P., Yang, Z., et al. (2015). Ex Vivo Copyright © 2022 Aparici Herraiz, Caires, Castillo-Fernań dez, Sima, Meń dez-Mora, Maintenance of Primary Human Multiple Myeloma Cells Through the Risueño, Sattabongkot, Roobsoong, Hernan ́ dez-Machado, Fernandez-Becerra, Optimization of the Osteoblastic Niche. PloS One 10 (5), e0125995. Barrias and del Portillo. This is an open-access article distributed under the doi: 10.1371/journal.pone.0125995 terms of the Creative Commons Attribution License (CC BY). The use, Zhang, W., Lee, W. Y., Siegel, D. S., Tolias, P., and Zilberberg, J. (2014). Patient- distribution or reproduction in other forums is permitted, provided the Specific 3D Microfluidic Tissue Model for Multiple Myeloma. Tissue Eng. Part original author(s) and the copyright owner(s) are credited and that the C. Methods 20 (8), 663–670. doi: 10.1089/ten.TEC.2013.0490 original publication in this journal is cited, in accordance with accepted Zheng, Y., Sun, Y., Yu, X., Shao, Y., Zhang, P., Dai, G., et al. (2016). Angiogenesis academic practice. No use, distribution or reproduction is permitted which in Liquid Tumors: An In Vitro Assay for Leukemic-Cell-Induced Bone Marrow does not comply with these terms. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 18 July 2022 | Volume 12 | Article 920204