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Experimental models of liver-stage malaria: Progress, gaps, and challenges

Abstract

For decades, achieving malaria eradication has proven difficult. Plasmodium parasites first multiply asymptomatically in the liver before causing cyclic erythrocytic infections and clinical symptoms. Unlike other species, Plasmodium vivax can remain dormant for months or years as hypnozoites within hepatocytes. When these latent parasites reactivate, they cause clinical episodes in most vivax malaria cases. Thus, targeting the liver stage is essential to prevent disease progression and relapse. Current therapies are effective but pose risks for individuals with glucose-6-phosphate dehydrogenase deficiency. Consequently, developing and evaluating new antimalarial agents requires in vitro models that accurately represent intrahepatic parasite growth. Existing in vitro models have improved over time, but still face limitations. Recent advancements in cell culture have introduced organoids, which are three-dimensional, self-organizing cellular structures that recreate the microarchitecture and functions of native tissues. This review highlights the strengths and weaknesses of current experimental platforms and identifies critical gaps. We discuss how organoid technology can address these shortcomings, guiding improvements in liver-stage malaria models. Such advances will facilitate the development of preventive strategies and radical cures.

Author summary

Ethical concerns regarding animal use and advances in cell culture have driven major improvements in in vitro disease models. Although often neglected, the liver stage is crucial for preventing and eradicating malaria. Modeling Plasmodium development in hepatocytes is more complex than modeling blood-stage parasites because preserving hepatocyte functions in cell culture is challenging. This review examines the progress and ongoing challenges associated with using three-dimensional cell culture systems to study liver-stage malaria.

Citation: Nitaramorn N, Kulkeaw K, Imwong M (2025) Experimental models of liver-stage malaria: Progress, gaps, and challenges. PLoS Pathog 21(12): e1013796. https://doi.org/10.1371/journal.ppat.1013796

Editor: Chetan E. Chitnis, Institut Pasteur, FRANCE

Published: December 18, 2025

Copyright: © 2025 Nitaramorn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by “Scholarships for PhD Students” from Mahidol University (PHD/0001/2565 to N. N. and K. K.) and the “Strategic Research Fund: Fiscal Year 2023,” Mahidol University (MU-SRF-PF-04B/66 to M. I.). The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation. N. N. received salary from the Scholarships for PhD Students.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Since Laveran’s discovery of blood parasites in 1880 [1,2], malaria has threatened millions of lives worldwide [2]. Malaria eradication is hindered by drug resistance and persistent latent infections in the liver, causing multiple relapses. Current therapies prevent clinical disease and relapse but raise safety concerns and show limited coherence. Only two drugs, primaquine and tafenoquine, have been available for over two decades, serving as prophylaxis and radical cures. Both can induce red blood cell lysis in individuals with glucose-6-phosphate dehydrogenase deficiency, which limits large-scale use. Consequently, new drug candidates and improved in vitro models capable of representing both active and dormant parasites in hepatocytes are essential. This review examines the usefulness and gaps in existing experimental platforms. Emerging three-dimensional (3D) cell culture systems, including organoids, better recapitulate native tissue organization and function. We summarize the progress in 3D liver-stage malaria modeling and discuss potential enhancements to accelerate drug discovery and support eradication efforts.

2. Challenges in malaria eradication

Plasmodium species are obligate intracellular protozoans that infect diverse vertebrates, including lizards, birds, and mammals. Five species infect humans: P. falciparum, P. vivax, P. ovale, P. malariae, and zoonotic P. knowlesi, and have complex life cycles (Fig 1). Nearly half of all countries remain malaria endemic, and about one-quarter of the global population is at risk, with over 200 million infections each year [3]. In 2015, the World Health Organization (WHO) deployed the Global Technical Strategy for Malaria. The strategy aimed to reduce malaria incidence and mortality by 90% between 2016 and 2030 [3,4]. While WHO-led campaigns achieved substantial reductions in global incidence between 2000 and 2019, progress has stalled. Annual cases plateaued at approximately 252 million from 2020 to 2023, highlighting the formidable challenges in achieving eradication [5].

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Fig 1. Cyclic development of Plasmodium species in three distinct niches of hosts.

In mammalian hosts, asexual development of all Plasmodium species begins in the liver. (A) Exo-erythrocytic (liver-stage) development: Following a blood meal, sporozoites are released from the salivary glands of a female Anopheles mosquito into the skin. They travel through blood vessels and invade hepatocytes. Inside the hepatocytes, sporozoites undergo multiple rounds of cell division—schizogony—to produce schizonts. In the case of P. vivax and P. ovale, some sporozoites enter a dormant, non-proliferative stage called a hypnozoite. Once schizonts fully mature, merosomes egress from the infected hepatocytes into the bloodstream. (B) Erythrocyte-stage development: Merozoites invade red blood cells and undergo schizogony to generate additional merozoites. Some ring trophozoite stages differentiate into male and female gametocytes, which are transmitted to a female Anopheles mosquito during a subsequent blood meal. (C) Sporogony-stage development: In the mosquito midgut, ingested gametocytes fertilize to form zygotes. These develop into ookinetes and then oocysts, which produce numerous sporozoites. The sporozoites migrate to the mosquito’s salivary glands and await transmission to a human host at the next blood meal. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/qbcziwc.

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Among the five major Plasmodium species infecting humans, P. falciparum malaria exhibits the highest prevalence, followed by P. vivax. P. falciparum occurs predominantly in Africa, whereas P. vivax is widespread across continents. Infections with P. falciparum can cause severe clinical manifestations, posing a life-threatening risk, especially to young children with limited immunity. Despite substantial efforts toward developing a protective vaccine targeting infective sporozoites, controlling P. vivax remains more challenging due to its latent liver-stage forms. P. vivax hypnozoites, which are non-proliferative, can persist in hepatocytes for months or years and may unexpectedly reinitiate cell division. Their reactivation leads to multiple relapses in vivax malaria [6–8]. These dormant stages remain undetectable with current laboratory tools. Most vivax malaria episodes result from hypnozoite reactivation. Hypnozoite reservoirs often evade immunity, remain asymptomatic, and maintain low parasitemia. Notably, P. vivax sexual stages can be transmitted from humans to Anopheles mosquitoes before symptoms arise. Thus, repeated relapses sustain malaria transmission [9] and exacerbate morbidity, particularly in infants, children, and pregnant women [10].

The updated Global Technical Strategy for Malaria (2016–2030) placed P. vivax hypnozoite reservoirs as a strategic target to achieve malaria-free status [10,11]. Mass administration of anti-hypnozoite drugs underlies these efforts. However, only two drugs-primaquine and tafenoquine, both 8-aminoquinolines-effectively eliminate P. vivax hypnozoites. These drugs can cause hemolytic anemia in individuals deficient in glucose-6-phosphate dehydrogenase (G6PD). Thus, G6PD deficiency testing is mandatory before administration, consuming additional resources and time. Moreover, these drugs are contraindicated for infants, pregnant women, and breastfeeding mothers, limiting large-scale implementation [11,12]. The urgent need to discover safer anti-relapse therapies targeting P. vivax hypnozoites is clear. Advancing our understanding of liver-stage malaria, including its molecular and cellular mechanisms, will facilitate the development of improved in vitro disease models.

3. Utility and drawbacks of the current liver-stage malaria models

In vivo and in vitro disease models have clarified many molecular and cellular mechanisms of sporozoite invasion and schizogony. Cancerous or immortalized cell lines and primary human hepatocytes often serve as in vitro models. Humanized rodent models provide an in vivo—relevant setting but still lack immune cells and human vasculature. Recognizing similarities and differences among these models is crucial for choosing the most appropriate platform for a given study.

4.1 Hepatic and non-hepatic cell lines

Cancerous cell lines, such as hepatocellular carcinoma lines, are commonly used due to simpler laboratory protocols (Fig 2). HepG2-A16, a HepG2 subclone, supports initial P. vivax sporozoite infection and schizogony [13,14]. In 1985, Hollingdale et al. first observed two intracellular forms of P. vivax in HepG2-A16 cells: small and large intrahepatic forms [13]. The small forms persisted, while the large schizonts eventually disappeared. In 2010, Chattopadhyay et al. used HepG2-A16 cells to test primaquine efficacy. The hepatic infection rates were 1.8% and 0.9% on days 3 and 9 post-infection, respectively. After three days of primaquine exposure, hepatic schizonts reduced by up to 40%. However, neither study confirmed complete intrahepatic development because detection relied on erythrocyte-infectious merozoites. Detecting P. vivax merozoites is more complex than detecting P. falciparum due to the need for human reticulocytes. The small P. vivax intrahepatic form is presumed to be a hypnozoite, but its reactivation has not been tested in HepG2-A16 cells. A major limitation of HepG2-A16 cells is their tendency to detach from cell culture surfaces, likely due to uncontrolled cancer cell growth. This instability complicates long-term culture required to study hypnozoite formation and reactivation. Moreover, HepG2-A16 cells have lower cytochrome enzyme activity than primary human hepatocytes [15], preventing primaquine bioactivation and possibly explaining the low observed efficacy [15]. In addition to the HepG2-A16 cell line, another hepatoma-derived cell line, HHS-102, exhibits characteristics more similar to normal human hepatocytes. Unlike HepG2 cells, HHS-102 cells can form bile canaliculi and halt their growth upon reaching confluence, thereby preventing overgrowth and detachment [16,17]. When these cells were tested for the intrahepatic development of P. falciparum, ring-stage parasites were observed in human erythrocytes, indicating that complete schizogony had occurred.

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Fig 2. Two-dimensional (2D) cell culture systems for modeling liver-stage malaria.

To study liver-stage development of Plasmodium spp., several human hepatic cell types are used: primary human hepatocytes (PHHs), hepatocellular carcinoma lines (e.g., HepG2-A16 and HHS-102), immortalized hepatic cell lines (e.g., HC-04), and induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells. Hepatocellular carcinoma and immortalized cells typically grow as monolayers on rigid plastic surfaces. In contrast, PHHs must be cultured on collagen type IV-coated surfaces or co-cultured with murine fibroblasts. Sporozoites are introduced into the culture well, often followed by centrifugation or reduced medium volume to enhance sporozoite-cell contact. Successful liver-stage development is commonly indicated by segmented schizonts, which serve as a proxy for parasite development. Detection of free merozoites in the culture medium is less frequent in most available liver-stage assays. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/3mgz0tq.

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The human Huh7 cell line originates from hepatoma cells. Although P. berghei naturally infects rodents, Huh7 cells also permit P. berghei infection. Using Huh7 cells has advanced our understanding of sporozoite invasion and productive infection. A study of P. berghei in Huh7 cells highlights membrane ruffles as crucial for sporozoite invasion [18]. Live cell imaging shows that membrane ruffles and filopodia extensions increase during invasion. Rho GTPase, a regulator of plasma membrane dynamics, is identified as a required host factor. Additionally, P. berghei sporozoite traversal triggers membrane ruffling. Treating Huh7 cells with a membrane ruffling inhibitor decreases the percentage of intrahepatic schizonts, suggesting reduced productive invasion. For complete schizogony, multidrug resistance-associated protein is essential for forming mature P. berghei schizonts in Huh7 cells [19]. P. berghei also hijacks host Golgi in Huh7 cells to complete intrahepatic development, confirmed in primary human hepatocyte cultures [20]. Furthermore, the parasite uses a nutrient transporter to establish the parasitophorous vacuole and maintain parasite load in Huh7 cells [21]. Huh7 cells also serve as a drug testing platform. For example, an antiretroviral drug used for HIV therapy impairs P. berghei development in Huh7 cells [22]. Genomic analysis indicates that the parasite relies on protein trafficking pathways. In the P. berghei-infected Huh7 model, knocking down genes involved in protein trafficking reduces schizont size but not infection rate [23].

Because HepG2 cells lack CD81, a key circumsporozoite protein (CSP)-binding receptor for P. falciparum sporozoite invasion [24], primary human hepatocytes were immortalized to generate the HC-04 cell line. HC-04 cells express albumin, transferrin, and other liver proteins under standard conditions [25]. They support complete schizogony of P. falciparum and P. vivax, as indicated by the presence of liver schizonts. Additionally, the HC-04.J7 cell line, a subclone of HC-04, shows higher infection efficiency. By limiting dilution, HC-04 subclones were obtained and screened for P. falciparum infectivity. The HC-04.J7 line had an infection rate threefold higher than HC-04. Comparing the transcriptome and proteome of HepG2, HC-04, and HC-04.J7 cells identified glypican-3 as a potential receptor for P. falciparum sporozoites [26]. Glypican-3 occurs only in HC-04 and HC-04.J7 cells, not in HepG2, suggesting HepG2’s lower susceptibility to P. falciparum. The HC-04.J7 line was later used as a sporozoite infectivity assay for anti-CSP monoclonal antibodies [18]. However, both HC-04 and HC-04.J7 proliferate rapidly, rendering them unsuitable for long-term studies, including hypnozoite formation and reactivation, due to eventual overgrowth and detachment.

To overcome the rapid proliferation issue, a mesenchymal-cell-derived immortalized hepatocyte-like cell line (imHC) was established, exhibiting a lower proliferation rate than HC-04 cells. The sporozoite infection rate in imHCs is comparable to that in HC-04 cells. The detection of parasitized erythrocytes in imHC cultures confirmed the production of merozoites, indicating that P. vivax undergoes complete hepatic schizogony in this system [27]. Despite the advantages of these cancer-derived or immortalized hepatic cell lines, they have inherent limitations. The uncontrolled growth of cancer cells often leads to detachment from the culture surface, complicating long-term maintenance and thus hindering studies of hypnozoite reactivation. Furthermore, the hepatic functions of cancer cells are generally reduced compared to mature hepatocytes. HepG2 cells, for instance, show lower transcript levels of most cytochrome enzymes than primary human hepatocytes [28], and lipid accumulation in hepatoma lines is lower than in primary human hepatocytes [29].

For non-hepatic cell lines, HeLa cells are epithelial carcinoma cells derived from human cervical cancer. They have been widely used to investigate fundamental cell biology. Despite not originating from hepatocytes, human HeLa cells are susceptible to P. berghei infection and contribute to liver-stage malaria research. Initially, HeLa cells helped reveal SPECT as a parasite factor for P. berghei cell traversal [30,31]. They also demonstrated how P. berghei exploits autophagy to survive [32]. HeLa cell cultures are not limited to studying host and parasite factors alone. Using transgenic P. berghei with fluorescent proteins, researchers identified stage-specific parasite transcripts from early to late schizonts in HeLa cells [33]. The resulting transcriptome database is accessible at the European Nucleotide Archive (http://www.ebi.ac.uk/ena/) under accession numbers PRJEB23770 and ERP105548. Finally, P. berghei parasites utilize host Golgi for complete intrahepatic schizogony in HeLa cells, echoing findings in Huh7 and primary human hepatocytes [20].

4.2 Primary human hepatocytes

Primary human hepatocytes (PHHs) can be isolated from adult donor liver biopsies or whole donated livers. Under optimal culture conditions, PHHs retain hepatic functions for approximately 1–2 weeks. Micropatterned co-cultures with fibroblasts extend these functions beyond 3 weeks, enabling studies of HCV, HBV, and Plasmodium (including P. falciparum and P. vivax) [34–36] (Fig 2). PHHs support the intrahepatic development of P. vivax, with schizonts visible around 5–6 days post-infection. Adding human reticulocytes at days 12–14 post-infection enables the detection of erythrocyte-infective merozoites, confirming complete schizogony [37]. Unlike cancerous or immortalized cell lines, PHH culture is more complex and expensive, often requiring growth factors, hormones, and a collagen layer for cell adherence. PHHs also tend to dedifferentiate over time, losing their hepatic functions. Coculturing PHHs with fibroblasts can mitigate dedifferentiation [35,38]. While long-term cultivation can be achieved, the infection rate varies significantly depending on the donor. Thus, prescreening frozen PHH batches to ensure adequate infection rates is often necessary before schizogony studies or drug testing [39]. In addition, a limited supply of PHHs and challenges in scaling them up pose constraints for large-scale drug screening. Using smaller culture wells can reduce the number of PHHs required and facilitate high-throughput drug testing [40]. Microscale cultures of PHHs combined with high-content imaging technologies have been shown to overcome these limitations, enabling efficient large-scale screening of antimalarial compounds [35,38,40].

4.3 Primary porcine hepatocytes

Parasite–host selectivity is commonly acknowledged. However, rodent P. yoelii sporozoites can infect and develop in both human and mouse hepatocytes, whereas P. falciparum sporozoites cannot invade mouse hepatocytes [24]. Intriguingly, P. falciparum sporozoites can infect porcine hepatocytes but exhibit atypical parasitophorous vacuoles, causing a mid-stage developmental arrest [41]. Thus, porcine hepatocytes are permissive to P. falciparum sporozoites but can’t support parasite development.

4.4 Induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-derived HLCs)

Because PHHs are finite and non-expandable, induced pluripotent stem cells (iPSCs) provide a promising alternative source of functional hepatocytes (Fig 2). iPSCs, derived from various somatic cell types, can proliferate indefinitely under optimal conditions, maintain genetic stability, and differentiate into multiple cell lineages. This capability allows the large-scale production of hepatocytes suitable for high-throughput testing. Si-Tayeb et al. demonstrated that mouse embryonic fibroblast-derived iPSCs could differentiate through distinct stages—endoderm, hepatic endoderm, hepatoblasts, and then hepatocyte-like cells—mimicking embryonic tissue formation [42]. These iPSC-derived hepatocyte-like cells (HLCs) express hepatocyte markers such as HNF4a, AFP, and ALB, and perform functions similar to PHHs, including albumin secretion, glycogen and lipid accumulation, and metabolic activities.

A study utilizing iPSC-derived HLCs showed that these cells, which express CD81 and SR-B1 receptors necessary for parasitophorous vacuole formation, support the intrahepatic development of P. falciparum and P. vivax at infection rates of 0.003%–0.02% [43]. This platform is suitable for testing atovaquone, a drug that is active without metabolic conversion. By contrast, primaquine requires cytochrome P450 2D6 metabolism, and the reduced cytochrome P450 activity in iPSC-derived HLCs impedes proper primaquine testing. Studies indicate that iPSC-derived HLCs resemble fetal hepatocytes, with significantly lower levels of urea production, albumin secretion, and cytochrome P450 activity compared to PHHs [44,45]. These cells also express lower levels of hepatic drug-metabolizing enzymes than PHHs [46], reflecting an immature, fetal-like state. To address this, strategies involving small molecules or specialized culture plates are used to enhance cytochrome P450 activity and thus enable primaquine sensitivity testing. However, although a non-replicative hypnozoite form was observed in iPSC-derived HLCs, no erythrocyte-infected parasites were detected [43]. Improving hepatocyte maturity remains essential before iPSCs can serve as a reliable PHH substitute [46,47].

4.5 Humanized mouse models

Mice and rats are natural hosts of P. yoelii and P. berghei, but neither form hypnozoites [48]. To model human liver-stage vivax malaria, researchers used commercially available human liver-chimeric mice [49,50]. These humanized mice lack functional mouse hepatocytes due to the deletion of the fumarylacetoacetate hydrolase (Fah) gene. Fah-knockout mice suffer from hepatocellular damage caused by the accumulation of toxic intermediates. Transplanting human hepatocytes into these mice replaces the damaged mouse hepatocytes, thereby creating a platform to study human liver-stage malaria in a physiologically relevant setting. To prevent immune rejection of xenografts, a severe combined immunodeficient (SCID) mouse line that lacks T and B lymphocytes (Rag2−/−IL2rg−/−) is combined with Fah−/− mice, creating FRG mice [51].

P. vivax sporozoites are intravenously injected into human hepatocyte-chimeric FRG mice. Two intrahepatic forms appear: a large form containing multiple merozoites that actively replicate, and a small, non-replicating form persisting up to 21 days post-infection, presumed to be a hypnozoite. After intravenous injections of human reticulocytes, blood-stage P. vivax parasites appear on day 9 post-infection. By day 14, hypnozoite-like forms remain visible, whereas the fully developed schizonts disappear. By day 21, most intrahepatic parasites persist as putative hypnozoites, with some increasing in size, suggesting possible reactivation. Recent studies show hypnozoite reactivation via secondary schizont formation at later time points [52,53]. Using FRG mice, atovaquone inhibits intrahepatic schizogony, while primaquine acts as prophylaxis, preventing relapse. Drug efficacy is evaluated via immunofluorescence and P. vivax 18S rRNA-based PCR [49]. By backcrossing the FRG mouse onto a non-obese diabetic (NOD) strain, Fah−/−Rag2−/−IL2rg−/−NOD (FGRN) mice are generated. These mice have defective macrophages that cannot engulf human grafts [54,55], allowing the successful repopulation of human erythrocytes. In these FGRN mice, all blood-stage forms of P. vivax, including gametocytes, have been observed. This system enabled testing of monoclonal antibodies targeting P. vivax Duffy Binding Protein to inhibit human reticulocyte invasion [56–60].

4.6 Three-dimensional (3D) culture models

Advances in cell culture technology have led to the improvement of in vitro models that better replicate human tissue pathology. Conventional two-dimensional (2D) cultures differ significantly from actual tissues. Many three-dimensional (3D) culture platforms, involving single or multiple cell types interacting with matrix proteins, now exist. Cells can form spheroids—aggregates that resemble some aspects of native tissues—or, using stem cells, can self-organize into organ-like structures known as organoids. Mellin and Boddey previously discussed using organoids for malaria research, but relatively few studies have been published [61]. Here, we update current applications and potential improvements of spheroids and organoids for modeling liver-stage malaria.

4.6.1 Hepatic spheroid models.

Hepatic spheroids can form by aggregation or proliferation of cancerous or immortalized hepatic cell lines. They may develop with or without scaffolds. A semi-solid gel scaffold, containing extracellular matrix proteins, supports cell adherence and nutrient diffusion, while scaffold-free methods rely on low-attachment surfaces to encourage cell aggregation. Both approaches produce spheroids that often develop hypoxic cores due to limited oxygen diffusion, creating zones of dead or unhealthy cells that mimic tumor-like conditions. Although spheroid use in infectious disease research is limited, these models are utilized for studying liver-stage malaria.

Chua and colleagues generated spheroids from primary simian and human hepatocytes using a disc-shaped, porous Cellusponge scaffold [62]. Compared to 2D collagen-coated cultures, simian hepatic spheroids produced higher concentrations of urea over 22 days, though urea levels gradually declined, indicating dedifferentiation over time. Immunofluorescence confirmed hepatocyte polarization, showing basolateral protein CD147 and apical protein MRP2 in human hepatic spheroids. A fluorescent substrate for esterase activity assessed membrane integrity and cell viability. Transcript levels of CYP1A2 and CYP3A4 in spheroids were similar to those in 2D human hepatocyte cultures.

These human hepatic spheroids expressed the sporozoite receptor CD81, a proposed host factor for hepatocyte infection. To improve infection rates, P. vivax or P. cynomolgi sporozoites were incubated with hepatocytes before forming 3D spheroids. In-suspension incubation with simian hepatocytes increased infection rates approximately threefold compared to direct inoculation into pre-formed 3D spheroids, suggesting that the tight spheroid structure may hinder sporozoite entry. In-suspension infection allowed P. cynomolgi to complete development, producing blood-stage parasites in simian reticulocytes. Applying this strategy to human hepatic spheroids increased P. vivax sporozoite infection rates compared to 2D cultures, but the release of infective intrahepatic merozoites was not observed. The compact spheroid structure may entrap merozoites inside. Dissociating spheroids into single cells or inducing cell lysis might release merozoites, enabling subsequent erythrocyte infection. However, this study lacks microscopic data to confirm the proper development of parasites. Whether the 3D Cellusponge-based spheroids support hypnozoite formation and reactivation remains unexplored.

4.6.2 Liver organoid.

Organoids arise from adult stem cells in a given tissue. Adult stem cells can divide and differentiate into mature, functioning cells to replace aged or damaged cells. Generally multipotent, these cells give rise to various specialized cell types. Under optimal biological stimuli, adult stem cells can self-organize and function in a manner similar to their tissue of origin. Organoids, three-dimensional structures formed from such stem cells, have emerged as valuable in vitro models to study tissue development and disease mechanisms [63,64]. Beyond multipotent adult stem cells, pluripotent stem cells can also be used to generate organoids (Fig 3A). Since organoid formation from both pluripotent and adult stem cells requires an understanding of embryogenesis and adult tissue maintenance, this section reviews current methods and assays for utilizing liver organoid platforms in the context of liver-stage malaria research, as well as their potential drawbacks.

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Fig 3. Liver development and differentiation of pluripotent stem cells (PSCs) into hepatocytes.

Insights into liver development largely arise from mouse embryonic studies. Applying these principles to human PSCs, such as embryonic stem cells or iPSCs, reveals that external stimuli (e.g., growth factors, hormones) drive PSCs to differentiate into hepatocytes. (A) External stimuli in hepatocyte generation from PSCs: PSCs are first induced to form definitive endoderm (DE) using signals from Nodal, bone morphogenic protein (BMP), fibroblast growth factor-a (FGFa), and Wnt pathways. Once committed, DE differentiates into hepatic endoderm (HE), which can give rise to pancreatic cells (P), cholangiocytes (Ch), and hepatocytes (H). Distinct sets of stimuli direct HE to differentiate into either hepatocytes or cholangiocytes. (B) Mouse liver budding: In mouse embryos, the foregut endoderm, derived from DE, forms the liver, pancreas, and gall bladder. The hepatic endoderm (HE) migrates into the septum transversum mesenchyme (STM), where it differentiates into hepatoblasts and then hepatocytes. Hepatic and endothelial cell formation in STM creates a bud-like structure, resembling a developing liver bud. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/nrtq41n.

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4.6.2.1 Hepatic organoids from adult and fetal liver: Although the presence of adult stem cells in the human liver is debated, isolating cell populations with stem cell-like properties has enabled the generation of human liver organoids. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), a Wnt pathway target gene, is used as a marker of adult stem cells in the intestine, colon, and stomach. In mice, Lgr5 is normally undetectable in the liver but appears in bile duct epithelial cells following liver injury. These damage-induced, Lgr5-positive cells can form organoids in the presence of a Wnt activator [65]. Huch et al. isolated EpCAM-positive cells from human bile ducts and cultured them in a three-dimensional (3D) gel-based system, enabling the formation of hepatic organoids [65,66]. These differentiated liver organoids expressed hepatocyte markers (HNF4a, albumin, and cytochrome proteins), secreted albumin and bile acids, and exhibited CYP3A3/4/5 activities as well as phase I and II detoxifying functions. In addition to isolating stem cell-like fractions, hepatic organoids can also be established using hepatocytes directly. Organoids derived from human fetal liver tissue (11–20 weeks of gestation) and from cryopreserved primary human hepatocytes (PHHs) can be generated without additional cell isolation steps. Compared to two-dimensional (2D) PHH cultures and fetal liver-derived organoids, PHH-derived organoids show higher CYP3A4 activity. Albumin secretion levels remain similar among PHH-derived organoids, fetal-liver-derived organoids, and 2D PHH cultures [67].

Two recent studies have demonstrated the utility of fetal and adult liver organoids for modeling the intrahepatic development of P. falciparum [68,69]. When fetal liver organoids were adapted to a 2D culture platform, they supported P. falciparum schizogony, allowing detection of small and large intrahepatic forms with parasite-specific antibodies. However, erythrocyte infection by released merozoites remained difficult to observe. Single-cell transcriptomic analysis of these organoids revealed increased lipid and cholesterol synthesis, along with upregulation of Scavenger Receptor B1 (SR-B1), consistent with enhanced HDL-mediated lipid uptake.

Another study generated liver organoids from adult donor intrahepatic ductal cells (cholangiocytes) [65]. By embedding these dissociated cells in basement membrane extract, 3D organoids were formed. To introduce P. falciparum sporozoites, the organoid was dissociated into small fragments and coincubated with sporozoites for three hours before re-embedding in the 3D matrix. After six days, infection and parasite maturation were assessed using antibodies against P. falciparum HSP70 and merozoite surface protein 1. Infection rates varied among donors, with a maximum of approximately 0.8%. Maturation of intrahepatic forms was indicated by an increase in size, although infective merozoites capable of invading human erythrocytes were not detected. Despite this limitation, this platform offers a valuable in vitro assay for identifying novel drugs targeting the liver-stage development of P. falciparum [69].

4.6.2.2 Hepatic organoids from iPSCs: Generating hepatic organoids from pluripotent stem cells (PSCs) is more complex than using adult stem cells, as it requires multiple lineage-specific differentiation steps that mirror embryonic hepatocyte development. After fertilization, the three germ layers—endoderm, mesoderm, and ectoderm—form. The endoderm gives rise to the gut tube, from which the liver develops. The hepatic endoderm migrates into the septum transversum mesenchyme (STM), ultimately differentiating into hepatoblasts and then hepatocytes. This process of hepatic endoderm migration and differentiation is called liver budding (Fig 3B). Maturation of the liver bud into a functional liver organ requires endothelial cells to establish proper vasculature.

iPSC-derived endodermal cells expressing EpCAM have been shown to form hepatic organoids resembling the fetal liver stage [70]. To achieve mature hepatocyte functions, an additional 10–14 days of in vitro culture are typically required. Our own experience confirms that iPSC-derived endoderm can also form hepatic organoids [47]. These organoids stably express the Plasmodium sporozoite receptor CD81 for up to 60 days, along with sustained albumin secretion and CYP4A3 expression. Moreover, intermediate endodermal cells obtained during the hepatic differentiation of human iPSCs in a 2D system can form spherical structures that, once embedded in Matrigel, gain mature hepatocyte functions, including CYP3A4 activity, urea production, and albumin secretion [71]. Whether these organoids also possess CYP3A4 and CYP2C19 activity necessary for primaquine metabolism remains to be investigated.

In 2013, researchers advanced the development of liver bud organoids by combining iPSC-derived hepatic endoderm with human umbilical vein endothelial cells and human mesenchymal stem cells [72]. Plating this three-cell mixture on Matrigel allowed self-organization into 3D cell clusters (Fig 4). Immunofluorescence staining showed clusters of AFP-expressing hepatoblasts surrounded by CD31-positive endothelial cells. Transplanting these liver bud organoids into severe combined immunodeficient (SCID) mice elevated human serum albumin levels in the host’s blood and improved survival in mice with induced hepatic injury. Thus, iPSC-derived liver bud organoids initially resemble fetal-stage liver in vitro but can acquire greater maturity upon in vivo engraftment. Recently, the protocol for hepatic organoid generation from human PSCs was further refined by optimizing the doses and exposure times of key factors [73].

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Fig 4. Methods for generation of liver organoids.

Various methods produce liver organoids, depending on the cell source and 3D structure formation strategy. Currently, liver organoids can be generated from primary human hepatocytes (PHHs) or PSCs. PSC-derived hepatic endoderm (HE) or hepatocytes (H) can form 3D organoids through stages involving definitive endoderm (DE), HE, hepatoblast (Hb), and eventually H. PHHs may originate from fetal or adult livers. To create a 3D structure, cells are embedded in a gel-like scaffold, such as Matrigel (an extracellular matrix-based medium), then covered with liquid medium. Following the principles of mouse liver budding, combining HE, endothelial cells (EC), and STM on top of solidified Matrigel can produce a liver bud-like structure. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/kkfupxg.

https://doi.org/10.1371/journal.ppat.1013796.g004

The hepatic organoids can be stably expanded in vitro while preserving hepatocyte functionality. Using this revised protocol, Jin et al. demonstrated that PSC-derived hepatic organoids are more suitable for in vitro drug toxicity testing than conventional 2D PSC-derived hepatocyte cultures. Recently, researchers employed an iPSC-derived liver bud model to study liver-stage malaria. The human liver bud is generated from hepatic endoderm, septum transversum mesenchyme, and endothelial cells, all originating from human iPSC lines without requiring donated tissue. Immunofluorescence staining with anti-UIS4 revealed extremely low infection rates, detecting only a small parasite form. PCR-based analysis of the parasite burden failed to detect the P. vivax 18S rRNA transcript. Nevertheless, evidence for complete schizogony arises from the detection of blood-stage parasites in human reticulocytes [74].

When comparing recent studies using human fetal and adult liver organoids, sporozoite infection methods vary. Yang et al. plated fetal liver organoids as 2D monolayers [68], whereas Rao et al. dissociated adult liver organoids from a gel scaffold before incubating with P. falciparum sporozoites [69]. Similarly, Nitaramorn et al. incubated P. vivax sporozoites with 3D human liver buds extracted from a gel scaffold. Low infection rates may stem from low sporozoite penetration since the compact liver bud lacks vasculature. Interestingly, directly exposing P. berghei to human hepatic cell line-derived compact spheroids in a stirring tank enhances infection, implying that dynamic force is needed during sporozoite infection [75]. Despite low infection, iPSC-derived liver buds display altered lipid metabolism at the transcript level. After P. falciparum sporozoite invasion, transcripts involved in lipogenesis increase [68]. In P. vivax-infected human liver buds, the cholesterol synthesis gene TM7SF2 is upregulated, mirroring the findings of Yang and colleagues [74].

5. Key characteristics of the organoid system for modeling liver-stage malaria

An ideal in vitro disease model should closely recapitulate the in vivo microarchitecture and functions of human tissues. This section outlines key biological molecules that can improve in vitro liver-stage malaria models. The discussion is divided into two phases: pre-sporozoite incubation and post-sporozoite infection.

5.1 Pre-sporozoite incubation

Determining which cellular components are needed and their required characteristics will guide the design of improved organoid models. In the human body, immune cells extravasate by capturing, adhering, and transmigrating to damaged sites. Similarly, a key question for modeling liver-stage malaria is how sporozoites identify where to halt their migration across the endothelial barrier and enter the liver parenchyma to infect hepatocytes.

Sporozoite surfaces are rich in CSP and thrombospondin-related anonymous protein, and binding these proteins to their ligands is crucial [76–78]. Known ligands include highly sulfated heparan sulfate proteoglycans (HSPGs) [79] and low-density lipoprotein receptor-related proteins [80]. Hepatocyte invasion is triggered when CSP interacts with highly sulfated HSPGs, activating calcium-dependent protein kinase 6 and inducing the invasive phenotype of the sporozoites. CSP-HSPG binding also leads to the release of P36 and P52 proteins, which are required for forming the parasitophorous vacuole. On the hepatocyte surface, CD81 and SR-B1 engage with P36 and P52 to facilitate rhoptry secretion at the sporozoite’s apical side. CD81 and SR-B1 are essential for hepatocyte infection by P. falciparum and P. vivax, respectively. Thus, critical components for successful sporozoite invasion and infection in liver organoids include: (1) HSPGs for sporozoite adhesion and (2) CD81 and SR-B1 for species-specific hepatocyte infection. However, hepatic organoids are often embedded in Matrigel, an extracellular matrix-based gel. This gel may act as a physical barrier that impedes direct sporozoite contact with hepatocytes. One potential solution is to use scaffold-free organoid platforms or apply the gel above (gel-topping) rather than embedding cells within it. Alternatively, dissociating the organoid into single cells may permit direct sporozoite-hepatocyte interactions. Following dissociation, the cells can be plated in a 2D culture or reassembled into a 3D organoid.

5.2 Post-sporozoite infection

Studies by Chua and colleagues and Rao and colleagues [62,69] confirm that 3D spheroid or organoid platforms can model liver-stage malaria. Yet, two major limitations persist. The first involves assessing complete parasite development where sporozoites form parasitophorous vacuoles multiply and release viable merozoites. Detecting these stages commonly relies on immunofluorescence, which often requires preparing cross-sections of spheroids or organoids and incubating them with specific antibodies. This approach only samples a portion of the culture, and low infection rates further complicate detection.

One attempt to improve detection involves dissociating hepatic spheroids and replating them as a monolayer on collagen before performing antibody-based assays. This method yields an overall infection rate but adds multiple steps, reducing the feasibility of high-throughput drug or compound screening. Unlike conventional 2D primary human hepatocyte cultures, high-content cell imaging can provide unbiased, highly sensitive quantification and staging of infecting sporozoites [40]. Another challenge is hypnozoite formation and reactivation. The hepatic organoid must remain functional for at least 10 days to support hypnozoite formation. Current protocols for hepatic organoid generation maintain hepatocyte functions for only about 3–27 days [47,66,72,81–87], limiting the study of dormant stages.

6. Challenges in the detection of hypnozoites

Modeling hypnozoite formation and reactivation is more complex than modeling schizogony. Rodent malaria parasites do not form hypnozoites, and simian malaria models, while informative, are costly and require highly regulated facilities. Historically, hypnozoites were first identified in P. cynomolgi, a simian parasite. In infected rhesus monkeys, an approximately 5 µm intracellular form persisted in liver sections at 7 days post-infection and remained unchanged at 105 days [88]. Whether these small forms eventually reactivate to cause blood-stage malaria remains unknown.

To date, hypnozoites are mainly inferred by parasite size and the time elapsed post-infection. In 2018, a recombinant monoclonal antibody against P. vivax UIS4 became available, enabling detection of the parasitophorous vacuole membrane [89]. A small, stable UIS4-positive vacuole now serves as a surrogate marker of hypnozoites [49]. Current in vitro models show evidence of hypnozoite reactivation. In micropatterned primary human hepatocyte co-cultures, Gural et al. observed spontaneous P. vivax hypnozoite reactivation, indicated by large schizonts emerging after the initial hypnozoite wave [38]. Using a transgenic P. cynomolgi line with a dual fluorescent reporter, Voorberg-van der Wel and colleagues showed that hypnozoites develop into schizonts in primary rhesus hepatocyte cultures [90]. Some studies suggest hypnozoite reactivation based on the disappearance of large forms and the persistence of small forms in human liver-chimeric mice and liver-on-chip systems [52,91]. Subsequently, the reappearance of large form schizonts implies that small forms may have reactivated. Both internal and external factors could influence hypnozoite activation, including geographic location, parasite strain characteristics, and host immunity [92]. Elucidating hypnozoite reactivation mechanisms demands a model that supports long-term culture and intervention testing.

7. Future perspective

Increasing ethical concerns about animal experimentation and improved cell culture methods have enhanced in vitro disease models. To replace existing models, future liver-stage malaria platforms must incorporate molecular and cellular components identified in prior studies. Findings from two-dimensional cell cultures and in vivo experiments will guide the design of three-dimensional (3D) models. Spheroid cultures improve cell functionality more than conventional two-dimensional hepatic cancer or immortalized cell lines. However, spheroids do not fully replicate native tissue microarchitecture. Organoids more closely mimic the structure and function of actual tissues. Yet, sporozoite invasion remains challenging due to gel-like extracellular barriers in organoid systems. Detecting schizonts and hypnozoites using antibodies in 3D models currently requires tissue-like analysis, producing only representative images. Employing transgenic Plasmodium strains with fluorescent reporters and whole-tissue analytical methods, such as tissue clearing, could improve infection quantification and treatment efficacy assessment. Advances in materials science may also enable microwell platforms with fluidic flow, further replicating the dynamic conditions of native tissues.

References

  1. 1. Laveran CL. Classics in infectious diseases: a newly discovered parasite in the blood of patients suffering from malaria. Parasitic etiology of attacks of malaria: Charles Louis Alphonse Laveran (1845-1922). Rev Infect Dis. 1982;4(4):908–11. pmid:6750753
  2. 2. Arrow KJ, Gelband H. A brief history of malaria. In: Arrow KJ, Gelband H, editors. Saving lives, buying time: Economics of malaria drugs in an age of resistance. Washington (DC): National Academies Press (US); 2004.
    • 3. World Health Organization. World malaria report 2023. 2023.
      • 4. Patouillard E, Griffin J, Bhatt S, Ghani A, Cibulskis R. Global investment targets for malaria control and elimination between 2016 and 2030. BMJ Glob Health. 2017;2(2):e000176. pmid:29242750
      • 5. World Health O. World malaria report 2024: addressing inequity in the global malaria response. Geneva: World Health Organization; 2024.
        • 6. Corder RM, de Lima ACP, Khoury DS, Docken SS, Davenport MP, Ferreira MU. Quantifying and preventing Plasmodium vivax recurrences in primaquine-untreated pregnant women: an observational and modeling study in Brazil. PLoS Negl Trop Dis. 2020;14(7):e0008526. pmid:32735631
        • 7. Commons RJ, Rajasekhar M, Edler P, Abreha T, Awab GR, Baird JK, et al. Effect of primaquine dose on the risk of recurrence in patients with uncomplicated Plasmodium vivax: a systematic review and individual patient data meta-analysis. Lancet Infect Dis. 2024;24(2):172–83. pmid:37748496
        • 8. Soto AM, González-Cerón L, Santillán-Valenzuela F, Parrales ME, Montoya A. Recurrent Plasmodium vivax cases of both short and long latency increased with transmission intensity and were distributed year-round in the most affected municipalities of the RACCN, Nicaragua, 2013-2018. Int J Environ Res Public Health. 2022;19(10):6195. pmid:35627730
        • 9. Fikadu M, Ashenafi E. Malaria: an overview. Infect Drug Resist. 2023;16:3339–47.
        • 10. Chu CS, White NJ. Management of relapsing Plasmodium vivax malaria. Expert Rev Anti Infect Ther. 2016;14(10):885–900. pmid:27530139
        • 11. Watson J, Taylor WRJ, Bancone G, Chu CS, Jittamala P, White NJ. Implications of current therapeutic restrictions for primaquine and tafenoquine in the radical cure of vivax malaria. PLoS Negl Trop Dis. 2018;12(4):e0006440. pmid:29677199
        • 12. Wattanakul T, Gilder ME, McGready R, Hanpithakpong W, Day NPJ, White NJ, et al. Population pharmacokinetic modelling of primaquine exposures in lactating women and breastfed infants. Nat Commun. 2024;15(1):3851. pmid:38719803
        • 13. Hollingdale MR, Collins WE, Campbell CC, Schwartz AL. In vitro culture of two populations (dividing and nondividing) of exoerythrocytic parasites of Plasmodium vivax. Am J Trop Med Hyg. 1985;34(2):216–22. pmid:2984950
        • 14. Chattopadhyay R, Velmurugan S, Chakiath C, Andrews Donkor L, Milhous W, Barnwell JW, et al. Establishment of an in vitro assay for assessing the effects of drugs on the liver stages of Plasmodium vivax malaria. PLoS One. 2010;5(12):e14275. pmid:21151554
        • 15. Dembélé L, Franetich J-F, Soulard V, Amanzougaghene N, Tajeri S, Bousema T, et al. Chloroquine potentiates primaquine activity against active and latent hepatic plasmodia ex vivo: potentials and pitfalls. Antimicrob Agents Chemother. 2020;65(1):e01416-20. pmid:33077656
        • 16. Karnasuta C, Pavanand K, Chantakulkij S, Luttiwongsakorn N, Rassamesoraj M, Laohathai K, et al. Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line. Am J Trop Med Hyg. 1995;53(6):607–11. pmid:8561262
        • 17. Laohathai NB. Culturing of human hepatocellular carcinoma. Am J Pathol. 1985;2:203–8.
        • 18. Schepis A, Kumar S, Kappe SHI. Malaria parasites harness Rho GTPase signaling and host cell membrane ruffling for productive invasion of hepatocytes. Cell Rep. 2023;42(1):111927. pmid:36640315
        • 19. Rijpma SR, van der Velden M, González-Pons M, Annoura T, van Schaijk BCL, van Gemert G-J, et al. Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites. Cell Microbiol. 2016;18(3):369–83. pmid:26332724
        • 20. De Niz M, Caldelari R, Kaiser G, Zuber B, Heo WD, Heussler VT, et al. Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites. J Cell Sci. 2021;134(10):jcs252213. pmid:34013963
        • 21. Hussain T, Linera-Gonzalez J, Beck JM, Fierro MA, Mair GR, Smith RC, et al. The PTEX pore component EXP2 is important for intrahepatic development during the Plasmodium liver stage. mBio. 2022;13(6):e0309622. pmid:36445080
        • 22. Machado M, Sanches-Vaz M, Cruz JP, Mendes AM, Prudêncio M. Inhibition of Plasmodium hepatic infection by antiretroviral compounds. Front Cell Infect Microbiol. 2017;7:329. pmid:28770176
        • 23. Raphemot R, Toro-Moreno M, Lu K-Y, Posfai D, Derbyshire ER. Discovery of druggable host factors critical to Plasmodium liver-stage infection. Cell Chem Biol. 2019;26(9):1253-1262.e5. pmid:31257182
        • 24. Silvie O, Rubinstein E, Franetich J-F, Prenant M, Belnoue E, Rénia L, et al. Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity. Nat Med. 2003;9(1):93–6. pmid:12483205
        • 25. Sattabongkot J, Yimamnuaychoke N, Leelaudomlipi S, Rasameesoraj M, Jenwithisuk R, Coleman RE, et al. Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. Am J Trop Med Hyg. 2006;74(5):708–15. pmid:16687667
        • 26. Tweedell RE, Tao D, Hamerly T, Robinson TM, Larsen S, Grønning AGB. The selection of a hepatocyte cell line susceptible to Plasmodium falciparum sporozoite invasion that is associated with expression of glypican-3. Front Microbiol. 2019;10.
        • 27. Pewkliang Y, Rungin S, Lerdpanyangam K, Duangmanee A, Kanjanasirirat P, Suthivanich P, et al. A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax. Malar J. 2018;17(1):50. pmid:29370800
        • 28. Westerink WMA, Schoonen WGEJ. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol In Vitro. 2007;21(8):1581–91. pmid:17637504
        • 29. Huggett ZJ, Smith A, De Vivo N, Gomez D, Jethwa P, Brameld JM. A comparison of primary human hepatocytes and hepatoma cell lines to model the effects of fatty acids, fructose and glucose on liver cell lipid accumulation. Nutrients. 2023;15(1).
        • 30. Ishino T, Yano K, Chinzei Y, Yuda M. Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol. 2004;2(1):E4. pmid:14737184
        • 31. Ishino T, Chinzei Y, Yuda M. A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell Microbiol. 2005;7(2):199–208. pmid:15659064
        • 32. Grützke J, Rindte K, Goosmann C, Silvie O, Rauch C, Heuer D, et al. The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network. Traffic. 2014;15(4):362–82. pmid:24423236
        • 33. Caldelari R, Dogga S, Schmid MW, Franke-Fayard B, Janse CJ, Soldati-Favre D, et al. Transcriptome analysis of Plasmodium berghei during exo-erythrocytic development. Malar J. 2019;18(1):330. pmid:31551073
        • 34. March S, Ramanan V, Trehan K, Ng S, Galstian A, Gural N, et al. Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. Nat Protoc. 2015;10(12):2027–53. pmid:26584444
        • 35. March S, Ng S, Velmurugan S, Galstian A, Shan J, Logan DJ, et al. A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax. Cell Host Microbe. 2013;14(1):104–15. pmid:23870318
        • 36. Ng S, March S, Galstian A, Hanson K, Carvalho T, Mota MM, et al. Hypoxia promotes liver-stage malaria infection in primary human hepatocytes in vitro. Dis Model Mech. 2014;7(2):215–24. pmid:24291761
        • 37. Mazier D, Landau I, Druilhe P, Miltgen F, Guguen-Guillouzo C, Baccam D, et al. Cultivation of the liver forms of Plasmodium vivax in human hepatocytes. Nature. 1984;307(5949):367–9. pmid:6363939
        • 38. Gural N, Mancio-Silva L, Miller AB, Galstian A, Butty VL, Levine SS, et al. In vitro culture, drug sensitivity, and transcriptome of Plasmodium vivax hypnozoites. Cell Host & Microbe. 2018;23(3):395–406.e4.
        • 39. Ruoß M, Vosough M, Königsrainer A, Nadalin S, Wagner S, Sajadian S, et al. Towards improved hepatocyte cultures: progress and limitations. Food Chem Toxicol. 2020;138:111188. pmid:32045649
        • 40. Roth A, Maher SP, Conway AJ, Ubalee R, Chaumeau V, Andolina C, et al. A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum. Nat Commun. 2018;9(1):1837. pmid:29743474
        • 41. van der Boor SC, van Gemert G-J, Hanssen AEJ, van Waardenburg YM, McCall MBB, Bousema T, et al. Mid-liver stage arrest of Plasmodium falciparum schizonts in primary porcine hepatocytes. Front Cell Infect Microbiol. 2022;12:834850. pmid:35252038
        • 42. Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. 2010;51(1):297–305. pmid:19998274
        • 43. Ng S, Schwartz RE, March S, Galstian A, Gural N, Shan J, et al. Human iPSC-derived hepatocyte-like cells support Plasmodium liver-stage infection in vitro. Stem Cell Reports. 2015;4(3):348–59. pmid:25660406
        • 44. Schwartz RE, Fleming HE, Khetani SR, Bhatia SN. Pluripotent stem cell-derived hepatocyte-like cells. Biotechnol Adv. 2014;32(2):504–13. pmid:24440487
        • 45. Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res. 2009;19(11):1233–42. pmid:19736565
        • 46. Shan J, Schwartz RE, Ross NT, Logan DJ, Thomas D, Duncan SA, et al. Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat Chem Biol. 2013;9(8):514–20. pmid:23728495
        • 47. Kulkeaw K, Tubsuwan A, Tongkrajang N, Whangviboonkij N. Generation of human liver organoids from pluripotent stem cell-derived hepatic endoderms. PeerJ. 2020;8:e9968. pmid:33133779
        • 48. Simwela NV, Waters AP. Current status of experimental models for the study of malaria. Parasitology. 2022;149(6):1–22. pmid:35357277
        • 49. Mikolajczak SA, Vaughan AM, Kangwanrangsan N, Roobsoong W, Fishbaugher M, Yimamnuaychok N, et al. Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice. Cell Host Microbe. 2015;17(4):526–35. pmid:25800544
        • 50. Vaughan AM, Mikolajczak SA, Wilson EM, Grompe M, Kaushansky A, Camargo N, et al. Complete Plasmodium falciparum liver-stage development in liver-chimeric mice. J Clin Invest. 2012;122(10):3618–28. pmid:22996664
        • 51. Strom SC, Davila J, Grompe M. Chimeric mice with humanized liver: tools for the study of drug metabolism, excretion, and toxicity. Methods Mol Biol. 2010;640:491–509. pmid:20645070
        • 52. Schäfer C, Dambrauskas N, Reynolds LM, Trakhimets O, Raappana A, Flannery EL, et al. Partial protection against P. vivax infection diminishes hypnozoite burden and blood-stage relapses. Cell Host Microbe. 2021;29(5):752–6.e4.
        • 53. Flannery EL, Kangwanrangsan N, Chuenchob V, Roobsoong W, Fishbaugher M, Zhou K, et al. Plasmodium vivax latent liver infection is characterized by persistent hypnozoites, hypnozoite-derived schizonts, and time-dependent efficacy of primaquine. Mol Ther Methods Clin Dev. 2022;26:427–40. pmid:36092359
        • 54. Wilson EM, Bial J, Tarlow B, Bial G, Jensen B, Greiner DL, et al. Extensive double humanization of both liver and hematopoiesis in FRGN mice. Stem Cell Res. 2014;13(3 Pt A):404–12. pmid:25310256
        • 55. Takenaka K, Prasolava TK, Wang JCY, Mortin-Toth SM, Khalouei S, Gan OI, et al. Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat Immunol. 2007;8(12):1313–23. pmid:17982459
        • 56. Foquet L, Schafer C, Minkah NK, Alanine DGW, Flannery EL, Steel RWJ, et al. Plasmodium falciparum liver stage infection and transition to stable blood stage infection in liver-humanized and blood-humanized FRGN KO mice enables testing of blood stage inhibitory antibodies (reticulocyte-binding protein homolog 5) in vivo. Front Immunol. 2018;9:524. pmid:29593746
        • 57. Luiza-Batista C, Thiberge S, Serra-Hassoun M, Nardella F, Claës A, Nicolete VC, et al. Humanized mice for investigating sustained Plasmodium vivax blood-stage infections and transmission. Nat Commun. 2022;13(1):4123. pmid:35840625
        • 58. Michailidis E, Vercauteren K, Mancio-Silva L, Andrus L, Jahan C, Ricardo-Lax I, et al. Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes. Proc Natl Acad Sci U S A. 2020;117(3):1678–88. pmid:31915293
        • 59. Minkah NK, Schafer C, Kappe SHI. Humanized mouse models for the study of human malaria parasite biology, pathogenesis, and immunity. Front Immunol. 2018;9:807. pmid:29725334
        • 60. Schäfer C, Roobsoong W, Kangwanrangsan N, Bardelli M, Rawlinson TA, Dambrauskas N, et al. A humanized mouse model for Plasmodium vivax to test interventions that block liver stage to blood stage transition and blood stage infection. iScience. 2020;23(8):101381. pmid:32739836
        • 61. Mellin R, Boddey JA. Organoids for liver stage malaria research. Trends Parasitol. 2020;36(2):158–69. pmid:31848118
        • 62. Chua ACY, Ananthanarayanan A, Ong JJY, Wong JY, Yip A, Singh NH, et al. Hepatic spheroids used as an in vitro model to study malaria relapse. Biomaterials. 2019;216:119221. pmid:31195301
        • 63. Zhao Z, Chen X, Dowbaj AM, Sljukic A, Bratlie K, Lin L. Organoids. Nat Rev Methods Primers. 2022;2.
        • 64. Lancaster MA, Huch M. Disease modelling in human organoids. Dis Model Mech. 2019;12(7):dmm039347. pmid:31383635
        • 65. Huch M, Dorrell C, Boj SF, van Es JH, Li VSW, van de Wetering M, et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature. 2013;494(7436):247–50. pmid:23354049
        • 66. Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MMA, et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell. 2015;160(1–2):299–312. pmid:25533785
        • 67. Hu H, Gehart H, Artegiani B, LÖpez-Iglesias C, Dekkers F, Basak O, et al. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell. 2018;175(6):1591-1606.e19. pmid:30500538
        • 68. Yang ASP, Dutta D, Kretzschmar K, Hendriks D, Puschhof J, Hu H, et al. Development of Plasmodium falciparum liver-stages in hepatocytes derived from human fetal liver organoid cultures. Nat Commun. 2023;14(1):4631. pmid:37532704
        • 69. Rao S, Romal S, Torenvliet B, Slotman JA, Huijs T, Mahmoudi T. A 3D organoid platform that supports liver-stage P. falciparum infection can be used to identify intrahepatic antimalarial drugs. Heliyon. 2024;10(10):e30740. pmid:38770342
        • 70. Akbari S, Sevinç GG, Ersoy N, Basak O, Kaplan K, Sevinç K, et al. Robust, long-term culture of endoderm-derived hepatic organoids for disease modeling. Stem Cell Reports. 2019;13(4):627–41. pmid:31522975
        • 71. Mun SJ, Ryu J-S, Lee M-O, Son YS, Oh SJ, Cho H-S, et al. Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids. J Hepatol. 2019;71(5):970–85. pmid:31299272
        • 72. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature. 2013;499(7459):481–4. pmid:23823721
        • 73. Jin Z-L, Xu K, Kim J, Guo H, Yao X, Xu Y-N, et al. 3D hepatic organoid production from human pluripotent stem cells. Differentiation. 2024;135:100742. pmid:38104501
        • 74. Nitaramorn N, Kobpornchai P, Tongkrajang N, Chaisri U, Imwong M, Kulkeaw K. Human liver organoids are susceptible to Plasmodium vivax infection. Malar J. 2024;23(1):368. pmid:39639330
        • 75. Arez F, Rebelo SP, Fontinha D, Simão D, Martins TR, Machado M, et al. Flexible 3D cell-based platforms for the discovery and profiling of novel drugs targeting Plasmodium hepatic infection. ACS Infect Dis. 2019;5(11):1831–42. pmid:31479238
        • 76. Manzoni G, Marinach C, Topçu S, Briquet S, Grand M, Tolle M, et al. Plasmodium P36 determines host cell receptor usage during sporozoite invasion. Elife. 2017;6:e25903. pmid:28506360
        • 77. Longley RJ, Bauza K, Ewer KJ, Hill AVS, Spencer AJ. Development of an in vitro assay and demonstration of Plasmodium berghei liver-stage inhibition by TRAP-specific CD8+ T cells. PLoS One. 2015;10(3):e0119880. pmid:25822951
        • 78. Zhao J, Bhanot P, Hu J, Wang Q. A comprehensive analysis of Plasmodium circumsporozoite protein binding to hepatocytes. PLoS One. 2016;11(8):e0161607. pmid:27560376
        • 79. Coppi ATR, Bishop JR, Bennett BL, Lawrence R, Esko JD, Billker O, et al. Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade cells. Cell Host Microbe. 2007;2.
        • 80. Shakibaei M, Frevert U. Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans. J Exp Med. 1996;184(5):1699–711. pmid:8920859
        • 81. De Crignis E, Hossain T, Romal S, Carofiglio F, Moulos P, Khalid MM, et al. Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. Elife. 2021;10:e60747. pmid:34328417
        • 82. Guan Y, Xu D, Garfin PM, Ehmer U, Hurwitz M, Enns G, et al. Human hepatic organoids for the analysis of human genetic diseases. JCI Insight. 2017;2(17):e94954. pmid:28878125
        • 83. Nie Y-Z, Zheng Y-W, Miyakawa K, Murata S, Zhang R-R, Sekine K, et al. Recapitulation of hepatitis B virus-host interactions in liver organoids from human induced pluripotent stem cells. EBioMedicine. 2018;35:114–23. pmid:30120080
        • 84. Ouchi R, Togo S, Kimura M, Shinozawa T, Koido M, Koike H, et al. Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids. Cell Metab. 2019;30(2):374-384.e6. pmid:31155493
        • 85. Ramli MNB, Lim YS, Koe CT, Demircioglu D, Tng W, Gonzales KAU, et al. Human pluripotent stem cell-derived organoids as models of liver disease. Gastroenterology. 2020;159(4):1471-1486.e12. pmid:32553762
        • 86. Wang S, Wang X, Tan Z, Su Y, Liu J, Chang M, et al. Human ESC-derived expandable hepatic organoids enable therapeutic liver repopulation and pathophysiological modeling of alcoholic liver injury. Cell Res. 2019;29(12):1009–26. pmid:31628434
        • 87. Shinozawa T, Kimura M, Cai Y, Saiki N, Yoneyama Y, Ouchi R, et al. High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids. Gastroenterology. 2021;160(3):831-846.e10. pmid:33039464
        • 88. Krotoski WA, Garnham PCC, Bray RS, Krotoski DM, Killick-Kendrick R, Draper CC, et al. Observations on early and late post-sporozoite tissue stages in primate malaria. Am J Trop Med Hyg. 1982;31(1):24–35.
        • 89. Schafer C, Dambrauskas N, Steel RW, Carbonetti S, Chuenchob V, Flannery EL, et al. A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages. Malar J. 2018;17(1):370. pmid:30333026
        • 90. Voorberg-van der Wel AM, Zeeman A-M, Nieuwenhuis IG, van der Werff NM, Klooster EJ, Klop O, et al. A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation. Commun Biol. 2020;3:7. pmid:31909199
        • 91. Maher SP, Conway AJ, Roth A, Adapa SR, Cualing P, Andolina C, et al. An adaptable soft-mold embossing process for fabricating optically-accessible, microfeature-based culture systems and application toward liver stage antimalarial compound testing. Lab Chip. 2020;20(6):1124–39. pmid:32055808
        • 92. Markus MB. Malaria: origin of the term “hypnozoite”. J Hist Biol. 2011;44(4):781–6. pmid:20665090