The use of organoids in infectious disease research represents a significant paradigm shift, offering a more physiologically relevant and ethically sound alternative to traditional cell cultures and animal models. Our analysis reveals their potential in elucidating complex host‒pathogen interactions, particularly in the context of emerging infectious diseases. This approach not only enhances our understanding of disease mechanisms at the cellular and molecular levels but also opens new avenues for the development of targeted therapeutics and vaccines.
1. The first stage: early exploration from 2012 to 2015
In 2009, the Hans Clevers group pioneered the development of the first organoid derived from murine intestines, inaugurating a novel domain of research in gastrointestinal diseases [19] (Fig. 5). Therefore, it is not surprising that our authorship and institutional analysis identified Hans Clevers and his collaborators as pioneers and central figures in the global network dedicated to infectious disease research using organoid models (Fig. 3a). An inaugural study utilizing an organoid model for infectious disease research emerged in 2012, elucidating the interaction of human intestinal organoids with rotaviruses [20]. This foundational work catalyzed subsequent research, including studies on Helicobacter pylori infections using gastric organoids [21], as well as research on human and murine intestinal organoids infected with Salmonella [22] and Clostridium difficile [23]. These initial investigations established the cornerstone for the expanding research domain of intestinal organoid infections, which emerged as the most prominent keyword cluster (depicted in red) in our analysis (Fig. 4a). This cluster, along with investigations into gastric organoids infected with Helicobacter pylori, features the earliest average publication year, highlighting its sustained significance in the field (Fig. 4b). A citation burst analysis delineates 'intestinal organoid' as exhibiting the highest burst strength, signifying its critical role in the initial phase of the field (Fig. 4c). This is further augmented by frequent studies on two bacterial pathogens, 'Helicobacter pylori' and 'Clostridium difficile', underscoring the seminal importance of these areas in early research.
Nevertheless, the aforementioned research encounters a significant challenge [24]. Although physiologically relevant, these organoids display polarized epithelial cells with a basolateral side facing outward and an apical side enclosed internally. Consequently, when bacteria approach these organoids externally, they interact with the basolateral surface, in contrast to in vivo conditions where the epithelial cells' apical surface engages with luminal components, including microorganisms. This arrangement complicates direct access to the apical side of the epithelial cells in organoid models.
To surmount this challenge, researchers have developed and employed various methodologies (Table 4). The microinjection technique, pioneered by Sina Bartfeld and Hans Clevers in 2015, introduces microorganisms directly into the luminal space of organoids, thereby facilitating interaction with the apical epithelium. This protocol, published in 2015, has garnered significant attention and citations [17]. It has catalyzed research on diverse pathogens, including Salmonella [25], Escherichia coli [26], Mycobacterium tuberculosis (Mtb), and Mycobacterium abscessus (Mabs) [15]. Despite its ingenuity, this method requires specific equipment and is labor intensive. The microinjection of pathogens in livestock intestinal organoids has also been reported by Beaumont et al. [27]. However, variability in injection volume and potential leakage into the extracellular medium challenge its reproducibility [27]. An alternative approach involves reversing the polarity of enteroid cells by removing the extracellular matrix [28]. However, polarity inversion is not observed in all organoids. This technique has been established with human organoids [29] and then adapted to pig [30], bovine [31], canine [32], and chicken [33] enteroids. Another method involving the exposure of intestinal organoids to an inflammatory cocktail has been shown to invert human organoid polarity [34], although its application in altering epithelial polarity in farm animal organoids remains unexplored. Additionally, researchers have cultivated murine organoid epithelial cells as 2D monolayers since 2014 [35], a technique applied to pig [36], rabbit [37], bovine [31], chicken [38] and equine [39] intestinal organoids. This method involves seeding dissociated organoid cells on surfaces precoated with diluted extracellular matrix components such as Matrigel™. This format promotes the formation of a cohesive epithelial barrier, as evidenced by increasing transepithelial electrical resistance measurements, indicative of barrier integrity. The apical surface in these monolayers is readily accessible for experimental interventions, with differentiation induced either by niche factor withdrawal or by establishing an air-liquid interface, as demonstrated in pig enteroids. However, while 2D monolayers provide easier access to the apical surface, they lack the complex three-dimensional architecture intrinsic to intestinal organoids, such as crypt-like structures, which are essential for mimicking the in vivo cellular organization of the intestine.
2. The second stage: ZIKV-related explosion from 2016 to 2019
First introduced in 2013, human cerebral organoids have served as research models for neural development [40], neurodegenerative disorders [41], neuropsychiatric conditions [42], and even patient-specific microcephaly models using induced pluripotent stem cells (iPSCs) [43]. However, their application in infectious disease research remained unexplored until 2016 (Fig. 5). Reacting swiftly to the ZIKV outbreak in Brazil, Patricia Pestana Garcez and her team from the University of São Paulo submitted a groundbreaking manuscript in March 2015 [44], at the same time with Brazil's official reporting of the outbreak. They were the first to investigate ZIKV-induced neural defects in cerebral organoids, offering invaluable insights into the challenges the ZIKV epidemic presented. In 2016, the outbreak led to a Public Health Emergency of International Concern (PHEIC) from February to November of that year. A group of high-impact articles emerged in leading journals since then (Fig. 1). Garcez and colleagues [45] continued to refine models that mimicked ZIKV-induced neural defects (Table 4). Concurrently, Qian et al. [46] developed a forebrain-specific organoid model from iPSCs using a miniaturized spinning bioreactor to study both African and Asian strains of ZIKV. Further deepening the field's understanding, Dang et al. [47] elucidated the activation of the TLR3 signaling pathway by ZIKV and its resultant depletion of neural progenitor cells, which led to reduced organoid size. In a novel approach, Xu et al. [48] deployed an ultrahigh-throughput drug screening platform based on cerebral organoids and discovered that Emricasan, a pan-caspase inhibitor, could mitigate ZIKV-induced caspase-3 activation.
Research fervor surrounding ZIKV persisted until 2019, as evidenced by the burst in citations for terms such as "Zika," "neural progenitor," and "neurons" (Fig. 4c). Although publication growth slowed down with the subsidence of the ZIKV epidemic, scholarly activity in this domain has not ceased. Recent studies continue to delve into the mechanisms underlying ZIKV-induced neurological defects [49, 50] and potential treatment avenues [51, 52]. Consequently, keywords associated with ZIKV form the smallest cluster in our analysis and a relatively aged average publication year, indicating its shifting priorities (Fig. 4a).
3. The third stage: SARS-CoV-2-related explosion from 2020 to the present
The initial airway organoid model, derived from murine sources, was established in 2009 [53], followed by human airway organoids reported in 2013 [43] (Fig. 5). Subsequent research on a human airway organoid model focused on various infectious agents, such as RSV [18] and influenza viruses [54], together forming a keyword cluster represented in blue. However, an advancement occurred in 2019 with the development of the first long-term pseudostratified airway epithelium organoid model using adult human tissues [18]. This pivotal innovation set the stage for numerous subsequent investigations, particularly those centering on SARS-CoV-2 and other respiratory pathogens. After the World Health Organization (WHO) declared COVID-19 a pandemic in March 2020, a wave of high-impact studies emerged, leveraging human-derived organoids from various organ systems (Fig. 1). The paper by Monteil et al. [55] stands as the most cited in this context, accumulating 1,410 citations thus far (Table 4). Their research validated that pre-existing human recombinant soluble ACE2 (hrsACE2) could inhibit SARS-CoV-2 infections in both human capillary and kidney organoids, which might be a potential treatment for COVID-19. Supporting these findings, Lamers et al. [56] confirmed that SARS-CoV-2 could infect human small intestinal organoids and demonstrated the effectiveness of hrsACE2 in preventing viral binding. Eric Song et al. explored SARS-CoV-2 infection in human cerebral organoids, shedding light on resultant metabolic alterations. Keywords from these more recent studies form a yellow keyword cluster, characterized by a notably recent average publication year.
While both are propelled by viral outbreaks, research into SARS-CoV-2 is notably more comprehensive than earlier studies on ZIKV. It goes beyond simply unraveling the mechanisms of infection to include vaccine and drug development. Given the species-specific nature of viral infections and the distinct differences in adaptive immunity between species, traditional animal models often prove inadequate. Many vaccine candidates that succeed in animal trials ultimately fail in humans. Consequently, innovative, human-specific testing methodologies could expedite drug and vaccine validation processes. Although human organoids lack an immune system, they offer unique advantages for understanding host‒virus interactions and for assessing the efficacy and neutralizing capabilities of potential vaccines [57]. Traditional cell lines are limited in replicating the physiologically relevant dynamics of in vivo SARS-CoV-2 infection, positioning human organoids as promising platforms for COVID-19 drug discovery. Specifically, human lung organoids have been employed to evaluate the effectiveness of drugs targeting viral entry and replication [58, 59]. This multifaceted approach facilitates a more direct translation from research to clinical application, offering prompt and effective interventions against the virus. Moreover, the broad impact of SARS-CoV-2 on various organ systems has been matched with a versatile range of well-established organoid models. Research has incorporated cerebral [60], gastrointestinal [56], liver [61], kidney [62], and tonsil [63] organoids, highlighting the solid groundwork and expansive potential of organoid-based research.
4. Organoid source and pathogen trends
In our analysis, it is evident that human is the primary specie for organoid generation (Fig. 2a). This preference is grounded in several key reasons. First, human organoids accurately mirror the structure and function of human organs, circumventing potential discrepancies due to species-specific physiological and disease susceptibility differences [4]. Second, advancements in biobanking and the refinement of ethical consent processes have enhanced the availability of human sources for research, making them a practical option for organoid creation [64]. This approach also mitigates ethical concerns often linked with animal model usage. Furthermore, human organoids have demonstrated superior predictability in assessing the efficacy and toxicity of new therapeutic agents, offering critical insights into drug development [65, 66].
Moreover, the majority of organoid research focuses on viruses, followed by bacteria (Fig. 2c). This emphasis on viruses is attributed to several factors. Firstly, organoids facilitate the cultivation of otherwise uncultivable viruses [67]. For instance, noroviruses, which cannot replicate in conventional cell lines, are capable of infecting human intestinal organoids [5]. Additionally, viruses can be directly amplified in organoid models from clinical isolates, negating the need for mutation or adaptation [68]. Secondly, the imperative created by recent significant epidemics has accelerated research in this area [69]. Similarly, organoids offer a promising approach to surmount current challenges of in vitro parasitic infections and to provide a mechanistic understanding of the impact of parasites on their host environments [70, 71]. However, due to the complexity of parasite life cycles and the size of parasites such as gastrointestinal nematodes that are generally 10 to 10,000-fold larger than viruses, bacteria and protists, the studies using organoids as model of parasite- host interaction have been considerably delayed.
While human and murine models have been the mainstay of organoid research, their application in animal models is expanding rapidly. Figure 5 illustrates the time lag between the establishment of species-specific organoids and their application in infectious disease research. Human and murine organoids, largely developed before 2016, usually had a lag of approximately five years before being applied to infectious disease research. In contrast, most animal organoids, apart from porcine [30, 72] and galline [73, 74] intestinal types, were developed after 2016. The integration of porcine and galline organoids into infectious disease research took approximately 7 and 5 years, respectively. Notably, a considerable number of organoids from other animals were quickly incorporated into infectious disease research upon development. Specifically, chiropteran airways [75] and intestinal organoids [76] have been developed to study the SARS-CoV-2 virus, given the suspected origin of COVID-19 in bats. Recent years have marked a notable increase in the development of animal organoids, signifying rapid advances in the establishment of animal organoids and their utilization in infectious disease research.
5. The quick response from scientists and publishers
The accelerated timelines for publishing pivotal articles related to SARS-CoV-2 underscore a remarkable synergy and urgency between academic researchers and publishing platforms. On January 31, 2020, a consortium of publishers and scholarly communication organizations issued a call to action, advocating for the rapid and open sharing of all COVID-19-related research and data [77]. The aim was to expedite the public health response and save lives. Specific measures include making all peer-reviewed research publications freely available at least for the duration of the outbreak; sharing research findings with the WHO immediately upon journal submission; using preprint servers or alternative platforms to disseminate research prior to formal peer review; sharing both interim and final research data, along with the protocols and standards used to collect said data, as quickly and widely as possible; and clarifying that sharing data or preprints ahead of formal submission would not preclude subsequent publication in journals.
This policy had an obvious effect, particularly when comparing the early pandemic stage publisher-responsiveness to that of publications about Middle East respiratory syndrome (MERS) [69]. Specifically, the time from submission to acceptance was 5 days for COVID-19 research, compared to 71.5 days for MERS, and the acceptance-to-publication time was also 5 days for COVID-19, compared to 22.5 days for MERS. Moreover, almost all of the papers were made open-access.
Facing a global health emergency, this rapid information exchange serves several indispensable roles. First, it accelerates the pace of scientific breakthroughs, enabling instantaneous scrutiny by the scholarly community—an indispensable process for confirming new data. The swift publication process also illustrates the resilience and adaptability of the scholarly publishing ecosystem. Hence, academic journals are prompted to expedite their peer-review workflows, all while upholding the integrity of scientific scrutiny. This enables the immediate dissemination of landmark findings that can potentially guide public health strategies. Moreover, the prompt timelines accentuate the importance of preprint archives, offering an early-access platform for researchers to circulate their discoveries before undergoing formal peer review. This is particularly beneficial in crisis scenarios, adding immediacy to the sharing of vital information. Collectively, the rapidity with which these seminal papers were publicized underscores a unified, community-focused response to a pressing global health dilemma. It epitomizes how both the scientific and publishing sectors can nimbly adjust and deploy their collective capabilities to confront urgent public health challenges.
6. Limitations and perspective
The research progress after the Zika virus (ZIKV) and SARS-CoV-2 outbreaks demonstrates a notably quicker and more comprehensive adoption of organoids in studies related to SARS-CoV-2, encompassing areas from fundamental mechanistic research to the development of vaccines and therapeutics. This rapid integration can largely be credited to the profound global impact of the SARS-CoV-2 pandemic and the pre-existing experience with airway organoids in various pathogenic investigations, including those involving viruses, bacteria, and other microbes [78]. This prior groundwork paved the way for the expedited adaptation and application of organoid infection models and methodologies in SARS-CoV-2 research.
Concurrently, it is vital to focus on developing more physiologically representative organoid models, particularly those incorporating the environment, such as mechanical force, microbiota, and immune cells co-cultures [79]. Such an advancement would not only improve the ability of these models to simulate physiological conditions but also deepen our understanding of the intricate interactions between pathogens and the host’s immune system. In the domain of animal research, the emphasis has predominantly been on organoids derived from stem cells or embryonic stem cells [80]. The exploration and utilization of iPSCs are essential. iPSCs can offer a wider array of organoid models, facilitating more detailed studies across different animal species and organ systems. This is critical for comprehending the variations in disease mechanisms and responses to treatments across species, which in turn would contribute significantly to the fields of comparative biology and translational medicine.
Moreover, the application of organoids should be expanded beyond just investigating infection mechanisms to encompass the areas of drug and vaccine development and screening [48, 59]. The establishment of extensive organoid biobanks would be instrumental in accelerating the identification and selection of patient-specific therapeutic agents, thereby improving preparedness for a range of pathogens [64].