Development trends of human organoid‐based COVID‐19 research based on bibliometric analysis

Abstract

Coronavirus disease 2019 (COVID‐19), a global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), has posed a catastrophic threat to human health worldwide. Human stem cell‐derived organoids serve as a promising platform for exploring SARS‐CoV‐2 infection. Several review articles have summarized the application of human organoids in COVID‐19, but the research status and development trend of this field have seldom been systematically and comprehensively studied. In this review, we use bibliometric analysis method to identify the characteristics of organoid‐based COVID‐19 research. First, an annual trend of publications and citations, the most contributing countries or regions and organizations, co‐citation analysis of references and sources and research hotspots are determined. Next, systematical summaries of organoid applications in investigating the pathology of SARS‐CoV‐2 infection, vaccine development and drug discovery, are provided. Lastly, the current challenges and future considerations of this field are discussed. The present study will provide an objective angle to identify the current trend and give novel insights for directing the future development of human organoid applications in SARS‐CoV‐2 infection.

Keyword co‐occurrence overlay visualization of the research of organoids for SARS‐CoV‐2 infection. More studies focused on pulmonary infection using organoid models at the early stages of the COVID‐19 pandemic. Recently, keywords included ‘drug repurposing’, ‘personalized medicine’, ‘repair’, ‘therapy’, ‘resistance’ and ‘therapeutics’ are more popular. Node size indicates the occurrence frequency; Node colour represents the keyword co‐occurrence based on their average publication year, purple means that keywords occurred earlier and marked with yellow stands that keywords are vigorous recently.

1. INTRODUCTION

The coronavirus disease 2019 (COVID‐19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, has posed a catastrophic threat to human health worldwide. Since its widely spread, as of 30 November 2022, roughly 639.1 million global cases were confirmed and 6.6 million deaths from COVID‐19. ¹ COVID‐19 cases could be asymptomatic or symptomatic, including fever or chills, cough, fatigue, shortness of breath and a loss of taste or sense of smell. Respiratory injury is the most common outcome in COVID‐19 patients, but SARS‐CoV‐2 can cause widespread damage to multi‐organs, including the eye, brain, heart, kidney, liver, lung and gastrointestinal tracts. ² To date, multiple SARS‐CoV‐2 variants have been described. For instance, the SARS‐CoV‐2 Omicron variant (B.1.1.529) was initially reported in South Africa and Botswana in November 2021. ³ , ⁴ Compared with the original coronavirus, the Omicron variant exhibits enhanced infectivity and transmissibility, high immune evasion ability and reduced pathogenicity. ³ , ⁵ , ⁶ Although several COVID‐19 vaccines can prevent viral infections, new coronavirus variant may escape from immunity induced by the existing vaccines, leading to a great number of breakthrough infections. ⁷ , ⁸ , ⁹ In this case, more efforts are needed to comprehensively decipher the pathophysiological processes of coronavirus infection and evaluate vaccine efficacy and safety.

Most current data on SARS‐CoV‐2 infection are limited to clinical data, classic cell experiments and restricted animal models. ¹⁰ , ¹¹ Given angiotensin‐converting enzyme 2 (ACE2) functions as the key SARS‐CoV‐2 receptor, transgenic animals expressing human ACE2 are good options. However, animal‐based studies fail to faithfully mimic human physiology and recapitulate the human response to SARS‐CoV‐2 infection because of species variants. In addition, limited data reported how genetic diversity will influence the responses to SARS‐CoV‐2. ¹² , ¹³ Human cell lines, including Caco‐2, Calu‐3, HEK293T, Huh7 and A549, have been utilized in viral infection experiments, whereas they are unable to adequately recapitulate the in vivo infection processes because monolayer cells lack cellular microenvironment, including cell–cell and cell–matrix interactions. Moreover, cancer cell lines carry the potential for malignant proliferation and uncontrollable mutations. Therefore, building human physiologically relevant model systems is dramatically demanded in the studies of viral infections.

Advanced stem cell technology has provided promising models in vitro to recapitulate human sophisticated, multicellular and physiological organs, namely organoids. They are three‐dimensional (3D) self‐organized tissues from human adult stem cells (ASCs) and pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Human adult stem cell‐ or progenitor cell‐derived organoids could retain their organ identity, such as containing various cell types present in the native organ, recapitulating the cellular architecture and spatial organization of the organ and maintaining genetic stability over a long period of time. ¹⁴ In contrast to the complicated and variable process of PSC differentiation, ASCs can be generated from biopsies isolated directly from healthy or patient tissue, thus providing a platform for personalized diagnosis and therapy. ¹⁵ , ¹⁶ ASC‐derived organoids are mainly generated from epithelial tissues (intestine, colon, stomach, liver, pancreas, lung, bladder and so forth) and need to isolate the tissue‐specific stem cell population, thus recapitulating only the epithelium of organs. ¹⁴ , ¹⁵ , ¹⁷ ASC‐derived organoids are typically cystic and highly polarized epithelium, lacking nerves, blood vessels and stromal elements. ¹⁸ , ¹⁹ , ²⁰ Compared with ASC‐derived organoids, PSC‐derived organoids present higher complexity in structure. ²⁰ PSC‐derived organoids can be established from non‐epithelial tissues, including blood vessels, the brain and the retina. ²¹ , ²² , ²³ PSC‐derived embryoid bodies (EBs) can be guided to differentiate into three primary germ layers (ectoderm, mesoderm and endoderm), which recapitulates early embryonic development and finally generate a large variety of organoids. ¹⁵ , ²⁴

Currently, various human organoids have been successfully established, such as brain, retinal, liver, kidney, stomach, intestinal, lung, cardiac and colon organoids and are widely applied for regenerative medicine and modelling human diseases, including infectious diseases. For instance, human brain organoid was utilized to understand Zika virus (ZIKV)‐induced adverse effects on the human brain. Qian's lab first generated brain organoids to elucidate ZIKV‐mediated neurodevelopmental disorder and model microcephaly. ²⁵ ZIKV was found to hijack host cells to increase viral replication and preferentially target neural progenitor cells, induce cell death, proliferation and premature differentiation of neural progenitors in human brain organoids. ²⁵ , ²⁶ , ²⁷ Furthermore, brain organoids were used for drug discovery against ZIKV infection. ²⁸ The study from Xu's group showed that Emricasan (a pan‐caspase inhibitor) and Niclosamide (The United States Food and Drug Administration (FDA)‐approved anthelmintic drug) were found to reduce ZIKV replication and protect neural progenitor cells from ZIKV‐induced cell death in brain organoids. ²⁹ Similarly, cholesterol‐25‐hydroxylase (CH25H) was observed to suppress viral infection and reduce tissue injuries in human ZIKV‐infected cortical organoids. ³⁰ These data stimulated the field to apply human organoids for studying viral infectivity, vaccine development and drug discovery of SARS‐CoV‐2.

Over the last 3 years, growing worldwide concerns regarding the application of human organoids for SARS‐CoV‐2 infection during the COVID‐19 epidemic scenario. Data from the Web of Science (WOS) Core Collection showed that the publication numbers dramatically increased, especially from 2020 to 2021 (Figure 1A). The major fields of organoid‐based COVID‐19 study are involved in Cell Biology, Biochemistry Molecular Biology, Cell Tissue Engineering, Medicine Research Experimental, Multidisciplinary Sciences, Microbiology, Immunology, Virology and so on (Figure 1B). Given the rapid development of organoid applications in SARS‐CoV‐2 infection, the research status and development trend of this field are systematically and comprehensively reviewed in the present study. Bibliometric analysis could be applied to determine the development, hotspots and trend directions of organoid applications in SARS‐CoV‐2 infection. ³¹ , ³² , ³³ , ³⁴ In this review, bibliometric method was used to reveal the research trends regarding the most influential countries and institutes, contributing journals and hotspots of the research field in organoid‐based COVID‐19 studies. We further provide a detail discussion on the application of human organoids in studying viral infectivity, vaccine development and drug discovery of SARS‐CoV‐2. At last, existing challenges and future perspectives in this field will be discussed. We hope this work will provide an objective angle to identify the current trend and give novel insights for guiding the future development of human organoid applications during the COVID‐19 pandemic scenario.

FIGURE 1.

Characteristics of publications regarding human organoids applied in the COVID‐19 study. (A) Semi‐annual publications of human organoid application for SARS‐CoV‐2 infection. (B) Major fields of organoid application in COVID‐19 research. (C) The top 10 contributing countries/regions in organoid‐based COVID‐19 study.

2. BIBLIOMETRIC ANALYSIS

2.1. Data source and search strategy

The WOS Core Collection Advanced Search based on Science Citation Index Expanded (SCI‐EXPANDED) and Social Sciences Citation Index (SSCI) was applied to retrieve records on organoid applications in SARS‐CoV‐2 research. The data was obtained from the period between 1 December 2019 and 27 November 2022, due to the first case occurred in Wuhan, China on 1 December 2019. Subsequently, WOS analysis and VOSviewer (version 1.6.18, from the Centre of Science and Technology Studies at Leiden University) application were applied to analyse the general information, including growing trends of publication and citation, the most productive/influential countries/regions and organizations, journal distribution, co‐cited references and sources and key research areas. The retrieval strategy was as follows: TS = (‘COVID‐19’ OR ‘coronavirus disease 2019’ OR ‘2019‐nCov’ OR ‘2019 novel coronavirus’ OR ‘SARS‐CoV‐2’ OR ‘Severe acute respiratory syndrome coronavirus 2’ OR ‘novel coronavirus disease 19’ OR ‘novel coronavirus disease‐19’ OR ‘SARS2’ OR ‘SARS‐2’ OR ‘COVID‐2019’ OR ‘COVID19’) AND (‘organoid*’). The inclusion criteria were publications written in English.

2.2. Publication and citation characteristics

A total of 284 records were retrieved, which included 154 original articles, 90 reviews, 18 editorials, 10 early access, 10 letters and 2 others. The articles and reviews constituted the largest share (54.22% and 31.69%, respectively) of the full share of the publications and were analysed in our subsequent analysis. As seen in Figure 1A, both the publications and citations of papers regarding organoid‐based COVID‐19 have increased rapidly over the last 3 years. This data suggests a growing interest in organoid applications in COVID‐19 research.

2.3. The most contributing countries/regions and organizations

Up to 45 countries or regions with publications related to organoid applications for SARS‐CoV‐2 infection. Figure 1C presents the top 10 contributing countries or regions in this field. As indicated, the United States ranks first with 101 publications and has the highest H‐index (25), which was normally utilized to evaluate the publication number and citation performance. ³⁵ , ³⁶ China (54 articles, 16) and Germany (28 articles, 11) rank second and third place, respectively. England, the Netherlands and India also are influential countries/regions in this field. Interestingly, in terms of average citations (citations per publication), Canada ranks first with 126.55 average times. The Netherlands (78.33) and England (50.06) rank second and third place, respectively. Regarding the most contributing organizations, 667 institutes around the world were identified associated with organoid‐based COVID‐19 study. As shown in Table S1, the University of California System and the Chinese Academy of Science rank first and second in terms of publication numbers, with 21 and 15 publications, respectively. The H‐indexes of the University of California System and the University of California San Diego are also in the leading place, reaching 12 and 9, respectively. Although the publications from the Karolinska Institutet put it in ninth place, it reached the highest average citations of 165.22. These top 10 contributing organizations are mainly from the United States, China and European countries. Taken together, scientists from the United States, China and European countries/regions paid great efforts in the application of organoids for SARS‐CoV‐2 infection.

2.4. Co‐citation analysis of references and sources

Co‐citation analysis will be helpful for the reader to know the frequency of co‐cited references and journals and can promote the development of the disciplines. ³⁷ , ³⁸ Hence, VOSviewer was applied to visualize the corresponding journals in the field of organoid applications in COVID‐19 research. A total of 69 journals were identified with more than 50 co‐cited papers, as shown in Figure 2A. The intensity of hotspots indicates the frequency of occurrences of that item. Warm colours represent hot items and cool colours represent cool items. The top 10 contributing journals based on citations are Nature, Cells, Science, Cell Stem Cell, New England Journal of Medicine, Proceedings of the National Academy of Sciences of the United States of America, Nature Communications, the Journal of Virology, Nature Methods and Lancet. In terms of co‐cited references, the top 5 references were listed in Figure 2B. The paper ‘SARS‐CoV‐2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor’ (https://doi.org/10.1016/j.cell.2020.02.052) was published in Cell in 2020 and received the highest co‐citation (119 times) in this field. The authors provided critical insights into the early stage of SARS‐CoV‐2 infection, which depends on the host cell factor ACE2 and co‐factor TMPRSS2 (transmembrane serine protease 2), thus identified potential targets for protection against the virus. ³⁹ Although the study did not focus on organoids, this groundbreaking will help decipher the pathomechanism of viral infection and drug candidates screening for COVID‐19 therapeutics. The flowing four publications were closely associated with human intestinal, kidney, liver, lung and colonic organoids, respectively. ¹¹ , ⁴⁰ , ⁴¹ , ⁴² These data indicate that human organoids contribute to the COVID‐19 study.

FIGURE 2.

Co‐citation analysis of the publications regarding organoid application for COVID‐19 study. (A) The density visualized network of the contributing journals (more than 50 citations). The intensity of hotspots indicates the frequency of occurrences of that item. Warm colours represent hot terms and cool colours represent cool items. (B) The top 5 co‐cited references for organoid‐based COVID‐19 research.

2.5. The hotspots of in organoid‐based COVID‐19 field

To better reveal the core topic in the organoid‐based COVID‐19 field, keyword co‐occurrence analysis was performed by the VOSviewer application. As shown in Figure 3, a total of 117 keywords were identified within the visualized network with a frequency of more than 3. The top 5 most frequently emerging keywords are ‘sarce‐cov‐2’ (110 co‐occurrence times), ‘covid‐19’ (99), ‘organoids’ (65), ‘ace2’ (50) and ‘infection’ (49). All 117 keywords were classified into eight clusters with distinct colours. The largest cluster, red colour, involved keywords associated with stem cells and disease models, such as ‘airway organoids’, ‘lung organoids’, ‘epithelial‐cells’, ‘transmission’, ‘disease’ and ‘virus‐infection’; Cluster 2 (green) involved in keywords associated with drug discovery and viral replication, such as ‘inhibitors’, ‘inhibition’, ‘resistance’ and ‘expression’; Cluster 3 (blue) involved in keywords associated with viral cell entry and virus–host interaction, such as ‘spike protein’, ‘entry’, ‘receptor’, ‘ace2’ and ‘tmprss2’; Cluster 4 (yellow) involved in keywords related to SARS‐CoV‐2 infection and immune response, such as ‘immunity’, ‘responses’, ‘t‐cells’ and ‘airway epithelium’; Cluster 5 (purple) involved in keywords associated with neural infection, such as ‘brain organoids’, ‘choroid‐plexus’, ‘neural progenitors’, ‘tau’ and ‘sars‐cov‐2 neurotropism’; and other clusters are associated with ‘stem cells’, ‘air–liquid interface’ and ‘intestinal organoids’.

FIGURE 3.

Keyword co‐occurrence (at least three times) network visualization of the research of organoids for SARS‐CoV‐2 infection. Node size indicates the occurrence frequency; node colour represents the cluster; cluster resolution = 1.00.

To obtain the emergence of keywords over time, the overlay visualization of keywords regarding organoid‐based COVID‐19 research was presented in Figure S1, a derivative of Figure 3. As indicated, the node colour stands for the average publication year (purple means that keywords occurred earlier and marked with yellow stands that keywords are vigorous recently). For example, keywords, such as ‘virus‐infection’, ‘sars‐cov‐2 infection’, ‘replication’, ‘airway’, ‘airway epithelium’, ‘acute respiratory syndrome’, ‘pneumonia’ and ‘ace2’ are active in the early era. This suggests more studies focused on pulmonary infection using organoid models at the early stages of the COVID‐19 pandemic. Recently, keywords included ‘drug repurposing’, ‘personalized medicine’, ‘repair’, ‘therapy’, ‘resistance’ and ‘therapeutics’ are more popular. Therefore, more strategies are urgently demanded for SARS‐CoV‐2 therapeutics based on organoid technology.

3. HUMAN ORGANOIDS FOR STUDYING SARS‐CoV‐2 INFECTIVITY

Human organoids are in vitro 3D tissues formed by the self‐assembly of human stem cells. Stem cell‐derived EBs can generate derivatives of three embryonic germ layers, including ectoderm, mesoderm and endoderm and thus various organoids are generated. Given human organoids can mimic the key features of tissues and organs in the human body, such as cell diversity, structural and functional properties and physiological microenvironment, organoid platforms have been applied for studying SARS‐CoV‐2 infectivity.

3.1. Respiratory organoids

The human airways comprise the nasal cavity, proximal and intermediate airways, respiratory bronchioles and alveoli. ⁴³ The airway epithelium is the primary target of SARS‐CoV‐2, which causes severe cough, excessive mucous production, shortness of breath, chest tightness and wheezing. ² , ⁴⁴ Studies regarding viral lung infection appeared relatively earlier in the early stages of the COVID‐19 pandemic (Figure S1). ACE2 and TMPRSS2, the key host proteins that promote cellular entry of SARS‐CoV‐2, were highly expressed on the lung epithelium. ⁴⁵ To date, nasal, bronchial, bronchioalveolar and alveolar organoids have been generated to investigate SARS‐CoV‐2 infectivity (Figure 4A–C). ² , ⁴⁸ , ⁵⁰ , ⁵¹ , ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ The upper airway, particularly the nasopharyngeal epithelium, is the entry portal and primary site for SARS‐CoV‐2 replication and transmission. ⁵¹ , ⁵⁶ , ⁵⁷ Recently, nasal epithelial cells procured via a non‐invasive procedure was used to generate 3D differentiated nasal organoids that adequately simulate the native nasal epithelium, accommodating all airway epithelial cell types, such as ciliated, basal, goblet and club cells. ⁵¹ The differentiated nasal organoids accurately recapitulated the differential infectivety of emerging variants. Moreover, the study revealed the potentially viral pathogenesis was closely associated with ciliary damage and tight junction disruption. ⁵¹

FIGURE 4.

Human lung and brain organoids for modelling SARS‐CoV‐2 infection. (A) Generation of human airway organoids (hAWOs) and human alveolar organoids (hALOs) from hESCs for SARS‐CoV‐2 infection. Reproduced with permission. ⁴⁶ Copyright 2020, Oxford University Press. (B) Generation of apical‐out distal lung organoids for modelling SARS‐CoV‐2 infection. Reproduced with permission. ⁴⁷ Copyright 2020, Springer Nature. (C) Human bronchial organoids were infected by SARS‐CoV‐2. Reproduced with permission. ⁴⁸ Copyright 2022, Springer Nature. (D–F) hiPSC‐derived cortical, hippocampal, hypothalamic, midbrain organoids (D,E) and choroid plexus organoids (F) could be infected by SARS‐CoV‐2. Reproduced with permission. ⁴⁹ Copyright 2020, Elsevier.

hPSC‐derived airway organoids (AWOs), containing functional multi‐ciliated cells, basal cells, mucus‐producing secretory cells and CC10‐secreting club cells, were also used to monitor SARS‐CoV‐2 infection, showing ciliated and club cells were infected. ⁴⁶ , ⁵⁸ , ⁵⁹ In the human lung, ciliated cells are decreased while club cells are increased from the proximal to the distal airway. ⁴⁶ Therefore, it was speculated that SARS‐CoV‐2 sequentially infected ciliated and club cells along the upper airway down to the alveoli. Moreover, SARS‐CoV‐2 infection could cause metabolic disorders, as illustrated by downregulated lipid metabolism and upregulated glycolysis. ⁴⁶ , ⁶⁰ The data of RNA‐sequence (RNA‐seq) of infected AWOs showed the upregulation of cytokine/chemokine signalling, which was consistent with exuberant inflammatory cytokine production in human COVID‐19 pulmonary infections. ⁶⁰ , ⁶¹ In this case, AWOs thus provide a promising platform for anti‐viral therapeutic drug discovery. Moreover, human bronchial organoids (BCOs), containing transient secretory, goblet and ciliated cells, were generated for SARS‐CoV‐2 research. ⁴⁸ , ⁶² The study from Sano's group proved that SARS‐CoV‐2 efficiently infected ciliated cells in BCOs, whereas not basal cells. It is probably due to ciliated cells expressing ACE2, but not basal cells, or club cells could dedifferentiate into basal cells. ⁴⁸ , ⁶³ The differentially expressed genes caused by viral infection were closely associated with immune response and cytokines. ⁶⁴

In addition to nasal organoids and AWOs, alveolar lung organoids (ALOs) were also used for SARS‐CoV‐2 studies. The ALO consists of type I cells (AT1) and alveolar type II cells (AT2), which functionally mimic the alveolar epithelium. ⁶⁵ hPSC‐derived ALOs have been applied to reveal the alveolar infection of SARS‐CoV‐2, showing that AT2 cells were permissive to SARS‐CoV‐2 infection. ⁴⁰ , ⁴⁷ , ⁶⁶ Given the complicated protocol and less reproducibility of PSC‐derived lung organoids, more ALOs have been established from purified primary alveolar stem cells. ⁶⁷ , ⁶⁸ For example, Salahudeen and colleagues established human distal lung organoids, including AT2 organoid and basal organoid, for exploring SARS‐CoV‐2 infection. ⁴⁷ The organoids with apical‐out polarity presenting ACE2 promoted viral infection in AT2, basal and club cells. Similarly, Chiu et al. proved that two‐dimensional (2D) ALOs sustained productive SARS‐CoV‐2, but the virus replicative fitness was lower than that in 2D AWOs. ⁶⁵ This could be due to more ACE2⁺ cells in AWOs compared to ALOs. This study suggests that higher viral transmissibility could occur in the lung airway than in the alveoli. More interestingly, it was reported that the Omicron variant only occurred in AWOs, not in ALOs. ⁶⁵ Consistently, researchers from the LKS Faculty of Medicine at the University of Hong Kong (HKUMed) proved that Omicron SARS‐CoV‐2 could infect and spread faster than ancestral strains in human bronchus but with lower severity in the lung. ⁶⁹ Another study from Chiu et al. showed that the differentiated nasal organoid monolayers accurately reproduced the highest transmissibility of the Omicron variant. ⁵¹ Moreover, the Omicron's replicative advantage was more remarkable in the nasal organoid monolayer than that in the AWOs. This evidence suggests that nasal organoids could more adequately recapitulate the variable replication capacity and transmissibility of emerging SARS‐CoV‐2 variants than AWOs.

3.2. Brain organoids

Besides respiratory insufficiency, existing studies have highlighted severe neurological complications in COVID‐19 patients, ranging from headache and loss of smell to confusion and disabling strokes. ⁷⁰ , ⁷¹ Despite viral RNA being detected in patient‐derived brain samples, it remains unclear the neurotropism of SARS‐CoV‐2 and its potential pathogenesis. ⁷² , ⁷³ , ⁷⁴ Given the great potential to elucidate the relationship between ZIKV infection and microcephaly, brain organoids show great promise for understanding the neurotropism and neurotoxic effects of SARS‐CoV‐2. ²⁵ , ⁶⁶ At the early onset of the COVID‐19 pandemic, SARS‐CoV‐2 (NRW‐42) isolated from a nasopharyngeal and oropharyngeal swab specimen of infected patients was found to enter human organoids with 2‐day viral exposure but did not appear to actively replicate. ⁷⁵ The study also showed that SARA‐CoV‐2 preferred relatively mature neurons in brain organoids. For instance, mature cortical neurons in the brain organoids were susceptible to SARA‐CoV‐2 exposure. ⁷⁵ , ⁷⁶ However, this is indeed markedly inconsistent with ZIKV infection, which was found to preferentially target neural progenitor cells and trigger them to premature differentiation. ²⁵ , ²⁶ , ⁷⁷ The authors also proved that SARS‐CoV‐2 infection was related to aberrant Tau localization from axons to soma and hyperphosphorylation, which might lead to neuronal death. ⁷⁵ High‐density viral regions have more apoptotic cells compared with the low‐density viral area. ⁷⁶ Moreover, SARS‐CoV‐2 infection was accompanied by metabolic alteration in infected and neighbouring neurons, which may create a resource‐restricted environment for neural cells.

Recently, region‐specific brain organoids, including cortical, hippocampal, hypothalamic and midbrain organoids, as well as choroid‐plexus organoids (CPOs) have been established to systematically test SARS‐CoV‐2 neurotropism (Figure 4D–F). ⁴⁹ , ⁷⁸ The study from Jacob's team showed that the virus exhibited high infectivity of choroid plexus epithelial cells while limited affinity with neurons and astrocytes. ⁴⁹ The transcriptional alteration in infected CPOs showed that SARS‐CoV‐2 caused inflammatory responses and damaged the choroid plexus, indicating that the choroid plexus epithelium might be a potential gateway for viral entry to the brain and contribute to neural infection. Consistently, Pellegrini et al. proved that SARS‐CoV‐2 could infect choroid plexus epithelial cells of the brain but not neurons, leading to a potential breakdown of blood‐cerebrospinal fluid barrier integrity and development of neurological complications. ⁷⁹ This could be closely associated with neural progenitors and neurons do not specifically express SARS‐CoV‐2 entry factors or co‐factors, ACE2 and TMPRSS2. ⁷¹ , ⁷⁹ , ⁸⁰ Conversely, in line with ZIKV infection, Zhang and colleagues proved that ACE2 and TMPRSS2 expressed in neural progenitor cells and SARS‐CoV‐2 directly infected neural progenitor cells in brain organoids. ⁸¹ Moreover, Wang et al. demonstrated that SARS‐CoV‐2 infected hiPSC‐derived neurons, astrocytes and brain organoids, especially ApoE4 astrocytes exhibited high susceptibility to SARS‐CoV‐2. ⁸² These discrepancies could be caused by the difference in differentiation protocol and differential neuronal phenotypes regulating SARS‐CoV‐2 susceptibility. These findings unambiguously demonstrate that brain organoids provide great promising platforms for probing the neurotropic features and neurotoxic effects of SARS‐CoV‐2.

3.3. Gastrointestinal organoids

Multiple studies have reported that COVID‐19 patients developed gastrointestinal symptoms, such as diarrhoea, vomiting, or abdominal pain, ¹¹ , ⁸³ , ⁸⁴ , ⁸⁵ suggesting that gastrointestinal tracts are considered targets of SARS‐CoV‐2. Given live virus was detected in the faeces of COVID‐19 patients, SARS‐CoV‐2 could be transmitted through the faecal‐oral route. ⁸⁴ , ⁸⁶ , ⁸⁷ Gastrointestinal organoids, such as intestinal organoids, colonic organoids and gastric organoids have been generated to unravel the susceptibility of gastrointestinal tracts to SARS‐CoV‐2 (Figure 5A). ⁴⁰ , ⁸⁷ , ⁸⁸ , ⁹³ , ⁹⁴ , ⁹⁵ Human and bat intestinal organoids were successfully generated by Zhou's group and first applied for modelling enteric SARS‐CoV‐2 infection. ⁹⁶ Active viral replication was found in intestinal organoids, which contain enterocytes, goblet cells, Paneth cells and enteroendocrine cells. The study showed that enterocytes were the main targets of SARS‐CoV‐2 probably due to ACE2 and TMPRSS2 being highly expressed in differentiated enterocytes. This finding is consistent with the data from Zhang's study, showing active SARS‐CoV‐2 replication in ACE2⁺ human mature enterocytes. ⁹⁷ The authors also proved that TMPRSS2 and TMPRSS4 facilitated SARS‐CoV‐2 entry into host cells by promoting viral spike fusogenic activity. Enterocytes in human colonic organoids were also sensitive to SARS‐CoV‐2 infection. ⁴⁰ Promoting enterocyte differentiation in the organoid could increase ACE2 expression. ⁹⁸ RNA‐seq analysis proved that viral infection in colonic organoids involved cytokines and chemokines signalling pathways. ⁴⁰

FIGURE 5.

Human organoids for modelling SARS‐CoV‐2 infection. (A) Generation of human intestinal organoids for modelling SARS‐CoV‐2 infection. Reproduced with permission. ⁸⁸ Copyright 2021, Elsevier. (B) SARS‐CoV‐2 infected liver organoids and biliary organoids. Reproduced with permission. ⁸⁹ Copyright 2022, BMJ Publishing Group Ltd. (C) Generation of human kidney organoids for modelling SARS‐CoV‐2 infection. Reproduced with permission. ⁴² Copyright 2020, (D) Diabetic conditions enhanced SARS‐CoV‐2 infections in human kidney organoids. Reproduced with permission. ⁹⁰ Copyright 2022, Elsevier. (E) SARS‐CoV‐2 infected and replicated in retinal organoids. Reproduced with permission. ⁹¹ Copyright 2022, Elsevier. (F) SARS‐CoV‐2 infected salivary gland organoids. Reproduced with permission. ⁹² Copyright 2022, Springer Nature.

Apart from intestinal and colonic organoids, gastric organoids are also susceptible to SARS‐CoV‐2 infection. Giobbe et al. generated gastric organoids from fetal, pediatric and adult biopsies for modelling SARS‐CoV‐2 infection and demonstrated that late fetal and pediatric organoids allowed higher viral replication. ⁹³ This could be caused by the lower levels of ACE2 and TMPRSS2 in the undifferentiated organoids, including early fetal and adult organoids, compared to organoids following differentiation. Consistently, several studies showed that differentiated intestinal organoids exhibited higher susceptibility to viral infection than undifferentiated ones. ⁹⁵ , ⁹⁹ SARS‐CoV‐2 infection was also found to induce mild innate antiviral responses, particularly involved in the interferon family in late fetal and pediatric organoids as illustrated by the transcription analysis. Likewise, single‐cell RNA‐seq (scRNA‐seq) analysis of viral‐infected intestinal organoids highlighted infected cells activated strong proinflammatory events and induced interferon production. ¹⁰⁰ These data indicate that human gastrointestinal organoids serve as a valuable platform for SARS‐CoV‐2 gastrointestinal infection and the development of therapeutic strategies.

3.4. Liver organoids

As epidemiologic studies reported that COVID‐19 patients had abnormal liver function mainly involved in hepatocytes and cholangiocytes, thus human hepatocyte and cholangiocyte organoids were used to validate these findings. ¹⁰¹ , ¹⁰² , ¹⁰³ ACE2 and TMPRSS2 were expressed in hepatocytes and cholangiocytes, making cells highly vulnerable to SARS‐CoV‐2 infection. ¹¹ , ¹⁰⁴ Therefore, viral replication was observed in both hepatocyte and cholangiocyte organoids after viral infection. ¹¹ Meanwhile, the SARS‐CoV‐2 pseudo‐entry virus was found to infect hepatocytes. The SARS‐CoV‐2 infection activated cellular cytokine–cytokine receptor interaction, IL‐17, chemokine, TNF (tumor necrosis factor) and NF‐κB signalling pathways and downregulated metabolism in infected organoids. ¹¹ Similarly, Zhao et al. revealed activated TNF signalling and apoptosis pathways in the infected liver and biliary organoids, implying that SARS‐CoV‐2 infection might trigger cell death of hepatocytes and cholangiocytes (Figure 5B). ⁸⁹ A subset of human‐specific ACE2⁺/TMPRSS2⁺ cholangiocytes was identified in human liver ductal organoids, thus the organoids were extremely susceptible to SARS‐CoV‐2, leading to robust viral replication was detected in organoids. ¹⁰⁴ Furthermore, SARS‐CoV‐2 exposure caused the disruption of the barrier and bile acid transporting functions of cholangiocytes through the dysregulation of gene expression, which was closely associated with a tight junction formation and bile acid transportation. Therefore, SARS‐CoV‐2 infection might induce cholangiocyte injuries and consequent bile acid accumulation in COVID‐19 patients.

3.5. Kidney organoids

Kidney failure was also frequently observed in COVID‐19 patients. ¹⁰⁵ Recent studies detected viral RNA in urine samples of COVID‐19 patients. ¹⁰⁶ ACE2 was expressed in kidney proximal convoluted tubules, indicating that renal tubule cells could be potential targets of SARS‐CoV‐2. ¹⁰⁷ , ¹⁰⁸ Thus, 3D human kidney organoids containing proximal tubule epithelial cells were generated for SARS‐CoV‐2 infection (Figure 5C). ⁴² , ¹⁰⁹ Compared with traditional 2D culture, ACE2 was higher expressed in organoids, indicating the organoids would be more permissive to SARS‐CoV‐2. ¹⁰⁹ Apart from proximal tubular cells, podocytes and stromal cells were also infected by SARS‐CoV‐2 in iPSC‐derived kidney organoids, which was confirmed by the results from the scRNA‐seq analysis. ¹¹⁰ Activated profibrotic signalling was also found in infected kidney organoids and could be inhibited by a protease blocker. More recently, Garreta and colleagues established diabetic human kidney organoids that exhibited higher susceptibility to SARS‐CoV‐2 compared to non‐diabetic control (Figure 5D). ⁹⁰ This was linked to diabetic‐induced upregulation of ACE2 and metabolic programming. Therefore, kidney organoids are useful tools for investigating kidney complications for SARS‐CoV‐2 infection and looking for therapeutic drugs.

3.6. Other organoids

SARS‐CoV‐2 infection can cause cardiac injury and dysfunction in patients and increase the risk of mortality. ¹¹¹ , ¹¹² Human cardiac organoids and vascular organoids have been established to explore SARS‐CoV‐2 infectivity in the cardiovascular system. ⁴² , ¹¹³ , ¹¹⁴ Mils et al. demonstrated that infected cardiac organoids could recapitulate key clinical features of diastolic malfunction in COVID‐19 patients. ¹¹⁵ The study also showed that cytokine‐induced cardiac dysfunction in cardiac organoids could be attenuated by using bromodomain and extraterminal family inhibitors (BETi).

To determine the ocular involvement and potential virus–host ocular interactions, Eriksen et al. generated a whole‐eye organoid, including the retina, retinal pigment epithelium, ciliary margin, iris, lens and cornea, for SARS‐CoV‐2 infection. ¹¹⁶ The study showed that higher viral replication was detected in limbus than in other ocular surface cells in eye organoids. Moreover, retinal organoids also were used to investigate retinal SARS‐CoV‐2 infection (Figure 5E). ⁹¹ , ¹¹⁷ Although relatively low expression of ACE2 in the retina, the study proved that SARS‐CoV‐2 infected retinal organoids, replicated in retinal cells, such as retinal ganglion cells and photoreceptors and induced the expression of inflammatory genes, such as interleukin 33. ⁹¹

It was recently reported that saliva could be a potential source of SARS‐CoV‐2 transmission due to the virus being identified in the salary glands of COVID‐19 patients. ⁶⁰ Indeed, salivary glands express ACE2 and TMPRSS2 and serve as reservoirs of viruses. ⁶⁰ , ⁹² , ¹¹⁸ Tanaka and colleagues generated salivary gland organoids (SGOs) from hiPSCs to model SARS‐CoV‐2 infection of salary glands (Figure 5F). ⁹² The human SGOs mimicked human embryonic salary gland characteristics and functions and expressed viral entry factors and co‐factors, including ACE2 and TMPRSS2. Thus, viral infection and replication were found in organoids after exposure to SARS‐CoV‐2. Although SGO is a prospective model for studying SARS‐CoV‐2 infection, it is not yet clear how SARS‐CoV‐2 replicated in salivary glands was secreted into saliva.

4. HUMAN ORGANOIDS FOR VACCINE DEVELOPMENT OF SARS‐COV‐2

Currently, COVID‐19 is still spreading and posing threats to widespread health, society and economy. ¹¹⁹ Although, as of 30 November 2022, approximately 13 billion vaccine doses have been administered, more effective vaccines are urgently demanded to prevent further spread and keep people from contracting the virus. ¹ , ¹²⁰ , ¹²¹ The development of a novel vaccine is still a lengthy process, including the initial vaccine design, preclinical testing in animal models and clinical trials (phases I–IV), which could take 15 years or more, great efforts have been paid in vaccine development responding to the COVID‐19 pandemic. ¹²¹ , ¹²² According to the WHO, as of 6 December 2022, 175 candidate vaccines are under clinical testing for treating COVID‐19 and 199 candidate vaccines are in pre‐clinical studying. ¹²³ Of the 175 candidates, 49 and 11 are undergoing further validations of safety and efficacy in phase III and phase IV clinical trials with a large number of volunteers, respectively. The technology platforms can be divided into traditional whole virus vaccines (inactivated or live attenuated vaccines), recombinant protein‐based vaccines (protein subunit vaccines, virus‐like particles), viral vector vaccines and nucleic acid vaccines (DNA‐ and RNA‐based vaccines). Among them, protein subunits and RNA are mainly candidate vaccines. ¹²³

As viral infections are the prototypic species‐specific diseases and pronounced species‐specific differences in adaptive immunity, many candidate vaccines that worked in animals while failing in human trails. ¹²⁴ , ¹²⁵ , ¹²⁶ , ¹²⁷ Therefore, novel animal‐free test methods may accelerate the testing programs for drug and vaccine development. ¹²⁴ Although in vitro human organoids are devoid of the host immune system, organoid‐based assays could provide unique opportunities for studying the host–virus interaction and testing the efficacy and neutralizing antibodies of candidate vaccines. ⁴⁰ Several studies have proved that antibodies were effective in neutralizing SARS‐CoV‐2. For instance, Pei and colleagues evaluated the inhibitory effect of a neutralizing antibody CB6 on SARS‐CoV‐2 infection in human lung organoids. ⁴⁶ The study showed that CB6 notably suppressed viral replication in infected lung organoids. This was consistent with the previous study that reported CB6 repressed SARS‐CoV‐2 infection in rhesus monkeys. ¹²⁸ Moreover, a new tetravalent neutralizing antibody (15033‐7) and a synthetic dipeptidyl peptidase‐4 (DPP4) peptide were observed to reduce SARS‐CoV‐2 entry in human lung organoids and modulate innate immunity and inflammatory response. ¹²⁹ These data will further aid vaccine and drug development for COVID‐19. Advanced engineering approaches, such as bioreactor or chip‐based engineering devices, showing great promise to generate immune cell organoids, but they cannot recapitulate the complex of human adaptive immunity. ¹³⁰ , ¹³¹ , ¹³² Importantly, the presence of immune elements, including B cells, T cells and microphages in organoid models might facilitate vaccine development. ⁴⁰

Recently, Wagar et al. established human tonsil organoids (TOs) from reaggregating the dissociated primary human tonsils to evaluate human adaptive immune responses to the SARS‐CoV‐2 vaccine. ¹²⁶ The specialized germinal centre (GC)‐like structure with distinct T cells and B cells was identified in human TOs, which is essential to produce antigen‐specific antibodies, affinity maturation, B cell differentiation and class‐switch recombination (Figure 6A). Increased B cell differentiation and influenza‐specific antibody production were observed in organoids after exposure to live attenuated influenza vaccine. To clear the responses of individual cells to influenza, the authors analysed the changes in plasmablast differentiation and antigen‐specific antibody production after antigen‐presenting cell (APC), T cell and B cell subsets depletion. The results showed that naive B cells, APCs, CD4⁺, or CD45⁻ stromal cells were minimally needed to constantly maintain plasmablast differentiation and naive antibody responses. ¹²⁶ Subsequently, TOs from different donors were used to investigate the response to the adenovirus‐based SARS‐CoV‐2 vaccine. Increased plasmablast differentiation, CD8⁺ T cell activation, and IgG and IgA antibody production were observed in TOs after 14‐day stimulation. Therefore, human TO application in COVID‐19 could help decipher immune responses to viruses and accelerate vaccine design. ¹³⁴ Although this model was not lacking in immune components, it is worth noting that human organoids used for viral vaccine development are in isolation. The organoid model could not well mimic the immune cell migration from pathogen entry sites to the lymph nodes, tonsils, or spleen, where GCs form in humans. ¹²⁶ Future studies are needed to incorporate more methods to capture features of human adaptive responses. Moreover, safety considerations are the most important things for vaccine development. ¹²⁰ It remains to be seen whether new SARS‐CoV‐2 vaccines could trigger aberrant immune responses and induce potential multi‐organ injuries.

FIGURE 6.

Human organoids for vaccine development and drug discovery of SARS‐CoV‐2. (A) Generation of human tonsil organoids for SARS‐CoV‐2 infection and vaccine development. Reproduced with permission. ¹²⁶ Copyright 2021, Springer Nature. (B) SOF (sofosbuvir) could inhibit SARS‐CoV‐2 replication in brain cells and rescue neurological impairments in human brain organoids. Reproduced with permission. ¹³³ Copyright 2022, Public Library of Science. (C,D) QNHC (quinacrine dihydrochloride), imatinib, and MPA (mycophenolic acid) blocked the entry of SARS‐CoV‐2 in both hPSC‐derived lung organoids (C) and colonic organoids (D). Reproduced with permission. ⁴⁰ Copyright 2021, Springer Nature. (E,F) EK1 and remdesivir inhibited infection of human intestinal organoids with SARS‐CoV‐2. Reproduced with permission. ⁸⁸ Copyright 2021, Elsevier.

5. HUMAN ORGANOIDS FOR DRUG DISCOVERY OF SARS‐COV‐2

Given traditional cell lines cannot recapitulate the physiologically relevant kinetics of in vivo SARS‐CoV‐2 infection, human organoids provide promising models for drug discovery of COVID‐19. The respiratory system is the initial viral target, thus human lung organoids were used to test the efficacy of drugs and compounds, focusing on viral entry and replication. ¹³⁵ , ¹³⁶ For instance, Xu et al. demonstrated that inhibition of receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1 kinase) caused by RIPK1 inhibitor Nec‐1s reduced viral load and inflammatory responses in infected lung organoids. ¹³⁷ The results suggest that the function of RIPK1 in SARS‐CoV‐2 propagation and suppression of RIPK1 might provide a strategy for COVID‐19 therapeutics and prevention. Since ACE2 serves as the entry receptor, a humanized decoy antibody (ACE2‐Fc fusion protein) has been designed to target the interaction between ACE2 and viral spike protein. ¹³⁸ The study proved that ACE2‐Fc effectively blocked the entry of SARS‐CoV‐2 spike‐expressing pseudotyped virus into lung organoids, thus suppressing viral replication, suggesting that ACE2‐Fc provides a latent prospect for COVID‐19 treatment. Samuel and colleagues demonstrated that androgen (AR) signalling regulated the entry factors and co‐factors of SARS‐CoV‐2 directly, ACE2 in addition to TMPRSS2. ¹³⁹ Thus, drugs inhibiting AR signalling were found to reduce viral infection in hESC‐derived lung organoids. The study underscores the critical roles of AR signalling in viral infection and lays the foundation for antiandrogenic drug application in COVID‐19 therapeutics.

Primary human lung epithelial infection models, consisting of differentiated air–liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells, were applied to validate the efficacy of remdesivir, hydroxychloroquine, and interferon beta 1 (IFN‐β1) in anti‐SARS‐CoV‐2. ¹⁴⁰ The results confirmed that three molecules significantly suppressed the infection and replication of SARS‐CoV‐2. Remdesivir, an inhibitor of the viral RNA‐dependent, is the most promising FDA‐approved drug in clinical use for hospitalized COVID‐19 patients. ¹²⁸ , ¹⁴¹ Treatment of remdesivir inhibited viral replication and alleviated alveolar‐capillary barrier disruption on bioengineered human alveolus chip. ¹⁴² Similarly, Pei et al. demonstrated remdesivir inhibited viral production in AWO and ALO models. ⁴⁶ Since the role of interferon lambda (IFN‐λ) in the innate antiviral immunity in the respiratory tract, Lamers and colleagues proved that SARS‐CoV‐2 replication and dissemination were abrogated by low‐dose IFN‐λ1 treatment in the organoid‐derived bronchioalveolar model. ⁵⁴ The study from Katsura's group also proved that pre‐treatment with a low dose of IFN alpha (IFN‐α) and IFN gamma (IFN‐γ) blocked SARS‐CoV‐2 replication in alveospheres. ⁶⁷ Moreover, pre‐treatment with both IFN‐λ and IFN‐β1 was observed to significantly impaired SARS‐CoV‐2 infection in colonic organoids. ¹⁴³ These data indicate interferons will be a viable therapeutic option for COVID‐19. ¹⁴⁴ , ¹⁴⁵

Human airway microfluidic devices, containing two parallel microchannels, lung bronchial‐airway basal stem cells located in an ‘airway channel’ exposed to air and lung endothelium cultured on the opposite side (‘vascular channel’) exposed to continuous fluid perfusion, have been established to test the efficacy of antiviral drug candidates (Figure 7A). ¹⁴⁶ The airway chip faithfully recapitulated key features of influenza‐induced human lung responses and the anti‐influenza efficacy of Oseltamivir. Moreover, the ‘airway channel’ of the airway chip was exposed to SARS‐CoV‐2 pseudoparticles to simulate viral airborne infection, confirming that SARS‐CoV‐2 pseudoparticles could infect human airway epithelial cells efficiently. Subsequently, the ‘vascular channel’ was continuously perfused by candidate drugs to simulate circulatory distribution after oral administration, and the results showed that amodiaquine and its metabolite inhibited SARS‐CoV‐2 infection. ¹⁴⁶ Although amodiaquine is used to prevent and treat malaria, this medicine showed great potential in antiviral therapeutics and prophylactics.

FIGURE 7.

Building advanced human organoids by integrating novel strategies and technologies. (A) A human‐airway‐on‐a‐chip for studying viral infection. Reproduced with permission. ¹⁴⁶ Copyright 2021, Springer Nature. (B) SARS‐CoV‐2 infection in brain organoids with innately developing microglia. Reproduced with permission. ¹⁴⁷ Copyright 2022, Springer Nature. (C,D) Pericyte‐like cell‐containing cortical organoids were generated by the integration of pericyte‐like cells into cortical brain organoids and were prone to be infected by SARS‐CoV‐2. Reproduced with permission. ¹⁴⁸ Copyright 2021, Springer Nature. (E,F) SARS‐CoV‐2 induced intestinal responses with a biomimetic human gut‐on‐chip. Reproduced with permission. ¹⁴⁹ Copyright 2021, Science China Press. (G) The human multi‐organ‐on‐a‐chip concept. Reproduced with permission. ¹⁵⁰ Copyright 2011, Elsevier. (H). Generation multisensor‐integrated organs‐on‐chips platform. Reproduced with permission. ¹⁵¹ Copyright 2017, National Academy of Sciences. (I,J). Single‐cell RNA‐seq analysis of hPSC‐derived airway organoids. Reproduced with permission. ⁶⁰ Copyright 2021, Elsevier.

hPSC‐derived lung and colon organoids were also applied for high‐throughput screening FDA‐approved drugs, such as imatinib, mycophenolic acid, and quinacrine dihydrochloride (Figure 6C,D). ⁴⁰ The study showed that all three drugs could inhibit SARS‐CoV‐2 infection in lung and colon organoids in a dose‐dependent manner. RNA‐seq data highlighted that imatinib‐treated infected organoids were closely associated with lipid metabolism. Tiwari et al. found that EK1 peptide (spike protein inhibitor) and camostat/nafamostat (TMPRSS2 inhibitors) could block SARS‐CoV‐2 entry into lung organoids. ⁶⁶ This result was consistent with previous studies, showing the antiviral efficacy of EK1 and camostat mesylate. ³⁹ , ¹⁵² Pei et al. also proved camostat exerted a slight suppressive effect on viral infection in AWOs, but not in ALOs. ⁴⁶ A recent study reported that human lung organoids were also applied to explore the antiviral activity of CH25H. ¹⁵³ Mechanistically, CH25H converted cholesterol to 25‐hydroxylase (25HC), triggering cholesterol depletion on the plasma membrane and affecting virus–cell membrane fusion, resulting in inhibiting viral entry. As 25HC is a natural product without toxicity at effective concentrations, it provides a potential antiviral agent to fight against SARS‐CoV‐2 infection.

As SARS‐CoV‐2‐infected intestinal organoids exhibited the disruption of intestinal integrity and organoid deterioration, mimicking SARS‐CoV‐2‐induced gastrointestinal damage, intestinal organoids were used to test drug efficacy in COVID‐19 patients. ⁸⁸ A study has shown that remdesivir could effectively inhibit SARS‐CoV‐2 infection dose‐dependently at low micromolar concentrations. However, famotidine did not impact viral infection and spread in intestinal organoids, which was inconsistent with a previous retrospective cohort study that proved famotidine use would be related to alleviative clinical outcomes in hospitalized COVID‐19 patients. ¹⁵⁴ Similar to lung organoids, EK1 peptide could inhibit viral infection of intestinal organoids (Figure 6E,F). ⁸⁸ Interferon‐inducible transmembrane (IFITM) proteins are highly expressed in intestinal organoids and function as entry co‐factors for efficient SARS‐CoV‐2 infection. ¹⁵⁵ Thus, IFITM‐derived peptides or antibodies targeting the N‐terminus of IFITMs were found to strongly reduce viral entry and replication in gut organoids.

A recent study showed that ACE2 expression was regulated by the farnesoid X receptor (FXR) signalling in the gastrointestinal and respiratory systems. ¹⁵⁶ Therefore, suppression of the FXR signalling by the off‐patent drug ursodeoxycholic acid or the over‐the‐counter compound z‐guggulsterone downregulated the expression of ACE2, subsequently reducing susceptibility to SARS‐CoV‐2 infection in cholangiocyte, airway, and intestinal organoids. The study identified a new regulation of ACE2 through the FXR signalling and provided a new therapeutic target for COVID‐19. More recently, clinical‐grade human recombinant soluble ACE2 (hrsACE2) has been found to inhibit SARS‐CoV‐2 infections of human kidney and capillary organoids dose‐dependently. ⁴² Similarly, Wysocki et al. also proved that a novel soluble ACE2 variant, consisting of 618 amino acids fused with an albumin binding domain (ABD), could neutralize SARS‐CoV‐2 in human kidney organoids. ¹⁵⁷ More intriguingly, Monteil and colleagues studied the additive effects of combination treatment with hrsACE2 and remdesivir on infected kidney organoids. ¹⁵⁸ The data showed that combining both drugs at low doses caused a notably reduced viral production. This finding lays the foundations for combinatorial regimes in future COVID‐19 clinical trials. MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase, also exhibited a high potency of anti‐SARS‐CoV‐2 activity in hESC‐derived kidney organoids. ¹⁵⁹

A study conducted by Mesci's group showed that SARS‐CoV‐2 infected brain organoids and caused neuron death, which was accompanied by excitatory synapses in neurons. ¹³³ SARS‐CoV‐2 regulated specific gene expression in neurons implicated in antigen presentation, viral entry via the endocytic pathway, neuronal projection development, oxidative stress, and the complement pathway. Treatment with sofosbuvir, a FDA‐approved antiviral drug, efficiently repressed SARS‐CoV‐2 replication and rescued viral‐induced neuronal damage in infected brain organoids (Figure 6B). ¹³³ In addition to lung and intestinal organoids, remdesivir was also found to effectively suppress SARS‐CoV‐2 infection and rescue disease phenotypes in neurons and astrocytes. ⁸² Therefore, brain organoids also provide invaluable in vitro tools for COVID‐19 drug discovery and validation. Moreover, the therapeutic efficacy of remdesivir against SARS‐CoV‐2 was also validated in the tonsil organoid model. ¹⁶⁰

6. CHALLENGES AND FUTURE CONSIDERATIONS

Although human organoids provide valuable platforms for deciphering the pathology of SRAS‐CoV‐2 infection and looking for effective vaccines and potential treatments, these models still have several problems that need to be addressed. One major remaining challenge is the absence of cellular complexity and cell–cell communications, thus organoid microenvironment cannot mimic in vivo organ situations because of lacking vascular, neural, and immune cells. COVID‐19 caused by SARS‐COV‐2 infection could induce multiple organ failure, whereas conventional organoid models are devoid of physiological relevance and organ‐organ interaction. ¹⁶¹ , ¹⁶² Therefore, advanced methods and technologies are urgently needed to improve organoid advancement.

6.1. Immune vascular organoids

The host immune system plays a pivotal role in anti‐SARS‐CoV‐2. ¹⁶³ Given most organoids lack immune cells, human organoids co‐culturing with immune cells have been applied to investigate the pathophysiology of COVID‐19 involved with immune responses. Recently, Zhang's group established a biomimetic human alveolus chip to recapitulate the alveolar‐capillary barrier by coculture of the alveolar epithelial cells, microvascular endothelial cells, and circulating immune cells with perfusing media flow. ¹⁴² Notably, SARS‐CoV‐2 caused immune cell recruitment and endothelium detachment and promoted inflammation, indicating the critical roles of immune cells in SARS‐CoV‐2‐induced lung alveolar‐capillary barrier injury and inflammatory response. Another study generated an immune‐cardiac coculture system containing cardiomyocytes and macrophages to study macrophage‐mediated host cell responses to SARS‐CoV‐2 infection. ¹⁶⁴ The results proved that macrophages induced reactive oxygen species production and apoptosis in cardiomyocytes after viral exposure. In this case, ranolazine and tofacitinib were then found to alleviate the macrophage‐induced cardiotoxicity. As astrocytes are permissive to SARS‐CoV‐2, brain organoids co‐culturing with astrocytes might boost the viral infection rate. ⁸² Microglia, the resident immune cells of the central nervous system, play vital roles in brain development, homeostasis, and diseases, incorporating microglia into neural organoids provides unprecedented opportunities to study infective neurological disorders. ¹⁶⁵ , ¹⁶⁶ , ¹⁶⁷ , ¹⁶⁸ , ¹⁶⁹ For instance, upon ZIKV infection, microglia became activated and targeted neurons and excessively pruned synapses in microglia‐containing brain organoids. ¹⁶⁸ Similarly, Samudyata et al. established a protocol for generating a brain organoid model with innately developing microglia for studying SARS‐CoV‐2 infection (Figure 7B). ¹⁴⁷ The authors proved that viral infection induced neuronal cell death and increased microglial engulfment of postsynaptic termini in infected microglia‐containing brain organoids. ScRNA‐seq analysis showed that upregulation of interferon‐responsive genes could promote microglia‐mediated synapse elimination secondary to SARS‐CoV‐2 exposure, subsequently causing disruption in circuit integrity, leading to potential cognitive impairments. The data indicate that microglia contribute to SARS‐CoV‐2‐induced neural damage. ¹⁴⁷ , ¹⁷⁰ , ¹⁷¹ , ¹⁷² Thus, microglia‐containing brain organoid models may provide an opportunity for studying SARS‐CoV‐2‐induced neuronal damage and preventing COVID‐19 neurological sequelae.

Notably, vascular networks play critical roles in organogenesis and organ identity. ¹⁷³ , ¹⁷⁴ Currently, evidence is accumulating regarding how to generate vascularized human organoids. ¹⁷⁵ , ¹⁷⁶ , ¹⁷⁷ Therefore, human organoid vascularization would promote their applications in studying SARS‐CoV‐2 infection. For instance, as brain pericytes are proposed as SARS‐CoV‐2 infection points, Wand et al. established pericyte‐like cell (PLC)‐containing cortical organoid (PCCO) ‘assembloid’ through the integration of PLCs into cortical organoids to model SARS‐CoV‐2‐induced neuropathology (Figure 7C,D). ¹⁴⁸ PLCs were found to promote astrocytic maturation and capture the features of PLC–basement membrane–astrocyte structure, mimicking human brain pericyte functions in vivo. Compared with conventional cortical organoids, PCCOs exhibited higher susceptibility to SARS‐CoV‐2 due to PLCs within PCCOs serving as viral ‘replication hubs’, promoting virus cell‐to‐cell spread. ¹⁴⁸ The study indicates that incorporating neurovascular unit components with brain organoids could provide a promising platform for modelling neural infection of SARS‐CoV‐2. As ACE2 is expressed throughout the vasculature of the body, virus–host interactions on the vascular barrier could contribute to virus access to multiple organ systems. ¹⁷⁸ , ¹⁷⁹ To date, 3D microfluidic chips modelling the human blood–brain barrier, ¹⁷⁹ respiratory vascular barrier, ¹⁸⁰ and intestinal epithelium–vascular endothelium barrier (Figure 7E, F) ¹⁴⁹ have been established to investigate the effect of SARS‐CoV‐2 on the endothelial barrier. Consistently, SARS‐CoV‐2 was found to disrupt vascular endothelial adherent junctions.

6.2. Multi‐organoids on a chip

In recent years, the microphysiological system, named the ‘human‐on‐a‐chip’ platform, provides specific compartments for multiple organoid interconnections through microchannels (Figure 7G). ¹⁵⁰ , ¹⁸¹ The fluidic flow in the chip is analogous to the vasculature through the organ and tissue residence time. ¹⁸² , ¹⁸³ Compared with ‘single‐organoid‐on‐a‐chip’ (single‐OoC) models, ‘muti‐organoid‐on‐a‐chip’ (multi‐OoC) models present advantages in providing a systemic approach to decipher cross‐organ communications. ¹⁸⁴ Multi‐OoC platforms could be classified into two groups, namely coupling of single‐OoC units or integration of multiple organoids into one dish. However, the latter offers higher throughput and is more valuable in identifying potential biomarkers and therapeutic targets when compared to the former. ¹⁸⁴ Multi‐OoCs hold great promise for systemic assessment of SARS‐CoV‐2 infection, determining organ susceptibility, and looking for anti‐viral therapeutic drugs. Indeed, SARS‐CoV‐2 infection presents multi‐organ injuries and acts at a high level of organ‐organ interaction. ¹⁶¹ , ¹⁶² Therefore, a multi‐organoid system could help to study the inter‐organ communications within organs after SARS‐CoV‐2 infection and the secondary effects of the infected organ on another organ. Since multi‐OoCs have been widely applied for toxicity assessment, thus multi‐OoC platforms could be used for the safety evaluation of COVID‐19 vaccines and drugs. ²⁴ , ¹⁸⁵ Moreover, muti‐OoCs provide promising models to evaluate the complex process of drug absorption (intestine), organ distribution (along with blood circulation), drug metabolism (liver), and excretion (kidney), as well as potentially reduce the cost of preclinical animal models before conducting clinical trials.

Although the muti‐OoC system has shown enormous potential as an advanced organoid model, muti‐OoC technology is still in its infancy and maintains scientific and technical challenges. First, it is important to optimize the culture medium for co‐culturing different organoids due to tissue‐specific small molecule and growth factor requirements through the differentiation process in guided stem cell differentiation. The culture medium should be provided to meet the needs of each organoid. Noteworthy, the flow rate of the medium needs to be optimized to ensure that sufficient nutrients and oxygen meet the requirements of the next organoids. Moreover, it is necessary to consider that the metabolites generated by the organoid could be toxic to other organoids through the distribution of flow. ¹⁸⁶ Therefore, the OoC platform needs a sophisticated programme for nutrient‐waste management. Second, highly integrated monitoring platforms could be used to real‐time monitor cellular activity and organoid behaviours, such as cell vitality, metabolites, inflammatory cytokines, and electrophysiology. ¹⁸⁷ , ¹⁸⁸ Multisensor‐integrated OoC platform will enhance their performance in drug screening by providing biomarker profiling (Figure 7H). ¹⁵¹ To overcome the high variability of organoids, multi‐organoids derived from the same hiPSC lines could resolve this problem and provide opportunities for an accurate model for personalized prediction and treatment. ¹⁸¹ To date, further improvements are still demanded until multi‐OoCs become a standard COVID‐19 evaluation tool.

6.3. Multi‐omics analysis

The increased resolution of scRNA‐seq technologies has led to great breakthroughs and improved our understanding of cellular antiviral responses, such as hepatitis B virus (HBV), ¹⁸⁹ , ¹⁹⁰ Ebola virus, ¹⁹¹ influenza virus, ¹⁹² , ¹⁹³ rabies virus, ¹⁹⁴ human immunodeficiency virus (HIV), ¹⁹⁵ , ¹⁹⁶ ZIKV, ¹⁹⁷ , ¹⁹⁸ , ¹⁹⁹ and SARS‐CoV‐2. ¹⁸⁹ , ²⁰⁰ , ²⁰¹ , ²⁰² , ²⁰³ , ²⁰⁴ , ²⁰⁵ , ²⁰⁶ , ²⁰⁷ scRNA‐seq enables transcriptome‐wide expression profile at a single‐cell resolution and provides a powerful method to investigate virus–host and host–host interactions and intercellular communication networks upon viral infection. ²⁰⁸ , ²⁰⁹ , ²¹⁰ Previous evidence has shown that early scRNA‐seq technology has been applied to investigate the expression profiling of the SARS‐CoV‐2 entry factors and co‐factors, such as ACE2, TMPRSS2 and FURIN. ⁴⁵ , ²¹¹ , ²¹² , ²¹³ As human organoids contain multiple differentiated cell types and allow to explore cell–cell interactions, organoid platforms combined with scRNA‐seq can be used to identify the gene expression of entry factors and co‐factors in diverse cell phenotypes. ²¹⁴ , ²¹⁵ For instance, Duan and colleagues proved that scRNA‐seq analysis validated that ACE2, TMPRSS2, and FURIN were expressed in the ciliated‐like cells, allowing SARS‐CoV‐2 entry in AWOs (Figure 7IJ). ⁶⁰ Similarly, the results of scRNA‐seq also showed that ACE2, TMPRSS2, and FURIN were enriched in the AT2‐like cell population in lung organoids. ⁴⁰ It is worth noting that the data from scRNA‐seq require to be validated by immunostaining and flow cytometry analyses. Although scRNA‐seq has gained tremendous attention, challenges in high sample preparation cost, severe batch effects, time‐consuming process, numerous sample requirements, and relatively low‐throughout manners limited the widespread adoption and scope of scRNA‐seq. ²¹⁰ , ²¹⁶ The existing strategies for sample multiplexing for scRNA‐seq with DNA‐based barcoding will help address these limitations. ²¹⁷ , ²¹⁸ Multiplexing of samples in scRNA‐seq in combination with human organoids could be successfully applied to study viral pathogenesis and develop viral vaccines and antiviral therapeutics.

In addition to transcriptomic analysis, epigenomic, proteomic, and metabolic analyses are needed to be integrated into exploring the wide dysregulation of tissues or organs upon SARS‐CoV‐2 infection. ²¹⁹ , ²²⁰ , ²²¹ The paired analyses empowered the study's ability to comprehensively reveal potential mechanisms and regulatory networks that respond to SARS‐CoV‐2 infection. More recently, novel single‐cell multi‐omics have been used to investigate to better understand SARS‐CoV‐2‐induced immune responses. ²²² , ²²³ , ²²⁴ , ²²⁵ , ²²⁶ For instance, scRNA‐seq, CITE‐seq (cellular indexing of transcriptomes and epitopes sequencing), TCR‐seq (T cell receptor sequencing) and BCR‐seq (B cell receptor sequencing) have been utilized to illustrate the dynamic responses of the innate and adaptive immune systems to SARS‐CoV‐2. ²²⁷ The high‐throughput immune profiling revealed progressive COVID‐19 was associated with dyssynchrony of the innate and adaptive immune responses. Similarly, Wilk's lab performed scRNA‐seq, scATAC‐seq (single‐cell assay for transposase‐accessible chromatin sequencing), and CyTOF (cytometry by time of flight) on the COVID‐19 patient‐derived peripheral immune cells. ²²¹ The epigenomic, proteomic and transcriptomic profiling revealed the aberrant peripheral immune responses to SARS‐CoV‐2, including neutrophil and natural killer cell hyperactivation. Regarding these, we foresee that multi‐omics technology integrated with organoid models will provide large‐scale information and improve the understanding of virus–host interactions.

7. CONCLUSION REMARKS

The present review aims to reveal the status and research trend of organoid applications systematically and comprehensively for COVID‐19 research, including studying the infection mechanism, vaccine development and drug discovery of SARS‐CoV‐2. Although fruitful data have been obtained, it cannot be denied that the organoid‐based COVID‐19 study is still in its infancy. Human organoids are closely resembling embryonic or fetal organs rather than mature adult organs. It remains to be seen whether organoid maturation could cause diverse cellular responses and susceptibility to viral infections between immature and mature organs. Moreover, differences in different organoid protocols might induce diverse susceptibility to SARS‐CoV‐2 infection. Building advanced human organoids by integrating novel strategies and technologies could address the challenges and promote the development of COVID‐19 research. We hope this study could fully present the status and research trend of organoid‐based SARS‐CoV‐2 study and aid in understanding COVID‐19 pathogenesis and facilitating vaccine development and drug discovery.

AUTHOR CONTRIBUTIONS

Minghui Li: Conceptualization, supervision, data curation, writing review, and editing. Yuhan Yuan, Ting Zou, and Zongkun Hou: Data curation, review, and editing. Liang Jin and Bochu Wang: Conceptualization, supervision, review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no potential conflicts of interest.

Supporting information

Appendix S1: Supporting information.

Li M, Yuan Y, Zou T, Hou Z, Jin L, Wang B. Development trends of human organoid‐based COVID‐19 research based on bibliometric analysis. Cell Prolif. 2023;56(12):e13496. doi: 10.1111/cpr.13496

DATA AVAILABILITY STATEMENT

The datasets analyzed during the current research are available from the corresponding author on reasonable request.