The emergence of novel viruses and virus mutations is inevitable and will only become a greater concern as the global population increases and areas get more densely populated (Arif and Sengupta, 2020; Whiting, 2020). Therefore, it is fundamental to establish and characterize robust pre-clinical models that can be applied instantly to study the virus-host interactions, to learn more about how emerging viruses transmit and interact with their human hosts to mediate human disease. Improved pre-clinical models may also be a useful tool for more reliable preclinical screening of potential prophylactic and therapeutic interventions, and improved selection of promising drug candidates for testing in clinical trials.

Respiratory viral infections are a leading cause of disease and mortality worldwide. Influenza viruses are negative-sense single-stranded RNA viruses of the family, Orthomyxoviridae (Bouvier and Palese, 2008). They are amongst the most common human respiratory viruses. According to the World Health Organization (WHO), seasonal influenza epidemics are estimated to result in about 3 to 5 million cases of severe illness and 290,000 to 650,000 deaths annually (World Health Organization, 2017). Another respiratory virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the ongoing COVID-19 pandemic, belongs to the virus family Coronaviridae, and is an enveloped positive-sense single-stranded RNA virus (Zhu et al., 2020). The SARS-CoV-2 virus spread rapidly across countries and the COVID-19 outbreak was declared a pandemic by the World Health Organization (WHO) on March 11^th 2020 (World Health Organization, 2020). As of 30 January 2022, over 370 million confirmed COVID-19 cases and more than 5.6 million deaths have been reported to World Health Organization (WHO) (World Health Organization, 2022). The mortality is primarily linked to acute respiratory distress syndrome (ARDS) (Huang et al., 2020) and the long-term effects of infection are still not known, highlighting the urgent need for rapid and extensive scientific research to limit further impact on societies and public health. Despite the intense research efforts since the outbreak of the ongoing COVID-19 pandemic, there are still a lot of uncertainties related to virus replication, pathogenesis, the long-term effects of COVID-19 and the significance of novel SARS-CoV-2 mutations.

Animal models have greatly contributed to our understanding of disease mechanisms and still constitute important pre-clinical tools that allows us to study in vivo replication of viruses and the viral interaction with the animal host defense system. However, biological processes occurring in humans may not always be faithfully reproduced in animal models, not even in non-human primate models, and this limits the applicability of the knowledge derived from animal models. One of the particular issues for virology is that several animal models that allow infection of human viruses rely on ectopic expression of entry factors, this implies that the expression pattern and the transcriptional regulation of viral entry factor expression is not identical to the human situation (van der Vaart et al., 2021; Yinda et al., 2021). Further, in virology, monolayers, also known as 2D cell cultures, of animal cells such as the African green monkey kidney cell line, VeroE6 and the canine kidney cell line, MDCK, are highly susceptible to infection by certain viruses and frequently applied to study viral replication and spread (Lugovtsev et al., 2013; Matsuyama et al., 2020). The human alveolar basal epithelial cell line, A549, derived from a human adenocarcinoma, and other human cancer cell lines, such as the human lung cancer cell line, Calu-3 2B4, are also frequently applied (Giard et al., 1973; Yoshikawa et al., 2010). However, although modified animal cell culture models or human cancer cell lines are optimized to support viral replication, they do not recapitulate the heterogeneity and complexity of human tissues, and not even the intracellular processes of primary cells of the organ they derive from. Since most of these in vitro models lack a functional Type 1 interferon response, they are also not suitable to assess how the epithelial cells communicate with the immune cells upon infection. Furthermore, primary human airway epithelial cells (pHAE) cultures are commonly used models for studying respiratory tract biology, respiratory tract diseases and therapeutic interventions. pHAE cultures functionally and morphologically resemble the epithelium of the upper conducting airways in vivo (Jonsdottir and Dijkman, 2015) and numerous human coronaviruses have been successfully propagated in pHAE cultures (Dijkman et al., 2013; Kindler et al., 2013). A recent study demonstrated the utility of pHAE cultures to model SARS-CoV-2 infection, and showed that SARS-CoV-2 infection of primary human airway epithelial cultures did not trigger an IFN response, but they are sensitive to the effect of both type I and type III IFNs (Vanderheiden et al., 2020). However, pHAE cultures have a restricted proliferative lifespan in culture, and may lose the ability to produce mucus or form cilia after only 3-4 population doublings (Gray et al., 1996; Martinovich et al., 2017). Improvements in the pHAE culture protocols are continuously being made, and include the culture of pHAE at the air-liquid interface for optimal differentiation (Bukowy-Bieryłło, 2021). pHAE cultures may only be applied to model infection of the upper respiratory tract, and not the alveoli, which are the functional units of the lung.

The human respiratory airways and lungs compose a complex vital organ system that can be divided into upper respiratory tract (nose, nasal cavity, and pharynx), and lower respiratory tract (trachea, bronchial tree, lungs) (Ross, 2019). Most of the conducting airways of the respiratory tree from the nasal cavity to the bronchioles consist of pseudostratified columnar epithelium where each region contains stem cell lineages crucial for maintaining tissue homeostasis and regeneration upon injury (Ross, 2019). The pseudostratified columnar epithelium in the conducting airways mainly consists of basal cells, club cells, goblet cells and ciliated cells. In addition, the bronchiolar epithelium may contain a low number of neuroendocrine cells, ionocytes and tuft cells (Montoro et al., 2018; Zepp and Morrisey, 2019). Basal cells are relatively undifferentiated cells and express the transcription factor Trp-63, and cytokeratins 5 and 14, in addition, basal cells are able to self-renew (Zepp and Morrisey, 2019). A subpopulation of basal cell has been found to function as airway epithelial stem cells that are able to give rise to goblet and multi-ciliated cells (Hong et al., 2004). Ciliated cells play an important role in trapping and removing inhaled pathogens, and motor proteins drive the coordinated directional movement of the beating cilia. Previous studies found that Influenza A virus infection immediately enhance cilia-driven flow and ciliary activity in the airway epithelium (Kamiya et al., 2020). The main secretory cells of the conducting airways include goblet cells and the non-ciliated club cells, also known as bronchiolar exocrine cells and formerly known as clara cells. Mucins such as Muc5AC and Muc5B are synthesized by goblet cells and are stored in vesicles that appear electron-lucent on transmission electron microscopy (TEM) images (Leishangthem et al., 2013). The proteins, Splunc1 and secretoglobins (Scgb1a1, Scgb3a2), are produced by mature club cells and are stored in apical granules that appear electron-dense in TEM images (Barkauskas et al., 2017). Lineage tracing studies have revealed that subpopulations of club cells are able to self-renew and may give rise to ciliated cells as well as goblet cells (Rawlins et al., 2009). Furthermore, studies have also found that Scgb1a1 expressing cells may differentiate to Type 1 pneumocytes (AT1) and Type 2 pneumocytes (AT2) cells (Zheng et al., 2012). More in depth studies of pulmonary pathologies are expected to shed light on this complexity, and the particular marker expression patterns associated with the various club cell phenotypes (Zuo et al., 2020).

In recent years a great amount of research has been allocated to the development of novel organotypic 3D culture models, namely organoids. Organoids are complex 3D multicellular constructs that can be derived from either induced pluripotent stem cells (iPSCs) or adult stem cells and grown in a supportive extracellular matrix (e.g. Matrigel) to mimic in particular basement membrane components. Cells grown in this 3D culture system have been found to resemble more closely the in vivo environment, where cells spontaneously self-organize into hierarchies of stem/progenitor cells and create a continuum of more differentiated and functional cell types. Several research groups have been successful in the development of adult stem cell derived human airway and lung organoids (Zhou et al., 2018; Sachs et al., 2019; Hoareau et al., 2021), and a long-term expansion culture of the bronchiolar pseudostratified epithelium that contain all major cellular elements was pioneered and described by Hans Clevers’ laboratory (Sachs et al., 2019). However, for unknown reasons, a representative model of human alveolar epithelial cells, the so-called alveolospheres, in vitro has proven to be more difficult to establish (Hoareau et al., 2021). The alveoli constitute the functional unit of the lungs and the site where the gas exchange between the air and the bloodstream occurs, and consist of a thin simple squamous epithelium with AT1 and AT2 cells forming the alveolar functional units (Ross, 2019). The lack of appropriate models of the alveoli represents an unmet need in this field. We have recently described a model where adult stem cell derived bronchiolospheres and alveolospheres were derived from the same patient tissue specimen (Hoareau et al., 2021). Here we expand the characterization of this model and assess the potential of this model to support viral infection and replication. Here we aimed to characterize a biologically relevant and reproducible human in vitro airway and lung model and subsequently assess its potential as a model for studies of respiratory virus infectivity and virus-mediated pathologies. The main benefit of this model is that bronchiolar and alveolar epithelium may be efficiently differentiated from adult stem cells in a single biopsy specimen, and thus favour downstream analysis of regional differences in the response to viral infection.

The interdisciplinary development and application of human airway and lung organoids in virology holds the potential to significantly aid in our understanding of virus- host interactions, pathogenesis and drug development. Thus, continued collaborative efforts to advance this development is urgently needed to increase global pandemic preparedness. The application of organoid cultures representing the epithelium from the various part of the respiratory tract is of particular importance to allow further insight into the viral tropism (van der Vaart and Clevers, 2021), and also, how various mutants affect cell type specific replication kinetics and communication with the immune system. Here, we present for the first-time a thorough characterization of a reproducible model where adult stem cell derived bronchiolospheres and alveolospheres are derived from single human biopsy specimens and conclude that the model is representative of the bronchiolar epithelium of the conducting airways and the respiratory epithelium of the alveoli, respectively. Furthermore, we confirmed the expression of viral entry factors for respiratory virus, including influenza virus and SARS-CoV-2, and conclude that the model system is easily infectable and well suited for virology studies. We propose that this representative model of human airways and lungs can be easily established in virology laboratories and applied in future studies where regional response to respiratory virus or other pathogens are needed.

The frequency and severity of viral pandemics are expected to increase as the global population increases and areas become more densely populated (Arif and Sengupta, 2020; Whiting, 2020). Therefore, it is of uttermost importance that we learn from the ongoing pandemic and improve global pandemic preparedness. Respiratory viruses are the most common acute infections (Boncristiani et al., 2009). Human respiratory virus belongs to several virus families that initially target airway epithelial cells, and it may be difficult to distinguish causative virus based on the clinical symptoms. Respiratory virus usually transmit via airway secretions from an infected individual, and may spread via direct or indirect contact, or via droplet and aerosol transmission (Siegel et al., 2007). Viral infections confined to the upper respiratory tract usually cause mild symptoms. When viral infections spread from the upper respiratory tract to the lungs it may cause severe morbidity and mortality. It is important to understand the modes of transmission, the viral entry, the regional specific virus-host interactions, and to gain a more complete understanding of how the virus mediates disease. The availability of improved and well-characterized pre-clinical models that resemble the various parts of the human respiratory airways and lungs are an important, as these models provide a superior tool to study virus-host interactions, test prophylactics and therapeutics for existing and emerging viruses, and to support the study of co-infections. More widespread application of these improved models in virology is expected to aid in the development of prophylactic measures as well as therapeutic options. This knowledge will allow a more rapid response when novel virus strains emerge. Here, we present a reproducible and representative adult stem cell derived model for virology that faithfully recapitulates the regional hallmarks of both bronchiolar and alveolar epithelium, and we show that the model is well suited to support viral infection and replication of selected respiratory viruses.

Murine models have been vital to study the in vivo replication of virus and the interaction of virus with the animal host defense system. However, significant differences between the murine and human immune systems may limit the value of their findings and mice lack appropriate receptors for SARS CoV-2. A recent study described use of transgenic mice with ectopic expression of human ACE2 to detect SARS CoV-2 antigens in bronchial epithelial cells, macrophages and alveolar epithelial cells (Bao et al., 2020). Further, the humanized mouse strain with induced human ACE2 expression, K18-hACE2 mice from Jackson labs, (McCray Jr et al., 2007) were shown to support SARS-CoV-2 replication in respiratory and brain tissues, and the authors showed that the model recapitulate many of the findings observed in patients with COVID-19 including changes in the expression of pro-inflammatory molecules similar to what have been observed in patients with COVID-19 post infection. A publication by Yinda and colleagues (Yinda et al., 2021) later showed that the rapid inflammatory response and observed pathology observed in this model bears resemblance to COVID-19. As the authors also point out, it should be noted that the murine K18-hACE2 model relies on artificial ectopic expression of hACE2 under the widely expressed Keratin 18 promotor, and this facilitates an expression pattern of hACE2 that does not precisely recapitulate the expression pattern in humans (Yinda et al., 2021).

There is no doubt that these mice are useful models to assess vaccines and antiviral agents against COVID-19, however, the limitation introduced by exogenous viral receptor expression remains, and may affect the tropism as well as replication kinetics. Due to the phylogenetic proximity to humans, non-human primate models have previously been utilized to study SARS and MERS (Haagmans et al., 2004; Wang et al., 2017; Chandrashekar et al., 2020; Munster et al., 2020; Rockx et al., 2020). During the current COVID-19 pandemic, there has been a significant increase in the demand and there is currently a large shortage of non-human primates (Zhang, 2020). SARS-CoV-2 infection were shown to induce protective immunity against re-challenge in rhesus macaques (Chandrashekar et al., 2020). However, the non-human primate models experience only the moderate disease and not the severe ARDS sometimes observed in humans (Munster et al., 2020).

Three dimensional (3D) organotypic models, or organoids, can be established from either induced pluripotent stem cells (iPSCs) or adult stem cells. Organoid cultures of human airway and lung tissue serve as a more relevant model of the heterogeneity and complexity of the human respiratory epithelium, compared to homogenous 2D cell cultures of immortalized animal cell lines or malignant human cell lines which are often not susceptible to multiple types of virus infection, and thus unsuited to study the pathogeneses caused by co-infections. Other research groups have pioneered the organoid technology, establishing organoids of bronchiolar differentiation from adult stem cells (Zhou et al., 2018; Sachs et al., 2019). We showed here that organoids displaying proper differentiation of epithelium characteristic of the conducting airways and the respiratory epithelium with respect to histo-architecture and cellular composition could be rapidly established from adult stem cells using the BRON protocol established by Hans Clevers laboratory (Sachs et al., 2019), and recently optimized by us for efficient differentiation into both bronchiolar and alveolar differentiation (Hoareau et al., 2021). This study shows that the organoids retain characteristic histological features and the expected cellular composition of the two epithelia throughout 4 passages. The ability for early differentiation as well as long-term expansion both represent huge experimental advantages. Patient-derived organoids have been found to retain similar frequencies of basal, club, multi-ciliated and secretory cells for over 1 year in culture in other studies (Sachs et al., 2019). Long term culture in combination with longitudinal sampling in this adapted model of BRON and ALV cultures would be required in this model also to validate if the robustness of the model is comparable to this. The fact that organoids recapitulate essential functional aspects of the lungs are of great importance as it contributes to a more complex and dynamic culturing system. In contrast, the frequently applied 2D cell cultures are easy to work with, but they do not recapitulate this functional complexity.

The alveoli are the functional units of the lung and consist mainly of AT1 cells, which are large and flat squamous cells that have an important role in gas exchange. In fact, 95% of the gas exchange surface of the lung is comprised of AT1 cells (Wu and Tang, 2021). In contrast, AT2 cells are smaller cells of a cuboidal shape. AT2 cells synthesize lung surfactant, which is composed of 90% lipids and helps to reduce the surface tension during breathing movements (Andreeva et al., 2007). Highly specialized organelles, termed lamellar bodies, are found in differentiated AT2 cells, and serves to package the surfactant (Sever et al., 2021). In addition, AT2 cells have been found to play a vital role in re-establishing homeostasis after lung damage. Under these challenging conditions, AT2 cells have been shown to display stem cell potential and the ability of AT2 cells to self-renew and differentiate into AT1 cells has been shown in several studies (Barkauskas et al., 2013; Desai et al., 2014; Katsura et al., 2019). However, it is still unknown whether all or just a subset of AT2 cells harbor this stem cell potential. In the alveolar organoids, we determined the presence of alveolar epithelium cells, Type 1 pneumocytes and Type 2 pneumocytes (AT1 and AT2 cells, respectively). AT1 cell studies have been restricted due to a lack of genes with expression unique to AT1 cells. PDPN was utilized as a AT1 cell marker in this study in order to be able to compare the expression levels with a previous report (Hoareau et al., 2021). However, PDPN is also expressed in basal cells (Van de Laar et al., 2014), among other cell types from other tissues, and it is therefore not an AT1 cell specific marker. Furthermore, the higher expression of KRT5 in the alveolar organoids compared to bronchiolar organoids requires further investigation as it calls the derivation of AT2 cells into question and suggests expansion of the niche-independent stem cells, namely basal cells. Nevertheless, KRT5 and PDPN expression profiles are not similar, suggesting a more complicated expression panel by different cell subtypes. The differentiation of alveolospheres with functional AT1 and AT2 cells have been a longstanding challenge in the field, and we acknowledge that there is still room for improvement in the differentiation protocols in order to generate a more robust ALV model.

The aim of this study was to determine whether the organotypic model described is suitable for infection by respiratory viruses. Influenza viruses are amongst the most common causes of human respiratory infections and are single-stranded, enveloped, negative-sense RNA viruses that belong to the Orthomyxoviridae family (Piret and Boivin, 2021). The Influenza A virus is responsible for most of the disease burden in humans, and its structural proteins include nucleoprotein, two matrix-proteins in addition to the two surface glycoproteins neuraminidase (NA) and hemagglutinin (HA) (Webster et al., 1992). A major concern with influenza viruses is their ability to undergo antigenic drift and subsequently acquire mutations in NA and HA that allows them to escape pre-existing immunity. Human influenza viruses preferentially bind to sialic acids (SA) attached to galactose via an α 2,6 linkage (Rogers et al., 1983; Connor et al., 1994), and these are known to be ubiquitously expressed in the human respiratory epithelium. As expected, high expression of SA and galactose were detected in both bronchiolar and alveolar organoids in this study. We further demonstrated the bronchiolar and alveolar organoids were susceptible to infection with pseudotype influenza H5N1 and H7N1 viruses and support active replication of influenza A H7N1 virus. Influenza virus infection has also been shown in human adult stem cells-derived airway organoids (Zhou et al., 2018).

SARS-CoV-2 preferably enters cells through binding via its Spike-protein (S-protein) to the angiotensin-converting enzyme 2 (ACE2) (Hoffmann et al., 2020). The receptor, ACE2, has been found to be expressed in organs such as the kidney, heart, testis, intestines and the lungs (Harmer et al., 2002). ACE2 expression has been shown to determine the host susceptibility to the SARS-CoV-2 (Lanjanian et al., 2021). However, the ACE2 protein expression is either low or lacking in the majority of cells found in the conducting and respiratory epithelium (Hikmet et al., 2020).

Artificial expression of human ACE2 is required to make human 2D cell lines susceptible to SARS-CoV-2 infection, such in the case of the human adenocarcinoma cell line A549. In contrast, VeroE6 cells, derived from African green monkey, and the human lung cancer cell line, Calu-3, endogenously express ACE2 (Ren et al., 2006). However, VeroE6 cells are not derived from humans and the cell line does not represent primary human airway cells, where expression was found to be either low or lacking in the majority of cells (Hikmet et al., 2020). In contrast to cell lines and animal models that depend on ectopic ACE2 expression to make them SARS-CoV-2 susceptible, we detected endogenous ACE2 expression in the alveolar and bronchiolar organoids using RT-qPCR and immunofluorescence. Heterogenous organoids with endogenous physiologically relevant levels of ACE2 expression may be more well suited to study viral tropism and particularly useful in studies of alternative modes of entry and alternative entry factors (van der Vaart and Clevers, 2021). ACE2 protein has been found to be heterogeneously expressed in the human respiratory tract, and in line with this, we observed a co-localization of ciliated cells and ACE2, which is consistent with previous reports (Lee et al., 2020; Mulay et al., 2020). The ACE2 expression in ciliated cells was very strong compared to the neighboring cells. SARS-CoV-2 has been found to predominantly infect ciliated cells, in models such as ALI culture systems and reconstructed human airway epithelium model (Mulay et al., 2020; Robinot et al., 2021). However, SARS-CoV-2 infection in mucus-secreting goblet cells, using 2D cultures, has not been observed in other studies (Hou et al., 2020; Lamers et al., 2020). From this we know that much is still to be learned regarding the tropism and replication kinetics of SARS-CoV-2 in human primary cells. This knowledge is particularly important for optimal rational selection and testing of potential antiviral drugs, and we envision that the organoid models will greatly facilitate this research in the future. Variation in ACE2 expression profiles also exists between individuals. In particular, ACE2 is found to be upregulated in the small airway epithelia of smokers (Zhang et al., 2020). Organoids derived from patients with co-morbidities and various ACE2 expression profiles, or in vitro simulation of these conditions, could serve as a valuable platform to study the effect on susceptibility, and this serves to be explored further.

In addition to ACE2, the serine protease, TMPRSS2, is important for ACE2 host entry as it cleaves the viral S-protein and consequently promotes SARS-CoV-2 cell entry at the plasma membrane (Hoffmann et al., 2020; Zang et al., 2020). We detected TMPRSS2 expression in alveolar and bronchiolar organoids. Our findings are consistent with previous studies, where in addition to TMPRSS2, TRMPRSS4 and TMPRSS11D expression was also detected (Zhou et al., 2018). In contrast, most 2D models have a low serine protease activity and require the addition of trypsin treated TPCK for the cleavage of viral glycoprotein HA to subsequently allow Influenza A virus to infect efficiently. Trypsin has been found to interfere with host pathogen-defense mechanisms, where in particular interferon signaling in MDCK cells was strongly inhibited in the presence of trypsin (Seitz et al., 2012). Therefore, organoid models that do not require exogenous trypsin may be more suitable for interferon-studies. The lack of TMPRSS2 could also indicate that these frequently applied cell lines are infected through viral entry via the endocytic pathway, and not by ACE2 and TMPRSS2 dependent cleavage at the cell membrane that is the preferred entry pathway in human primary respiratory epithelium (van der Vaart and Clevers, 2021). Thus, highlights yet another vital difference between traditionally used virology models and the human respiratory epithelium (Bohan et al., 2021; van der Vaart and Clevers, 2021). Furthermore, the endosomal cysteine protease, cathepsin L, has also been found to be involved in SARS-CoV-2 entry through the endosomal route (Padmanabhan et al., 2020), but via a different pathway. Having confirmed endogenous expression of ACE2 and TMPRSS2 in both bronchiolar and alveolar organoids, we aimed to evaluate infection of the organoids by a clinical SARS-CoV-2 isolate, Washington strain. Successful infection of the organoid cultures was confirmed using RT-qPCR to detect the presence of SARS-CoV-2 N gene mRNA in infected organoids of bronchiolar as well as alveolar differentiation. Next, we aimed to explore whether the infected organoids were permissive to SARS-CoV-2 i.e. if they were able to produce infectious viral particles. Of note, so called abortive infection, i.e. infection without successful replication, was recently shown for SARS-CoV infected macrophages (Yilla et al., 2005) and dendritic cells (Law et al., 2005). Despite abortive infection, the SARS-CoV infection were able to induce expression of proinflammatory, but not antiviral cytokine production in the infected macrophages (Cheung et al., 2005; Tseng et al., 2005). For human respiratory epithelium, the innate recognition mechanisms and response to viral infection is being explored at the single cell level, and there is still an urgent need to identify biomarkers that can predict which patients will experience a severe disease course upon infection (Abassi et al., 2020; Yamada et al., 2021). By TCID50 assays we were able to show that a clinical isolate of SARS-CoV-2 (hCoV-19/Norway/Bergen-01/2020), could efficiently replicate in organoids of bronchiolar as well as alveolar differentiation. Virus production was detected in organoids infected at an estimated MOI of 0.5 at both 72h and 96h post-infection. Dysmorphic cells of SARS-CoV-2 infected human lungs were characterized by the frequent appearance of syncytia, characterized by several nuclei with an ample cytoplasm surrounded by a single plasma membrane (Bussani et al., 2020). Histopathological examination of the infected organoids in this study also revealed the resemblance to the human COVID19 lungs with the presence of dysmorphic pneumocytes and prominent syncytia formation, indicating viral infection. Immunostaining to detect the SARS-CoV-2 Nucleocapsid Protein (NP) is one of the core components of the SARS-CoV-2, and the NP oligomerizes to form the closed capsule protecting the viral RNA. Immunofluorescent staining of the infected organoid cultures of bronchiolar and alveolar differentiation in this study confirmed successful infection of SARS-CoV-2 (hCoV-19/Norway/Bergen-01/2020).

Much remains to be learnt about the immunological outcome of human cell type specific infection, and the respiratory airway and lung organoid model presented in this study may serve as a useful tool to learn gain insight into the cell type specific kinetics of viral infection and replication, and the initial communication from infected cells to the innate immune cells. This is important as more simplistic models, such as permissive cell lines, although more robust and easier to infect, cannot be applied to address remaining questions related to cell type specific and regional differences in the viral life cycle and the immune interactions. Due to the physiologically relevant heterogeneity of the pHAE and organoid models, their application is particularly useful in combination with spatially resolved single cell high-dimensional methodologies like mass cytometry (CyToF), imaging mass cytometry (IMC) or single-cell RNA seq. Furthermore, compared to cell line-based models, organoid models require careful validation of differentiation status and expression of viral entry factors prior to use, which in turn encourage the development of true interdisciplinary collaborations.

In conclusion, we have developed and characterized an organoid model from adult stem cells with efficient bronchiolar as well as alveolar differentiation, characterized the presence of the various cell types expected in the differentiated human tissues in situ, as well as the endogenous expression of viral entry factors required for respiratory influenza and SARS-CoV-2 viruses. The particular strength of this model is that bronchiolospheres and alveolospheres may be generated efficiently from a single tissue specimen, and the organoids represent the histoarchitecture and the cell types representative of the upper and lower respiratory epithelium, respectively. Successful infection in the model were confirmed by two clinical isolates of SARS-CoV-2 (WA-1 and hCoV-19/Norway/Bergen-01/2020), and successful replication of SARS-CoV-2 in the organoids of bronchiolar and alveolar differentiation were determined by TCID50 assays. Thus, our findings strongly support application of the model in future studies of viral tropism and replication kinetics, as well as screening of antiviral compounds in available human pre-clinical organoid models. If successfully implemented, the knowledge and application of human organoid models in the study of existing and emerging respiratory viruses are important to reduce the impact of future pandemics.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Regional Ethics Committee Vest (REK Vest), Vestland, Norway. The patients/participants provided their written informed consent to participate in this study.

All authors contributed to the conception and design of experiments. Performed the experiments: CE, FZ, DB, ML, LH, NLu, AE. Analyzed the data: CE, FZ, DB, MR, GR, NLu, LA, NL, RC, WM, LS, JL, and AE. Contributed materials/reagents/ analysis tools: MR, MA, FG, PS, MB; TH, HR, LA. Wrote the first draft of the paper: CE, ML, LS, JL, AE. All authors were involved in article revision and approved the submitted version.

This study was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 223250 (CCBIO affiliates). The Influenza Centre is funded by the University of Bergen, Ministry of Health and Care Services, Helse Vest (F-11628), the Trond Mohn Foundation (TMS2020TMT05), the European Union (EU IMI115672 FLUCOP, H2020 874866 INCENTIVE, H2020 101037867 VACCELERATE, EU IMI101007799 Inno4Vac) and Nanomedicines Flunanoair (ERA-NETet EuroNanoMed2, JTC2016), and the Research Council of Norway GLOBVAC program (284930). WM was supported by NIH/NIAID grant R01 AI134733 and a contract from BerGenBio ASA. DB was supported by NIH/NIAID grant T32 AI007511. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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