Special Issue "The Application of 3D Tissue Culture Systems in Virology"

A special issue of Viruses (ISSN 1999-4915).

Deadline for manuscript submissions: 15 August 2021.

Special Issue Editor

Dr. Margaret Scull
E-Mail Website
Guest Editor
Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA
Interests: airway biology; virus-host interactions; innate antiviral defense; species tropism; tissue engineering; primary cell systems; respiratory viruses

Special Issue Information

Dear Colleagues,

Since viruses are obligate intracellular parasites, the advent and subsequent innovations in cell culture technology have significantly impacted the field of virology. In recent years, the incorporation of tissue architecture and the integration of relevant cell–cell interactions, together with additional biochemical and mechanical cues that recapitulate the in vivo microenvironment, have yielded more physiologically relevant systems that bridge the gap between standard monolayer cell cultures and animal models. These novel models provide unique opportunities to further analyze virus–host interactions, culture previously hard-to-grow pathogens, identify novel therapeutic targets, and advance drug development.  

In this Special Issue, we will showcase recent advancements in the engineering and/or application of three-dimensional culture systems with emergent properties for virus infection. We welcome submissions reporting any aspect of virus–host interactions in these systems, especially those focusing on pathogens with patterns of infection that are not easily recapitulated in cell lines.

Dr. Margaret Scull
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2200 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • emergent properties
  • tissue engineering
  • organoids
  • spheroids
  • persistent infections
  • virus-host interactions
  • spatiotemporal dynamics
  • multiple organ components
  • high-throughput applications

Published Papers (13 papers)

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Research

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Open AccessArticle
Targeted Profiling of Immunological Genes during Norovirus Replication in Human Intestinal Enteroids
Viruses 2021, 13(2), 155; https://doi.org/10.3390/v13020155 - 21 Jan 2021
Viewed by 533
Abstract
Norovirus is the leading cause of acute gastroenteritis worldwide. The pathogenesis of norovirus and the induced immune response remain poorly understood due to the lack of a robust virus culture system. The monolayers of two secretor-positive Chinese human intestinal enteroid (HIE) lines were [...] Read more.
Norovirus is the leading cause of acute gastroenteritis worldwide. The pathogenesis of norovirus and the induced immune response remain poorly understood due to the lack of a robust virus culture system. The monolayers of two secretor-positive Chinese human intestinal enteroid (HIE) lines were challenged with two norovirus pandemic GII.4 Sydney strains. Norovirus RNA replication in supernatants and cell lysates were quantified by RT-qPCR. RNA expression levels of immune-related genes were profiled using PCR arrays. The secreted protein levels of shortlisted upregulated genes were measured in supernatants using analyte-specific enzyme-linked immunosorbent assay (ELISA). Productive norovirus replications were achieved in three (75%) out of four inoculations. The two most upregulated immune-related genes were CXCL10 (93-folds) and IFI44L (580-folds). Gene expressions of CXCL10 and IFI44L were positively correlated with the level of norovirus RNA replication (CXCL10: Spearman’s r = 0.779, p < 0.05; IFI44L: r = 0.881, p < 0.01). The higher level of secreted CXCL10 and IFI44L proteins confirmed their elevated gene expression. The two genes have been reported to be upregulated in norovirus volunteer challenges and natural human infections by other viruses. Our data suggested that HIE could mimic the innate immune response elicited in natural norovirus infection and, therefore, could serve as an experimental model for future virus-host interaction and antiviral studies. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessArticle
RSV and HMPV Infections in 3D Tissue Cultures: Mechanisms Involved in Virus-Host and Virus-Virus Interactions
Viruses 2021, 13(1), 139; https://doi.org/10.3390/v13010139 - 19 Jan 2021
Viewed by 614
Abstract
Respiratory viral infections constitute a global public health concern. Among prevalent respiratory viruses, two pneumoviruses can be life-threatening in high-risk populations. In young children, they constitute the first cause of hospitalization due to severe lower respiratory tract diseases. A better understanding of their [...] Read more.
Respiratory viral infections constitute a global public health concern. Among prevalent respiratory viruses, two pneumoviruses can be life-threatening in high-risk populations. In young children, they constitute the first cause of hospitalization due to severe lower respiratory tract diseases. A better understanding of their pathogenesis is still needed as there are no approved efficient anti-viral nor vaccine against pneumoviruses. We studied Respiratory Syncytial virus (RSV) and human Metapneumovirus (HMPV) in single and dual infections in three-dimensional cultures, a highly relevant model to study viral respiratory infections of the airway epithelium. Our investigation showed that HMPV is less pathogenic than RSV in this model. Compared to RSV, HMPV replicated less efficiently, induced a lower immune response, did not block cilia beating, and was more sensitive to IFNs. In dual infections, RSV-infected epithelia were less permissive to HMPV. By neutralizing IFNs in co-infection assays, we partially prevented HMPV inhibition by RSV and significantly increased the number of co-infected cells in the tissue. This suggests that interference in dual infection would be at least partly mediated by the host immune response. In summary, this work provides new insight regarding virus-host and virus-virus interactions of pneumoviruses in the airway epithelium. This could be helpful for the proper handling of at-risk patients. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessArticle
A Novel In Vitro Culture Model System to Study Merkel Cell Polyomavirus–Associated MCC Using Three-Dimensional Organotypic Raft Equivalents of Human Skin
Viruses 2021, 13(1), 138; https://doi.org/10.3390/v13010138 - 19 Jan 2021
Viewed by 550
Abstract
Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro [...] Read more.
Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessArticle
Patient-Derived Organotypic Epithelial Rafts Model Phenotypes in Juvenile-Onset Recurrent Respiratory Papillomatosis
Viruses 2021, 13(1), 68; https://doi.org/10.3390/v13010068 - 06 Jan 2021
Cited by 1 | Viewed by 681
Abstract
Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is driven by human papillomavirus (HPV) low-risk strains and is associated with significant morbidity. While previous studies of 2D cultures have shed light on disease pathogenesis and demonstrated the utility of personalized medicine approaches, monolayer cultures lack the [...] Read more.
Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is driven by human papillomavirus (HPV) low-risk strains and is associated with significant morbidity. While previous studies of 2D cultures have shed light on disease pathogenesis and demonstrated the utility of personalized medicine approaches, monolayer cultures lack the 3D tissue architecture and physiology of stratified, sequentially differentiated mucosal epithelium important in RRP disease pathogenesis. Herein we describe the establishment of JoRRP-derived primary cell populations that retain HPV genomes and viral gene expression in culture. These were directly compared to cells from matched adjacent non-diseased tissue, given the known RRP patient-to-patient variability. JoRRP papilloma versus control cells displayed decreased growth at subconfluency, with a switch to increased growth after reaching confluency, suggesting relative resistance to cell-cell contact and/or differentiation. The same papilloma cells grown as 3D organotypic rafts harbored hyperproliferation as compared to controls, with increased numbers of proliferating basal cells and inappropriately replicating suprabasal cells, mimicking phenotypes in the patient biopsies from which they were derived. These complementary model systems provide novel opportunities to elucidate disease mechanisms at distinct stages in JoRRP progression and to identify diagnostic, prognostic and therapeutic factors to personalize patient management and treatment. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessCommunication
The Potentials and Pitfalls of a Human Cervical Organoid Model Including Langerhans Cells
Viruses 2020, 12(12), 1375; https://doi.org/10.3390/v12121375 - 01 Dec 2020
Viewed by 423
Abstract
Three-dimensional cell culturing to capture a life-like experimental environment has become a versatile tool for basic and clinical research. Mucosal and skin tissues can be grown as “organoids” in a petri dish and serve a wide variety of research questions. Here, we report [...] Read more.
Three-dimensional cell culturing to capture a life-like experimental environment has become a versatile tool for basic and clinical research. Mucosal and skin tissues can be grown as “organoids” in a petri dish and serve a wide variety of research questions. Here, we report our experience with human cervical organoids which could also include an immune component, e.g., Langerhans cells. We employ commercially available human cervical keratinocytes and fibroblasts as well as a myeloid cell line matured and purified into langerin-positive Langerhans cells. These are then seeded on a layer of keratinocytes with underlying dermal equivalent. Using about 10-fold more than the reported number in healthy cervical tissue (1–3%), we obtain differentiated cervical epithelium after 14 days with ~1% being Langerhans cells. We provide a detailed protocol for interested researchers to apply the described “aseptic” organoid model for all sorts of investigations—with or without Langerhans cells. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessArticle
Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection
Viruses 2020, 12(11), 1298; https://doi.org/10.3390/v12111298 - 12 Nov 2020
Viewed by 1118
Abstract
Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In [...] Read more.
Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus–host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessArticle
Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture
Viruses 2020, 12(11), 1294; https://doi.org/10.3390/v12111294 - 12 Nov 2020
Viewed by 664
Abstract
Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from [...] Read more.
Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Review

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Open AccessReview
Bioengineered Liver Cell Models of Hepatotropic Infections
Viruses 2021, 13(5), 773; https://doi.org/10.3390/v13050773 - 27 Apr 2021
Viewed by 257
Abstract
Hepatitis viruses and liver-stage malaria are within the liver infections causing higher morbidity and mortality rates worldwide. The highly restricted tropism of the major human hepatotropic pathogens—namely, the human hepatitis B and C viruses and the Plasmodium falciparum and Plasmodium vivax parasites—has hampered [...] Read more.
Hepatitis viruses and liver-stage malaria are within the liver infections causing higher morbidity and mortality rates worldwide. The highly restricted tropism of the major human hepatotropic pathogens—namely, the human hepatitis B and C viruses and the Plasmodium falciparum and Plasmodium vivax parasites—has hampered the development of disease models. These models are crucial for uncovering the molecular mechanisms underlying the biology of infection and governing host–pathogen interaction, as well as for fostering drug development. Bioengineered cell models better recapitulate the human liver microenvironment and extend hepatocyte viability and phenotype in vitro, when compared with conventional two-dimensional cell models. In this article, we review the bioengineering tools employed in the development of hepatic cell models for studying infection, with an emphasis on 3D cell culture strategies, and discuss how those tools contributed to the level of recapitulation attained in the different model layouts. Examples of host–pathogen interactions uncovered by engineered liver models and their usefulness in drug development are also presented. Finally, we address the current bottlenecks, trends, and prospect toward cell models’ reliability, robustness, and reproducibility. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessReview
From Submerged Cultures to 3D Cell Culture Models: Evolution of Nasal Epithelial Cells in Asthma Research and Virus Infection
Viruses 2021, 13(3), 387; https://doi.org/10.3390/v13030387 - 28 Feb 2021
Viewed by 581
Abstract
Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and [...] Read more.
Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and spheroid cell culture models of nasal epithelial cells, which were used to study asthma and allergy with a special focus on virus infection. In detail, this review summarizes the importance, benefits, and disadvantages of patient-derived cell culture models of nasal- and bronchial epithelial cells, including a comparison of these cell culture models and a discussion on why investigators should consider using nasal epithelial cells in their research. Exposure experiments, simple virus transduction analyses as well as genetic studies can be performed in these models, which may provide first insights into the complexity of molecular signatures and may open new doors for drug discovery and biomarker research. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessReview
Three-Dimensional Cell Culture Systems for Studying Hepatitis C Virus
Viruses 2021, 13(2), 211; https://doi.org/10.3390/v13020211 - 30 Jan 2021
Viewed by 474
Abstract
Hepatocytes, the major target of hepatitis C virus (HCV), are highly polarized. HCV infection requires extensive trafficking to distinct subcellular domains in the polarized hepatocyte. Polarized cells and three-dimensional organoids are commonly used to study liver functions and differentiation. Researchers have begun adapting [...] Read more.
Hepatocytes, the major target of hepatitis C virus (HCV), are highly polarized. HCV infection requires extensive trafficking to distinct subcellular domains in the polarized hepatocyte. Polarized cells and three-dimensional organoids are commonly used to study liver functions and differentiation. Researchers have begun adapting these cell culture models that morphologically and physiologically resemble hepatocytes in vivo to study HCV infection. This review summarizes the use of three-dimensional cell culture systems in studies of HCV infection. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessReview
Leveraging 3D Model Systems to Understand Viral Interactions with the Respiratory Mucosa
Viruses 2020, 12(12), 1425; https://doi.org/10.3390/v12121425 - 11 Dec 2020
Cited by 1 | Viewed by 762
Abstract
Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus–host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture [...] Read more.
Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus–host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air–liquid interface, organoids, or ‘on-chip’ technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium—the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Open AccessReview
Put Some Guts into It: Intestinal Organoid Models to Study Viral Infection
Viruses 2020, 12(11), 1288; https://doi.org/10.3390/v12111288 - 11 Nov 2020
Cited by 1 | Viewed by 855
Abstract
The knowledge about enteric viral infection has vastly increased over the last eight years due to the development of intestinal organoids and enteroids that suppose a step forward from conventional studies using cell lines. Intestinal organoids and enteroids are three-dimensional (3D) models that [...] Read more.
The knowledge about enteric viral infection has vastly increased over the last eight years due to the development of intestinal organoids and enteroids that suppose a step forward from conventional studies using cell lines. Intestinal organoids and enteroids are three-dimensional (3D) models that closely mimic intestinal cellular heterogeneity and organization. The barrier function within these models has been adapted to facilitate viral studies. In this review, several adaptations (such as organoid-derived two-dimensional (2D) monolayers) and original intestinal 3D models are discussed. The specific advantages and applications, as well as improvements of each model are analyzed and an insight into the possible path for the field is given. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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Other

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Open AccessOpinion
A Perspective on Organoids for Virology Research
Viruses 2020, 12(11), 1341; https://doi.org/10.3390/v12111341 - 23 Nov 2020
Cited by 2 | Viewed by 633
Abstract
Animal models and cell lines are invaluable for virology research and host–pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to [...] Read more.
Animal models and cell lines are invaluable for virology research and host–pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to study viral infections in the human context. This perspective explores the potential of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and shaping the virology landscape. Full article
(This article belongs to the Special Issue The Application of 3D Tissue Culture Systems in Virology)
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