State-of-the-art analytical methods of viral infections in human lung organoids

• Published in: PloS one Vol. 17; no. 12; p. e0276115
• Main Authors: Baumgardt, Morris, Hülsemann, Maren, Löwa, Anna, Fatykhova, Diana, Hoffmann, Karen, Kessler, Mirjana, Mieth, Maren, Hellwig, Katharina, Frey, Doris, Langenhagen, Alina, Voss, Anne, Obermayer, Benedikt, Wyler, Emanuel, Dökel, Simon, Gruber, Achim D., Tölch, Ulf, Hippenstiel, Stefan, Hocke, Andreas C., Hönzke, Katja
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.12.2022; Public Library of Science (PLoS)
• Subjects: ACE2; Alveoli; Analysis; Analytical methods; Angiotensin; Angiotensin-converting enzyme 2; Angiotensin-Converting Enzyme 2 - metabolism; Biology and life sciences; Cell culture; Cell cycle; Control; Coronaviruses; COVID-19; COVID-19 - metabolism; Diagnosis; E protein; Epidemics; Flow cytometry; Furin; Furin - metabolism; Gene expression; Gene sequencing; Germany; Human performance; Humans; Hybridization; Immunohistochemistry; Impact analysis; Infections; Lab Protocol; Lung - metabolism; Medical research; Medicine and health sciences; mRNA; Organoids; Pandemics; Peptidyl-dipeptidase A; Plaque assay; Proteins; Replication; Research and Analysis Methods; Ribonucleic acid; RNA; RNA processing; SARS-CoV-2; Serine proteinase; Severe acute respiratory syndrome coronavirus 2; Tropism; Viral diseases; Viral infections; Virulence; Virulence (Microbiology); Virus diseases; Viruses; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0276115

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2 , TMPRSS2 , and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.