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State-of-the-art analytical methods of viral infections in human lung organoids.
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.
Afiliación
  • Baumgardt M; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Hülsemann M; Berlin Institute of Health at Charité (BIH), BIH QUEST Center for Responsible Research, Berlin, Germany.
  • Löwa A; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Fatykhova D; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Hoffmann K; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Kessler M; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Mieth M; Department of Gynecology and Obstetrics, University Hospital, LMU, Munich, Germany.
  • Hellwig K; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Frey D; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Langenhagen A; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Voss A; Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.
  • Obermayer B; Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.
  • Wyler E; Core Unit Bioinformatics, Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany.
  • Dökel S; Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC) and IRI Life Sciences, Institute for Biology, Humboldt Universität zu Berlin, Berlin, Germany.
  • Gruber AD; Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.
  • Tölch U; Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.
  • Hippenstiel S; Berlin Institute of Health at Charité (BIH), BIH QUEST Center for Responsible Research, Berlin, Germany.
  • Hocke AC; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
  • Hönzke K; Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Berlin, Germany.
PLoS One ; 17(12): e0276115, 2022.
Article en En | MEDLINE | ID: mdl-36538516
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.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: COVID-19 Tipo de estudio: Prognostic_studies Límite: Humans Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2022 Tipo del documento: Article País de afiliación: Alemania

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: COVID-19 Tipo de estudio: Prognostic_studies Límite: Humans Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2022 Tipo del documento: Article País de afiliación: Alemania