Collection: State-of-the-Art Analytical Methods of Viral Infections in Human Lung Organoids
Morris Baumgardt, Katharina Hellwig, Alina Langenhagen, Anne Voss, Simon Dökel, Achim D. Gruber, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke, Maren Hülsemann, Anna Löwa, Benedikt Obermayer, Emanuel Wyler, Karen Hoffmann, Mirjana Kessler, Maren Mieth, Doris Frey, Diana Fatykhova, Ulf Tölch
Human Lung Organoids
Protein Extraction
Western Blot
RNA Extraction
RT-qPCR
SARS-CoV-2. Infection
Plaque Assay
viral qPCR
Single Cell Isolation
Single Cell RNA Sequencing
Immunohistochemistry
in situ Hybridization
Abstract
Organ models have received widespread attention in the study of SARS-CoV-2, the pathogen causing the current COVID-19 pandemic. Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens.
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 e.g., ACE2 or other host factors of SARS-CoV-2 on RNA 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.
Before start
Grow the virus stock (SARS-CoV-2 B.1) on Vero E6 cells (RRID:CVCL_0574), please work with maximum passage 3 and sequence the virus stock initially.
