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BRAIN Publication-derived

SARS-CoV-2 Infection of 3D Human Brain Organoids

Source Ramani et al., 2020 · Institute of Human Genetics, University Hospital Düsseldorf, Heinrich-Heine-Universität · 10.15252/embj.2020106230

👤 Anand Ramani, Lisa Müller, Philipp N Ostermann, Elke Gabriel, Pranty Abida-Islam, Andreas Müller-Schiffmann, Aruljothi Mariappan, Olivier Goureau, Henning Gruell, Andreas Walker, Marcel Andrée, Sandra Hauka, Torsten Houwaart, Alexander Dilthey, Kai Wohlgemuth, Heymut Omran, Florian Klein, Dagmar Wieczorek, Ortwin Adams, Jörg Timm, Carsten Korth, Heiner Schaal, Jay Gopalakrishnan ⏱ 60 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of iPSC-derived 3D brain organoids and their exposure to SARS-CoV-2 virus to model neurotropic infection. The organoids develop mature neuronal populations in cortical regions and exhibit viral entry into neurons, Tau pathology, and neuronal cell death upon infection.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

42 steps across 8 phases

iPSC-Derived Brain Organoid Generation Day 0-60
  1. 1 iPSC Preparation
  2. 2 Neural Induction
  3. 3 Neurosphere Formation and Embedding
  4. 4 Organoid Maturation in Spinner Flasks
  5. 5 Organoid Culture to Day 15
  6. 6 Organoid Culture to Day 60
SARS-CoV-2 Isolation and Propagation Preparation (0-4 days)
  1. 1 Clinical Specimen Collection
  2. 2 Specimen Preparation for Inoculation
  3. 3 Vero Cell Inoculation
  4. 4 Viral Replication Monitoring
  5. 5 Viral Propagation
  6. 6 Viral Titer Determination (TCID50 Assay)
SARS-CoV-2 Infection of Brain Organoids Day 15 or 60 organoid age + 0-6 days post-infection
  1. 1 Organoid Transfer to Infection Plates
  2. 2 Viral Inoculation
  3. 3 Mock Control Infection
  4. 4 Sampling at 2 Days Post-Infection (dpi)
  5. 5 Sampling at 4-6 Days Post-Infection (dpi)
Organoid Slice Culture (Optional Enhanced Maturation) Day 60 organoids + 0-2 days exposure
  1. 1 Organoid Sectioning
  2. 2 Slice Plating
  3. 3 Viral Exposure of Organotypic Slices
Tissue Processing and Immunofluorescence Post-infection sampling
  1. 1 Organoid Fixation
  2. 2 Sucrose Infiltration
  3. 3 Tissue Embedding and Sectioning
  4. 4 Antigen Retrieval (for SOX2 staining)
  5. 5 Permeabilization
  6. 6 Blocking
  7. 7 Primary Antibody Incubation
  8. 8 Secondary Antibody Incubation
  9. 9 Nuclear Staining
  10. 10 Mounting
Confocal Microscopy and Image Analysis Post-mounting
  1. 1 Confocal Image Acquisition
  2. 2 Deconvolution and 3D Reconstruction
  3. 3 Image Processing and Visualization
  4. 4 Quantitative Analysis
Viral RNA Quantification (qRT-PCR) At each sampling timepoint
  1. 1 RNA Extraction
  2. 2 qRT-PCR Preparation
  3. 3 Real-Time PCR Cycling
  4. 4 Standard Curve Generation
  5. 5 Sample Analysis
TUNEL Assay for Apoptosis Detection At 2-6 dpi
  1. 1 Assay Preparation
  2. 2 TUNEL Staining
  3. 3 Quantification

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ramani et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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