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BRAIN Publication-derived

SARS-CoV-2 targets glial cells in human cortical organoids

Source McMahon et al. · <UNKNOWN>

👤 Courtney L. McMahon, Hilary Staples, Michal Gazi, Ricardo Carrion, Jenny Hsieh ⏱ 43 days 📋 6 phases 🧫 Human ESC (H9)

Abstract

This protocol generates human cortical organoids from H9 embryonic stem cells using dual SMAD inhibition and characterizes SARS-CoV-2 infection patterns, particularly in glial cells, using immunofluorescence and molecular analysis. The method enables investigation of viral tropism and cellular responses in a 3D human brain tissue model.

Cell source
Human ESC (H9)
Application
Disease modeling, Viral infection study

Protocol overview

25 steps across 6 phases

ESC Expansion and Neural Induction Days 0-6
  1. 1 H9 ESC Culture to Confluence
  2. 2 Dissociation to Single Cells
  3. 3 Transfer to Ultralow-Attachment Plates
  4. 4 Medium Replacement at Day 6
Organoid Growth and Maturation Days 6-43
  1. 5 EGF and FGF-2 Supplementation (Days 6–24)
  2. 6 Switch to BDNF and NT3 (Day 24–43)
  3. 7 Unsupplemented Medium (Day 43+)
Vero E6 Cell Culture and SARS-CoV-2 Virus Amplification Preparation phase
  1. 8 Vero E6 Cell Culturing
  2. 9 P5 Virus Generation from P4
  3. 10 P6 Working Stock Generation from P5
RNA Isolation and Quantification Experimental phase
  1. 11 Virus Inactivation with TRIzol
  2. 12 RNA Extraction Using Liquid Handler
  3. 13 RNA Quantification
RT-qPCR for Genome Equivalents and Subgenomic RNA Experimental phase
  1. 14 RT-qPCR Setup for Genomic RNA Quantification
  2. 15 Standard Curve and GE Calculations
  3. 16 Subgenomic RNA qPCR (E Region/Envelope)
Immunofluorescence and Confocal Microscopy Analysis phase
  1. 17 Organoid Fixation
  2. 18 Cryoprotection in Sucrose
  3. 19 OCT Embedding and Cryosectioning
  4. 20 Blocking and Permeabilization
  5. 21 Primary Antibody Incubation
  6. 22 Secondary Antibody Incubation
  7. 23 Streptavidin Incubation
  8. 24 TBS Washes
  9. 25 Confocal Microscopy Imaging

Full SOP

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Attribution

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

This wording is awaiting legal review.

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