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BRAIN Publication-derived

SARS-CoV-2 Infection of iPSC-Derived Human Brain Cortical Organoids and Sofosbuvir Treatment

Source Mesci et al., 2022 · University of California San Diego · 10.1371/journal

👤 Pinar Mesci, Janaina S. de Souza, Laura Martin-Sancho, Angela Macia, Aurian Saleh, Xin Yin, Cedric Snethlage, Jason W. Adams, Simoni H. Avansini, Roberto H. Herai, Angels Almenar-Queralt, Yuan Pu, Ryan A. Szeto, Gabriela Goldberg, Patrick T. Bruck, Fabio Papes, Sumit K. Chanda, Alysson R. Muotri ⏱ 52 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of eight-week-old human brain cortical organoids (BCO) from healthy donor iPSCs, their infection with SARS-CoV-2, and treatment with the FDA-approved antiviral Sofosbuvir. The protocol enables assessment of viral replication, neural cell death, synaptic loss, and antiviral efficacy in a human brain tissue model.

Cell source
Human iPSC
Application
Disease modeling and drug screening

Protocol overview

44 steps across 10 phases

iPSC Culture and Maintenance Prior to differentiation
  1. 1 Culture iPSC lines on Matrigel-coated plates
Neural Induction and Brain Organoid Differentiation Days 1–52
  1. 1 Initiate neural induction with growth factor inhibition
  2. 2 Apply neural induction medium
  3. 3 Switch to neural proliferation medium with bFGF
  4. 4 Continue neural proliferation with EGF supplementation
  5. 5 Apply neuronal maturation medium
  6. 6 Maintain organoids in maintenance medium
Viral Infection Day 52
  1. 1 Prepare SARS-CoV-2 inoculum
  2. 2 Infect brain organoids with SARS-CoV-2
  3. 3 Remove inoculum and wash organoids
Post-Infection Treatment and Sample Collection Days 0–7 post-infection
  1. 1 Apply Sofosbuvir treatment
  2. 2 Collect supernatant for viral quantification
  3. 3 Harvest organoids for analysis at day 7 post-infection
Tissue Fixation and Processing for Immunostaining Day 7 post-infection (or as scheduled)
  1. 1 Fix organoids with paraformaldehyde
  2. 2 Cryoprotect organoids in sucrose
  3. 3 Embed organoids in optimal cutting temperature (OCT) compound
  4. 4 Cryosection organoids
Immunofluorescence Staining Post-sectioning
  1. 1 Permeabilize tissue sections
  2. 2 Block non-specific binding
  3. 3 Apply primary antibodies
  4. 4 Wash primary antibodies
  5. 5 Apply secondary antibodies
  6. 6 Apply TUNEL stain (for apoptosis detection)
  7. 7 Apply nuclear counterstain and mount
Microscopy Image Acquisition and Analysis Post-mounting
  1. 1 Acquire confocal images
  2. 2 Analyze images with ImageJ software
  3. 3 Generate 3D reconstructions
RNA Extraction and qRT-PCR Analysis As scheduled (e.g., 8, 24, 48 hours, or 7 days post-infection)
  1. 1 Extract total RNA
  2. 2 Quantify viral RNA by qRT-PCR
  3. 3 Verify viral RNA peak kinetics
Western Blot Analysis Day 7 post-infection
  1. 1 Extract total protein from organoids
  2. 2 Prepare protein samples for gel electrophoresis
  3. 3 Separate proteins by SDS-PAGE
  4. 4 Transfer proteins to nitrocellulose membrane
  5. 5 Block and incubate with primary antibody
  6. 6 Wash and incubate with secondary antibody
  7. 7 Image and quantify bands
Plaque Assay for Infectious Viral Quantification 16 hours post-infection (sample collection) + 3 days for plaque formation
  1. 1 Collect and freeze organoid supernatants
  2. 2 Prepare Vero E6 cell monolayers
  3. 3 Perform 10-fold serial dilutions of supernatants
  4. 4 Infect cell monolayers and incubate
  5. 5 Add overlay medium
  6. 6 Incubate and visualize plaques
  7. 7 Count and calculate viral titer

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Mesci et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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