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BRAIN Publication-derived

COVID-19 Cortical Organoids for Drug Screening

Full SOP with Academic or a plan

Source Mesci et al., 2022 · University of California San Diego · 10.1371/journal.pbio.3001845

Authors: 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 Duration: 52 days 10 phases Cell source: 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

Phase 1 · iPSC Culture and Maintenance Prior to differentiation
  1. a Culture iPSC lines on Matrigel-coated plates
Phase 2 · Neural Induction and Brain Organoid Differentiation Days 1-52
  1. a Initiate neural induction with growth factor inhibition
  2. b Apply neural induction medium
  3. c Switch to neural proliferation medium with bFGF
  4. d Continue neural proliferation with EGF supplementation
  5. e Apply neuronal maturation medium
  6. f Maintain organoids in maintenance medium
Phase 3 · Viral Infection Day 52
  1. a Prepare SARS-CoV-2 inoculum
  2. b Infect brain organoids with SARS-CoV-2
  3. c Remove inoculum and wash organoids
Phase 4 · Post-Infection Treatment and Sample Collection Days 0-7 post-infection
  1. a Apply Sofosbuvir treatment
  2. b Collect supernatant for viral quantification
  3. c Harvest organoids for analysis at day 7 post-infection
Phase 5 · Tissue Fixation and Processing for Immunostaining Day 7 post-infection (or as scheduled)
  1. a Fix organoids with paraformaldehyde
  2. b Cryoprotect organoids in sucrose
  3. c Embed organoids in optimal cutting temperature (OCT) compound
  4. d Cryosection organoids
Phase 6 · Immunofluorescence Staining Post-sectioning
  1. a Permeabilize tissue sections
  2. b Block non-specific binding
  3. c Apply primary antibodies
  4. d Wash primary antibodies
  5. e Apply secondary antibodies
  6. f Apply TUNEL stain (for apoptosis detection)
  7. g Apply nuclear counterstain and mount
Phase 7 · Microscopy Image Acquisition and Analysis Post-mounting
  1. a Acquire confocal images
  2. b Analyze images with ImageJ software
  3. c Generate 3D reconstructions
Phase 8 · RNA Extraction and qRT-PCR Analysis As scheduled (e.g., 8, 24, 48 hours, or 7 days post-infection)
  1. a Extract total RNA
  2. b Quantify viral RNA by qRT-PCR
  3. c Verify viral RNA peak kinetics
Phase 9 · Western Blot Analysis Day 7 post-infection
  1. a Extract total protein from organoids
  2. b Prepare protein samples for gel electrophoresis
  3. c Separate proteins by SDS-PAGE
  4. d Transfer proteins to nitrocellulose membrane
  5. e Block and incubate with primary antibody
  6. f Wash and incubate with secondary antibody
  7. g Image and quantify bands
Phase 10 · Plaque Assay for Infectious Viral Quantification 16 hours post-infection (sample collection) + 3 days for plaque formation
  1. a Collect and freeze organoid supernatants
  2. b Prepare Vero E6 cell monolayers
  3. c Perform 10-fold serial dilutions of supernatants
  4. d Infect cell monolayers and incubate
  5. e Add overlay medium
  6. f Incubate and visualize plaques
  7. g Count and calculate viral titer

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Attribution

This SOP was written by Organthis based on the published method in Mesci et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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