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BRAIN Publication-derived

COVID-19 Choroid Plexus Organoids for Infection Studies

Full SOP with Academic or a plan

Source Pellegrini et al., 2020 · MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK · 10.1016/j.stem.2020.10.001

Authors: Laura Pellegrini, Anna Albecka, Donna L. Mallery, Max J. Kellner, David Paul, Andrew P. Carter, Leo C. James, Madeline A. Lancaster Duration: 55 days 9 phases Cell source: Human ESC (H9)

Abstract

This protocol generates human brain organoids with choroid plexus (ChP) identity and characterizes SARS-CoV-2 infection dynamics in this tissue. The protocol demonstrates that SARS-CoV-2 productively infects ChP epithelial cells but not neurons, and that viral infection disrupts the blood-cerebrospinal fluid barrier through tight junction damage and leakage.

Cell source
Human ESC (H9)
Application
Disease modeling - Viral neurotropism and barrier dysfunction

Protocol overview

50 steps across 9 phases

Phase 1 · Cerebral and Choroid Plexus Organoid Generation Days 0-30
  1. a Generate embryoid bodies (EBs)
  2. b Replace EB media with neural induction media
  3. c Embed EBs in Matrigel
  4. d Transfer to expansion culture
  5. e Apply BMP4 and CHIR for ChP specification (for ChP organoids only)
  6. f Add dissolved Matrigel to Maturation media (from day 30 onward)
Phase 2 · Viral Preparation and Pseudovirus Generation Days (variable - virus production)
  1. a Prepare HEK293T cells and transfect with viral plasmids
  2. b Harvest viral supernatant
  3. c Concentrate viral particles by ultracentrifugation
  4. d Resuspend pelleted virions
  5. e Quantify viral titer by qRT-PCR
  6. f Determine pseudovirus titer in 293T ACE2 cells
Phase 3 · Live SARS-CoV-2 Virus Production and Titration Days (variable - virus production)
  1. a Generate Vero-hACE2-TMPRSS2 cells
  2. b Infect Vero-hACE2-TMPRSS2 monolayer with SARS-CoV-2
  3. c Harvest and prepare virus stock
  4. d Determine viral titer by plaque assay
Phase 4 · Organoid Infection with Pseudovirus and Live Virus Days 55+ (organoid maturation dependent)
  1. a Prepare organoids for infection
  2. b Add virus to organoid culture
  3. c Incubate organoids with virus
Phase 5 · Air-Liquid Interface (ALI) Cerebral Organoid Preparation Days 55+ (prior to infection)
  1. a Collect and prepare mature organoids for sectioning
  2. b Embed organoids in low-gelling-temperature agarose
  3. c Vibratome sectioning
  4. d Transfer sections to culture inserts
  5. e Transition to serum-free culture
Phase 6 · Immunostaining and Confocal Microscopy Post-infection (variable)
  1. a Fix organoids in paraformaldehyde
  2. b Cryoprotect with sucrose gradient
  3. c Embed and section organoids
  4. d Permeabilize and block
  5. e Primary antibody incubation
  6. f Secondary antibody and DAPI staining
  7. g Optional: LipidTOX staining for lipid droplets
  8. h Mount slides
  9. i Confocal microscopy imaging
  10. j Image processing and analysis
Phase 7 · Viral Replication Analysis by qRT-PCR Post-infection (1-4 days)
  1. a Collect culture supernatant
  2. b Extract viral RNA
  3. c Prepare qRT-PCR reaction mix
  4. d Run one-step RT-qPCR
  5. e Analyze and report results
Phase 8 · Barrier Integrity and Leakage Assays Post-infection (typically days 2-4)
  1. a Assess organoid morphology
  2. b Measure internal and external fluid volumes
  3. c Measure total media protein concentration
  4. d Immunoblot for CSF-specific protein (APOJ/clusterin)
Phase 9 · Immunoblotting for ACE2 and Lipoprotein Markers Days 33-53 (organoid lysates) or post-infection
  1. a Prepare organoid lysates
  2. b Prepare protein samples
  3. c Run SDS-PAGE
  4. d Transfer to PVDF membrane
  5. e Block and incubate with primary antibodies
  6. f Wash and incubate with secondary antibodies
  7. g Image membrane

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Attribution

This SOP was written by Organthis based on the published method in Pellegrini et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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