Skip to content
← Back to browse
BRAIN Publication-derived

SARS-CoV-2 Infection of Brain Choroid Plexus Organoids and Blood-CSF Barrier Disruption

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

👤 Laura Pellegrini, Anna Albecka, Donna L. Mallery, Max J. Kellner, David Paul, Andrew P. Carter, Leo C. James, Madeline A. Lancaster ⏱ 55 days 📋 9 phases 🧫 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

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

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

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

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence