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BRAIN Publication-derived

SARS-CoV-2 infection of human neural progenitor cells and brain organoids

Source Chu et al. · <UNKNOWN> · 10.1021/acsinfecdis.9b00526

👤 Chu, H., <UNKNOWN> ⏱ 72 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human neural progenitor cells (hNPCs), neurospheres, and brain organoids from iPSCs, and their infection with SARS-CoV-2 to study viral permissiveness and replication in neural tissues. The protocol includes characterization of infected cultures via immunostaining, electron microscopy, qRT-PCR, and plaque assays.

Cell source
Human iPSC
Application
Disease modeling; viral infection studies

Protocol overview

72 steps across 12 phases

Generation and maintenance of human iPSCs Days 1–7
  1. 1 Maintain iPSCs in culture
Differentiation of iPSCs to human neural progenitor cells (hNPCs) Days 1–9
  1. 1 Prepare differentiation medium
  2. 2 Add differentiation cocktail to basal medium
  3. 3 Begin differentiation
  4. 4 Refresh medium (first 4 days)
  5. 5 Refresh medium (days 5–9)
  6. 6 Seed differentiated hNPCs for infection
Formation of neurospheres Days 1–21
  1. 1 Dissociate iPSCs
  2. 2 Prepare neurosphere induction medium
  3. 3 Form embryoid bodies (EBs)
  4. 4 Incubate EBs
  5. 5 Plate EBs for neural rosette formation
  6. 6 Generate neurospheres
Formation of brain organoids Days 1–35
  1. 1 Maintain feeder-free iPSCs
  2. 2 Dissociate iPSC colonies
  3. 3 Centrifuge dissociated cells
  4. 4 Prepare medium with inhibitors
  5. 5 Transfer cells and add ROCK inhibitor
  6. 6 Form free-floating spheres
  7. 7 Replace medium with neurobasal Media I
  8. 8 Maintain in Media I
  9. 9 Replace with neurobasal Media II
  10. 10 Maintain in Media II
  11. 11 Replace with neurobasal Media III
  12. 12 Maintain in Media III
  13. 13 Transfer to Media II for maturation
Virus propagation and biosafety Pre-infection preparation
  1. 1 Propagate SARS-CoV-2 and SARS-CoV
  2. 2 Titer viruses by plaque assay
Infection of neurospheres and organoids with SARS-CoV-2 Single time point
  1. 1 Prepare viral inoculum
  2. 2 Inoculate neurospheres or organoids
  3. 3 Remove inoculum
  4. 4 Wash organoids/spheres
  5. 5 Continue culture until harvest
Infection of hNPCs with SARS-CoV-2 or SARS-CoV Single time point
  1. 1 Challenge hNPCs with virus
  2. 2 Harvest supernatant samples
  3. 3 Lyse samples for viral RNA extraction
Immunofluorescence staining and confocal microscopy Single time point
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Incubate in sucrose solution
  3. 3 Embed in cryomold
  4. 4 Cryosection organoids
  5. 5 Wash sections in PBS
  6. 6 Permeabilize tissue
  7. 7 Block nonspecific binding
  8. 8 Apply primary antibodies
  9. 9 Wash after primary antibodies
  10. 10 Apply secondary antibodies and DAPI
  11. 11 Wash after secondary antibodies
  12. 12 Mount sections
  13. 13 Acquire confocal images
  14. 14 Process images
Transmission electron microscopy (TEM) Single time point
  1. 1 Wash samples in PBS
  2. 2 Primary fixation with glutaraldehyde
  3. 3 Post-fixation with osmium tetroxide
  4. 4 Dehydrate and embed
  5. 5 Prepare ultrathin sections
  6. 6 Stain ultrathin sections
  7. 7 Examine by transmission electron microscopy
Viral RNA extraction and qRT-PCR quantification 0–72 hours post-infection
  1. 1 Harvest supernatant samples
  2. 2 Extract viral RNA
  3. 3 Quantify viral titer by qRT-PCR
  4. 4 Analyze qRT-PCR data
  5. 5 Measure host gene expression
Plaque assay for viral titration 0–72 hours post-infection
  1. 1 Harvest supernatant for titration
  2. 2 Inoculate Vero E6 cells
  3. 3 Fix infected cells
  4. 4 Stain plaques with crystal violet
  5. 5 Count plaques and calculate titer
Cell viability assay Post-infection time points
  1. 1 Prepare cells for viability assessment
  2. 2 Apply CellTiter-Glo reagent
  3. 3 Incubate samples
  4. 4 Measure luminescence
  5. 5 Analyze viability data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Chu 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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