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BRAIN Publication-derived

Brain Organoids for Infection Studies (No. 2)

Full SOP with Academic or a plan

Source Chu et al. · 10.1021/acsinfecdis.9b00526

Authors: Chu, H. Duration: 72 days 12 phases Cell source: 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

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

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Attribution

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

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