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BRAIN Publication-derived

Influenza Brain Organoids

Full SOP with Academic or a plan

Source Zhang et al., 2022 · Department of Chemical Biology, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University · 10.7150/thno.75123

Authors: Xiaodong Zhang, Haishuang Lin, Liangzhen Dong, Qing Xia Duration: 40 days 11 phases Cell source: Human ESC

Abstract

This protocol describes the generation of human pluripotent stem cell (hPSC)-derived brain organoids and their use as an in vitro model to study influenza virus infection, viral neuropathogenesis, and to screen antiviral compounds. The organoids recapitulate key aspects of human brain development and are infected with H1N1-WSN virus to model virus-induced neuroinflammation, apoptosis, and identify therapeutic targets.

Cell source
Human ESC
Application
Disease modeling; Antiviral and neuroprotective drug screening

Protocol overview

42 steps across 11 phases

Phase 1 · Neural Stem Cell Differentiation Day 0-7
  1. a Prepare hESCs for differentiation
  2. b Initiate neural differentiation
Phase 2 · Brain Organoid Generation and Culture Day 0-10+ (40+ days)
  1. a Form embryoid bodies (EBs)
  2. b Transfer EBs to neural induction medium
  3. c Embed EBs in Matrigel and initiate organoid maturation
  4. d Transfer organoids to orbital shaker and continue differentiation
Phase 3 · Virus Preparation and Titration Preparation phase (before infection)
  1. a Prepare virus stocks
  2. b Determine virus titer by TCID50
Phase 4 · Virus Infection of Brain Organoids Day 40 organoids; infection at 1 and 4 dpi
  1. a Prepare organoids for infection
  2. b Remove inoculum and continue culture
  3. c Harvest samples at 24 h and 96 h post-infection
Phase 5 · Immunofluorescence Staining of Brain Organoids Post-infection (1 dpi and 4 dpi)
  1. a Fix organoids
  2. b Cryoprotect and freeze organoids
  3. c Obtain cryosections
  4. d Permeabilize and block sections
  5. e Incubate with primary antibodies
  6. f Incubate with secondary antibodies
  7. g Mount and image sections
  8. h Apoptosis detection by TUNEL assay
Phase 6 · RNA Isolation and Quantitative RT-PCR Post-infection (1 dpi and 4 dpi)
  1. a Extract total RNA
  2. b Perform quantitative RT-PCR
  3. c Calculate relative expression
Phase 7 · Enzyme-Linked Immunosorbent Assay (ELISA) Post-infection (1 dpi and 4 dpi)
  1. a Measure inflammatory factors in organoid supernatants
  2. b Read optical density
Phase 8 · Whole Transcriptome RNA-Seq Analysis Post-infection (1 dpi and 4 dpi)
  1. a Extract RNA and prepare samples
  2. b Construct RNA libraries
  3. c Sequence libraries
  4. d Trim adapters and align reads
  5. e Quantify gene expression
Phase 9 · Microelectrode Array (MEA) Recording Day 40 organoids; recording over 13 days post-seeding on MEA plates
  1. a Seed organoids on MEA plates
  2. b Perform MEA recordings
  3. c Apply test compounds
  4. d Image organoids
Phase 10 · Antiviral Drug Screening Day 40 organoids; drug co-treatment with virus for 1 and 4 dpi
  1. a Pre-treat organoids with compounds
  2. b Co-treat with virus and compounds
  3. c Continue compound treatment post-infection
  4. d Assess antiviral efficacy
Phase 11 · Neurotrophic Factor Treatment Day 40 organoids; co-treatment with virus for 1 and 4 dpi
  1. a Prepare neurotrophic factor solutions
  2. b Pre-treat organoids with neurotrophic factors
  3. c Co-treat with virus and neurotrophic factors
  4. d Continue neurotrophic factor treatment
  5. e Assess antiviral and neuroprotective efficacy

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Attribution

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

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