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BRAIN Publication-derived

Brain Organoids for Infection Studies (2016)

Full SOP with Academic or a plan

Source Garcez et al., 2016 · D'Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil · 10.1126/science.aad5252

Authors: Patricia P. Garcez, Erick Correia Loiola, Rodrigo Madeiro da Costa, Luiza M. Higa, Pablo Trindade, Rodrigo Delvecchio, Juliana Minardi Nascimento, Rodrigo Brindeiro, Amilcar Tanuri, Stevens K. Rehen Duration: 46 days 7 phases Cell source: Human iPSC-derived neural stem cells (NSCs)

Abstract

This protocol establishes methods to study Zika virus (ZIKV) infection in human iPSC-derived neural stem cells (NSCs) cultured as neurospheres and brain organoids. ZIKV-infected NSCs, neurospheres, and organoids exhibit reduced viability, altered morphology, and impaired growth compared to mock controls, providing an in vitro model to investigate ZIKV-induced microcephaly during early human brain development.

Cell source
Human iPSC-derived neural stem cells (NSCs)
Application
Disease modeling

Protocol overview

17 steps across 7 phases

Phase 1 · NSC Preparation and ZIKV Exposure Day 0-1
  1. a Culture human iPSC-derived neural stem cells (NSCs)
  2. b Expose NSCs to ZIKV at specified multiplicity of infection (MOI)
  3. c Incubate infected NSCs for 24 hours
Phase 2 · Neurosphere Formation and Analysis Day 3-6
  1. a Culture NSCs as neurospheres for initial assessment
  2. b Observe neurosphere morphology at 3 days in vitro (DIV)
  3. c Continue culture to 6 DIV and count neurospheres
Phase 3 · Brain Organoid Preparation and ZIKV Exposure Day 35 of organoid development
  1. a Prepare human iPSC-derived brain organoids at day 35 of development
  2. b Expose brain organoids to ZIKV or mock conditions
Phase 4 · Organoid Growth Monitoring and Analysis Day 35-46 of organoid development
  1. a Monitor organoid growth for 11 days in vitro (DIV) post-exposure
  2. b Measure organoid growth area before and after 11 DIV exposure
Phase 5 · Viability and Cell Death Assessment Day 0-6 (NSC and neurosphere phase)
  1. a Perform qRT-PCR to detect ZIKV RNA in culture supernatants
  2. b Assess ZIKV viral protein localization by immunocytochemistry
  3. c Evaluate cell death markers in neurospheres
Phase 6 · Ultrastructural Analysis by Electron Microscopy Day 6 post-infection (neurospheres)
  1. a Prepare neurosphere samples for transmission electron microscopy (TEM)
  2. b Examine ultrastructural features by electron microscopy
Phase 7 · Dengue Virus Control Experiments Day 0-11
  1. a Prepare dengue virus 2 (DENV2) control infection
  2. b Compare DENV2 and ZIKV effects on NSC infection and cell death

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Attribution

This SOP was written by Organthis based on the published method in Garcez et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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