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BRAIN Publication-derived

Zika Virus Infection Brain Organoids for Disease Modelling

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 6 phases Cell source: Human iPSC-derived Neural Stem Cells (NSCs)

Abstract

This protocol describes the infection of human iPSC-derived neural stem cells, neurospheres, and brain organoids with Zika virus (ZIKV) to investigate the effects of ZIKV infection during neurogenesis. The study demonstrates that ZIKV targets human brain cells, reduces their viability and growth, and induces cell death in neural stem cells, providing evidence of how ZIKV may impair human brain development during early fetal stages.

Cell source
Human iPSC-derived Neural Stem Cells (NSCs)
Application
Disease modeling

Protocol overview

21 steps across 6 phases

Phase 1 · Neural Stem Cell Culture and Viral Exposure Day 0-1
  1. a Culture human iPS-derived NSCs
  2. b Expose NSCs to Zika virus
  3. c Incubate infected NSCs for 24 hours
Phase 2 · Neurosphere Formation and Assessment Day 0-6
  1. a Culture mock- and ZIKV-infected NSCs as neurospheres
  2. b Assess neurosphere morphology at 3 days in vitro (DIV)
  3. c Count neurosphere numbers at 6 DIV
Phase 3 · Viral Detection and Characterization Day 1-3
  1. a Perform immunocytochemistry for ZIKV detection
  2. b Quantify ZIKV-infected SOX2-positive cells
  3. c Detect ZIKV RNA by quantitative RT-PCR
Phase 4 · Ultrastructural Analysis of Infected Neurospheres Day 6
  1. a Prepare neurospheres for electron microscopy
  2. b Image and characterize ultrastructure
Phase 5 · Brain Organoid Culture and ZIKV Exposure Day 35-46
  1. a Culture human iPS-derived brain organoids
  2. b Expose 35-day-old organoids to mock or ZIKV
  3. c Measure organoid area before and after 11 DIV of viral exposure
  4. d Document organoid morphology and cell detachment
Phase 6 · Control Experiment: Dengue Virus 2 (DENV2) Comparison Day 1-11
  1. a Culture NSCs infected with Dengue virus 2 (DENV2)
  2. b Assess DENV2 infection rate at 24 hours
  3. c Measure caspase 3/7-mediated cell death at 3 DIV
  4. d Compare cell viability at 6 DIV
  5. e Assess DENV2-exposed neurosphere morphology at 6 DIV
  6. f Measure growth of DENV2-exposed brain organoids at 11 DIV

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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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