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BRAIN Publication-derived

Zika virus impairs growth in human neurospheres and brain organoids

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

👤 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 ⏱ 46 days 📋 7 phases 🧫 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

NSC Preparation and ZIKV Exposure Day 0–1
  1. 1 Culture human iPSC-derived neural stem cells (NSCs)
  2. 2 Expose NSCs to ZIKV at specified multiplicity of infection (MOI)
  3. 3 Incubate infected NSCs for 24 hours
Neurosphere Formation and Analysis Day 3–6
  1. 4 Culture NSCs as neurospheres for initial assessment
  2. 5 Observe neurosphere morphology at 3 days in vitro (DIV)
  3. 6 Continue culture to 6 DIV and count neurospheres
Brain Organoid Preparation and ZIKV Exposure Day 35 of organoid development
  1. 7 Prepare human iPSC-derived brain organoids at day 35 of development
  2. 8 Expose brain organoids to ZIKV or mock conditions
Organoid Growth Monitoring and Analysis Day 35–46 of organoid development
  1. 9 Monitor organoid growth for 11 days in vitro (DIV) post-exposure
  2. 10 Measure organoid growth area before and after 11 DIV exposure
Viability and Cell Death Assessment Day 0–6 (NSC and neurosphere phase)
  1. 11 Perform qRT-PCR to detect ZIKV RNA in culture supernatants
  2. 12 Assess ZIKV viral protein localization by immunocytochemistry
  3. 13 Evaluate cell death markers in neurospheres
Ultrastructural Analysis by Electron Microscopy Day 6 post-infection (neurospheres)
  1. 14 Prepare neurosphere samples for transmission electron microscopy (TEM)
  2. 15 Examine ultrastructural features by electron microscopy
Dengue Virus Control Experiments Day 0–11
  1. 16 Prepare dengue virus 2 (DENV2) control infection
  2. 17 Compare DENV2 and ZIKV effects on NSC infection and cell death

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Garcez et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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