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BRAIN Publication-derived

Zika Virus Infection-Induced Microcephaly Cerebral Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Dang et al., 2016 · University of California San Diego, Department of Pediatrics · 10.1016/j.stem.2016.04.014

Authors: Jason Dang, Shashi Kant Tiwari, Gianluigi Lichinchi, Yue Qin, Veena S. Patil, Alexey M. Eroshkin, Tariq M. Rana Duration: 30 days 7 phases Cell source: Human ESC (H9 hESC, WA09)

Abstract

This protocol describes the generation of human embryonic stem cell-derived cerebral organoids to model early fetal brain development and investigate Zika virus (ZIKV) infection mechanisms. The protocol demonstrates ZIKV infection of neural progenitor cells via TLR3-mediated innate immune activation, leading to dysregulation of neurogenesis and increased apoptosis, recapitulating microcephaly-like phenotypes.

Cell source
Human ESC (H9 hESC, WA09)
Application
Disease modeling - Zika virus-induced microcephaly

Protocol overview

20 steps across 7 phases

Phase 1 · hESC Preparation and Embryoid Body Formation Day 0 to Day 2
  1. a hESC Culture and Passaging
  2. b Hanging Drop Embryoid Body Formation
  3. c Transfer Embryoid Bodies to Culture Dishes
Phase 2 · Neural Induction and Organoid Formation Day 2 to Day 15
  1. a Transfer to Neural Induction Media
  2. b Matrigel Embedding and Differentiation Media Transfer
  3. c Transfer to Bioreactors and Long-Term Culture
Phase 3 · ZIKV Expansion and Viral Inoculation Day -3 to Day 0 (before infection experiment)
  1. a ZIKV Stock Expansion in Vero Cells
  2. b Viral Titer Quantification
  3. c Organoid Infection with ZIKV
Phase 4 · Post-Infection Monitoring and Analysis Day 0 to Day 5 (post-infection)
  1. a Organoid Size Tracking
  2. b Viral Kinetics Assessment by RT-qPCR
  3. c Immunohistochemistry for ZIKV and Neural Markers
Phase 5 · TLR3 Activation and Inhibition Studies Concurrent with Phase 4, Day 0 to Day 5
  1. a TLR3 Agonist Treatment with poly(I:C)
  2. b TLR3 Competitive Inhibitor Treatment
  3. c Size Quantification of Neurospheres and Organoids
Phase 6 · Gene Expression Analysis Throughout infection (Day 0 to Day 5)
  1. a Total RNA Extraction
  2. b RT-qPCR Analysis of TLR3 and Target Genes
  3. c RNA-Seq Transcriptome Analysis
  4. d Pathway and Gene Ontology Analysis
Phase 7 · Functional Validation - Calcium Imaging Day 20-30 of organoid culture (optional, for functional characterization)
  1. a Calcium Dye Imaging of Neural Activity

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Attribution

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

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