Skip to content
← Back to browse
BRAIN Publication-derived

Zika Virus Infection of Human Cerebral Organoids and Analysis of TLR3-Mediated Immune Response

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

👤 Jason Dang, Shashi Kant Tiwari, Gianluigi Lichinchi, Yue Qin, Veena S. Patil, Alexey M. Eroshkin, Tariq M. Rana ⏱ 30 days 📋 11 phases 🧫 Human ESC (H9 hESCs)

Abstract

This protocol describes the generation of human embryonic stem cell-derived cerebral organoids and their infection with Zika virus (MR766 strain) to model early fetal brain development and study virus-induced neural progenitor depletion through TLR3 activation.

Cell source
Human ESC (H9 hESCs)
Application
Disease modeling (Zika virus-induced microcephaly)

Protocol overview

44 steps across 11 phases

hESC Culture and Embryoid Body Formation Day 0–2
  1. 1 Prepare hESCs for dissociation
  2. 2 Secondary dissociation with dispase
  3. 3 Generate single-cell suspension
  4. 4 Form embryoid bodies by hanging drop method
  5. 5 Transfer embryoid bodies to petri dishes
Neural Induction and Organoid Differentiation Day 6–15
  1. 1 Transfer to neural induction media
  2. 2 Embed organoids in Matrigel
  3. 3 Transfer to stir flask bioreactors
  4. 4 Maintain organoid cultures
ZIKV Expansion and Viral Titer Determination Prior to infection
  1. 1 Prepare Vero cells for virus expansion
  2. 2 Change medium 24 hours post-inoculation
  3. 3 Harvest viral supernatant
  4. 4 Quantify viral titer
Organoid Infection with ZIKV Day 10 post-organoid generation
  1. 1 Prepare organoids for infection
  2. 2 Inoculate organoids with ZIKV
  3. 3 Monitor organoid growth post-infection
  4. 4 Quantify viral copy number in supernatant
TLR3 Activation and Inhibition Studies Parallel to infection studies (Day 10+)
  1. 1 Treat organoids with TLR3 agonist poly(I:C)
  2. 2 Treat organoids with TLR3 competitive inhibitor
  3. 3 Co-treat with ZIKV and TLR3 inhibitor
  4. 4 Measure organoid size changes
Sample Collection and RNA Extraction Throughout infection and treatment studies
  1. 1 Harvest organoids for immunostaining
  2. 2 Fix organoids
  3. 3 Prepare organoids for cryosectioning
  4. 4 Extract total RNA for qPCR
  5. 5 Synthesize cDNA
Immunohistochemistry and Imaging Following sample collection
  1. 1 Block and permeabilize cryosections
  2. 2 Incubate with primary antibodies
  3. 3 Wash and incubate with secondary antibodies
  4. 4 Mount and image samples
Quantitative RT-qPCR Analysis Following cDNA synthesis
  1. 1 Set up qPCR reactions
  2. 2 Perform qPCR
  3. 3 Analyze expression data
RNA-seq Transcriptome Analysis Following RNA extraction
  1. 1 Prepare RNA for sequencing
  2. 2 Perform RNA sequencing
  3. 3 Map reads and normalize data
  4. 4 Identify differentially expressed genes
  5. 5 Perform pathway and ontology analysis
Supplementary: Neurosphere Culture and Analysis Parallel to organoid studies
  1. 1 Culture mouse neurospheres
  2. 2 Treat neurospheres with ZIKV and TLR3 modulators
  3. 3 Measure neurosphere size changes
Supplementary: Functional Calcium Imaging Day 20–30 of organoid culture
  1. 1 Load organoids with Fluo-4 calcium indicator
  2. 2 Stimulate with glutamate
  3. 3 Analyze calcium dynamics

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

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

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence