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BRAIN Publication-derived

Microcephaly Cerebral Organoids for Infection Studies

Full SOP with Academic or a plan

Source Dang et al., 2016 · University of California San Diego · 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 11 phases Cell source: 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

Phase 1 · hESC Culture and Embryoid Body Formation Day 0-2
  1. a Prepare hESCs for dissociation
  2. b Secondary dissociation with dispase
  3. c Generate single-cell suspension
  4. d Form embryoid bodies by hanging drop method
  5. e Transfer embryoid bodies to petri dishes
Phase 2 · Neural Induction and Organoid Differentiation Day 6-15
  1. a Transfer to neural induction media
  2. b Embed organoids in Matrigel
  3. c Transfer to stir flask bioreactors
  4. d Maintain organoid cultures
Phase 3 · ZIKV Expansion and Viral Titer Determination Prior to infection
  1. a Prepare Vero cells for virus expansion
  2. b Change medium 24 hours post-inoculation
  3. c Harvest viral supernatant
  4. d Quantify viral titer
Phase 4 · Organoid Infection with ZIKV Day 10 post-organoid generation
  1. a Prepare organoids for infection
  2. b Inoculate organoids with ZIKV
  3. c Monitor organoid growth post-infection
  4. d Quantify viral copy number in supernatant
Phase 5 · TLR3 Activation and Inhibition Studies Parallel to infection studies (Day 10+)
  1. a Treat organoids with TLR3 agonist poly(I:C)
  2. b Treat organoids with TLR3 competitive inhibitor
  3. c Co-treat with ZIKV and TLR3 inhibitor
  4. d Measure organoid size changes
Phase 6 · Sample Collection and RNA Extraction Throughout infection and treatment studies
  1. a Harvest organoids for immunostaining
  2. b Fix organoids
  3. c Prepare organoids for cryosectioning
  4. d Extract total RNA for qPCR
  5. e Synthesize cDNA
Phase 7 · Immunohistochemistry and Imaging Following sample collection
  1. a Block and permeabilize cryosections
  2. b Incubate with primary antibodies
  3. c Wash and incubate with secondary antibodies
  4. d Mount and image samples
Phase 8 · Quantitative RT-qPCR Analysis Following cDNA synthesis
  1. a Set up qPCR reactions
  2. b Perform qPCR
  3. c Analyze expression data
Phase 9 · RNA-seq Transcriptome Analysis Following RNA extraction
  1. a Prepare RNA for sequencing
  2. b Perform RNA sequencing
  3. c Map reads and normalize data
  4. d Identify differentially expressed genes
  5. e Perform pathway and ontology analysis
Phase 10 · Supplementary: Neurosphere Culture and Analysis Parallel to organoid studies
  1. a Culture mouse neurospheres
  2. b Treat neurospheres with ZIKV and TLR3 modulators
  3. c Measure neurosphere size changes
Phase 11 · Supplementary: Functional Calcium Imaging Day 20-30 of organoid culture
  1. a Load organoids with Fluo-4 calcium indicator
  2. b Stimulate with glutamate
  3. c Analyze calcium dynamics

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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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