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BRAIN Publication-derived

Organoid modeling of Zika and Herpes Simplex virus 1 infections reveals virus-specific responses leading to microcephaly

Source Krenn et al., 2021 · Institute of Molecular Biotechnology (IMBA), Vienna BioCenter (VBC), Vienna, Austria · 10.1016/j.stem.2021.03.004

👤 Veronica Krenn, Camilla Bosone, Thomas Rainer Burkard, Julia Spanier, Ulrich Kalinke, Arianna Calistri, Cristiano Salata, Raissa Rilo Christoff, Patricia Pestana Garcez, Ali Mirazimi, Jürgen Arthur Knoblich ⏱ 65 days 📋 11 phases 🧫 Human ESC (H9)

Abstract

This protocol describes the generation of human brain organoids from embryonic stem cells and their infection with Zika virus (ZIKV) or Herpes Simplex virus 1 (HSV-1) to model virus-induced microcephaly. The organoid models reproduce key phenotypic features of microcephaly, including growth attenuation, neural progenitor cell depletion, and distinct viral- and host-specific transcriptional signatures.

Cell source
Human ESC (H9)
Application
Disease modeling

Protocol overview

28 steps across 11 phases

hESC Maintenance and Preparation Variable, pre-organoid
  1. 1 Culture feeder-free H9 cells
Cerebral Organoid Generation (Days 0–15) Days 0–15
  1. 1 Initiate organoid formation in ultra-low binding 96-well plates
  2. 2 Replace medium (Day 3)
  3. 3 Begin neural induction (Days 5–6)
  4. 4 Embed organoids in Matrigel droplets (Days 10–11)
  5. 5 Transition to differentiation medium (Day 15)
Cerebral Organoid Maturation (Days 15–65+) Days 15–65+
  1. 1 Continue differentiation and medium changes
  2. 2 Enhanced maturation from day 65 onwards
Viral Inoculation and Infection (Days 10–12 organoids) Day 10 (organoid age)
  1. 1 Prepare virus inoculum
  2. 2 Expose day-10 organoids to virus inoculum
  3. 3 Embed infected organoids in Matrigel and culture
Interferon and Poly(I:C) Treatments Days 2–8 post-infection or stimulation
  1. 1 Prepare recombinant human interferons
  2. 2 Apply interferon treatments
  3. 3 Stimulate organoids with poly(I:C)
Sample Collection and Analysis (Days 4, 8, 12 post-infection) Days 4, 8, 12 post-infection
  1. 1 Collect supernatants for viral titer and cytokine assays
  2. 2 Harvest organoids for histology and RNA extraction
Immunostaining and Microscopy Variable (after sample collection)
  1. 1 Cryopreservation and sectioning of fixed organoids
  2. 2 Immunostaining of organoid sections
  3. 3 Imaging and quantification
RNA Extraction and RT-qPCR Variable (same day as sample collection or from frozen lysates)
  1. 1 Extract total RNA from organoid lysates
  2. 2 Synthesize cDNA
  3. 3 Perform qPCR analysis
Viral Titer Determination (Vero Cell Bioassay) 5 days (performed on Vero cells)
  1. 1 ZIKV TCID₅₀ assay
  2. 2 HSV-1 plaque assay
Cytokine Quantification by ELISA Same day as supernatant analysis or within a few days
  1. 1 Prepare and concentrate supernatants
  2. 2 Perform ELISA for IFN-α and IFN-β
RNA-Sequencing (Optional, for transcriptional profiling) Variable (sample collection to data analysis)
  1. 1 Prepare RNA samples for sequencing
  2. 2 Sequence and analyze

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Krenn et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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