Skip to content
Back to browse
BRAIN Publication-derived

Herpes Simplex Virus Infection Brain Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 65 days 11 phases Cell source: 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

Phase 1 · hESC Maintenance and Preparation Variable, pre-organoid
  1. a Culture feeder-free H9 cells
Phase 2 · Cerebral Organoid Generation (Days 0-15) Days 0-15
  1. a Initiate organoid formation in ultra-low binding 96-well plates
  2. b Replace medium (Day 3)
  3. c Begin neural induction (Days 5-6)
  4. d Embed organoids in Matrigel droplets (Days 10-11)
  5. e Transition to differentiation medium (Day 15)
Phase 3 · Cerebral Organoid Maturation (Days 15-65+) Days 15-65+
  1. a Continue differentiation and medium changes
  2. b Enhanced maturation from day 65 onwards
Phase 4 · Viral Inoculation and Infection (Days 10-12 organoids) Day 10 (organoid age)
  1. a Prepare virus inoculum
  2. b Expose day-10 organoids to virus inoculum
  3. c Embed infected organoids in Matrigel and culture
Phase 5 · Interferon and Poly(I:C) Treatments Days 2-8 post-infection or stimulation
  1. a Prepare recombinant human interferons
  2. b Apply interferon treatments
  3. c Stimulate organoids with poly(I:C)
Phase 6 · Sample Collection and Analysis (Days 4, 8, 12 post-infection) Days 4, 8, 12 post-infection
  1. a Collect supernatants for viral titer and cytokine assays
  2. b Harvest organoids for histology and RNA extraction
Phase 7 · Immunostaining and Microscopy Variable (after sample collection)
  1. a Cryopreservation and sectioning of fixed organoids
  2. b Immunostaining of organoid sections
  3. c Imaging and quantification
Phase 8 · RNA Extraction and RT-qPCR Variable (same day as sample collection or from frozen lysates)
  1. a Extract total RNA from organoid lysates
  2. b Synthesize cDNA
  3. c Perform qPCR analysis
Phase 9 · Viral Titer Determination (Vero Cell Bioassay) 5 days (performed on Vero cells)
  1. a ZIKV TCID₅₀ assay
  2. b HSV-1 plaque assay
Phase 10 · Cytokine Quantification by ELISA Same day as supernatant analysis or within a few days
  1. a Prepare and concentrate supernatants
  2. b Perform ELISA for IFN-α and IFN-β
Phase 11 · RNA-Sequencing (Optional, for transcriptional profiling) Variable (sample collection to data analysis)
  1. a Prepare RNA samples for sequencing
  2. b Sequence and analyze

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Krenn et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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