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BRAIN Publication-derived

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids

Source Rybak-Wolf et al., 2023 · Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association · 10.1038/s41564-023-01405-y

👤 Agnieszka Rybak-Wolf, Emanuel Wyler, Tancredi Massimo Pentimalli, Ivano Legnini, Anna Oliveras Martinez, Petar Glažar, Anna Loewa, Seung Joon Kim, Benedikt B. Kaufer, Andrew Woehler, Markus Landthaler, Nikolaus Rajewsky ⏱ 60 days 📋 5 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human cerebral organoids from induced pluripotent stem cells and their infection with HSV-1 to model herpes simplex encephalitis. The protocol enables investigation of viral pathogenesis, neuronal dysfunction, and evaluation of therapeutic interventions including antiviral and anti-inflammatory agents.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

18 steps across 5 phases

Embryoid body formation and neural induction Day 0–11
  1. 1 Prepare iPS cell suspension and seed into embryoid body medium
  2. 2 Replace medium (day 4)
  3. 3 Transition to neural induction medium (day 5)
  4. 4 Embed organoids in Matrigel (days 7–9)
Organoid maturation and culture Day 11–60
  1. 5 Transfer organoids to ultralow-attachment plates and culture on shaker
  2. 6 Culture organoids until day 60
HSV-1 infection Day 60 (infection initiation)
  1. 7 Prepare HSV-1 inoculum
  2. 8 Infect organoids with HSV-1
  3. 9 Replace medium 24 hours post-infection
  4. 10 Culture infected organoids to 1 or 3 days post-infection (dpi)
Antiviral and anti-inflammatory treatment 8–12 hours post-infection onwards
  1. 11 Prepare treatment reagents
  2. 12 Initiate treatment 8–12 hours post-infection
  3. 13 Continue culture with treatment for 1 or 3 days
Analysis and characterization 1 and 3 days post-infection
  1. 14 Harvest organoids for molecular analysis
  2. 15 Perform immunostaining on cryosections
  3. 16 Perform western blot analysis
  4. 17 Perform calcium imaging of neuronal activity
  5. 18 Perform single-cell RNA sequencing (scRNA-seq)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Rybak-Wolf et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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