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BRAIN Publication-derived

Herpes Simplex Encephalitis Cerebral Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 60 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Rybak-Wolf et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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