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BRAIN Publication-derived

Neonatal Herpes Encephalitis Brain Organoids

Full SOP with Academic or a plan

Source D'Aiuto et al., 2022 · Western Psychiatric Institute and Clinic, Department of Psychiatry, School of Medicine, University of Pittsburgh · 10.3390/cells11223539

Authors: Leonardo D'Aiuto, Jill K. Caldwell, Callen T. Wallace, Tristan R. Grams, Maribeth A. Wesesky, Joel A. Wood, Simon C. Watkins, Paul R. Kinchington, David C. Bloom, Vishwajit L. Nimgaonkar Duration: 35 days 12 phases Cell source: Human iPSC

Abstract

This protocol models neurodevelopmental impairment caused by HSV-1 infection of early-stage brain organoids derived from human induced pluripotent stem cells (iPSCs). Early-stage organoids composed of neural rosettes are infected with HSV-1 and cultured in the presence or absence of acyclovir to investigate neuropathological effects on neural differentiation and the emergence of antiviral-resistant viral mutants.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

73 steps across 12 phases

Phase 1 · Cell Culture Maintenance Ongoing
  1. a Maintain Vero cells for plaque assay
  2. b Culture human iPSC line SC0000020 (subclone SF)
Phase 2 · Virus Preparation Days 1-3
  1. a Prepare HSV-1 virus stock
  2. b Harvest infected cells
  3. c Centrifuge cell suspension
  4. d Resuspend cell pellet
  5. e Freeze-thaw cycle
  6. f Remove debris
  7. g Store viral stock
Phase 3 · Virus Titration by Plaque Assay Days 1-4
  1. a Prepare Vero cell monolayers
  2. b Infect with serial dilutions of HSV
  3. c Overlay with CMC medium
  4. d Incubate plates
  5. e Remove overlay and fix cells
  6. f Stain and count plaques
Phase 4 · Acyclovir Resistance Assay Days 1-3
  1. a Prepare rabbit skin cells
  2. b Pretreat with acyclovir
  3. c Infect with culture supernatant
  4. d Replace medium with ACV-containing medium
  5. e Incubate and fix cells
  6. f Enumerate plaques
Phase 5 · Neural Rosette Generation from iPSCs Days 1-7
  1. a Prepare hiPSCs for neural induction
  2. b Withdraw ROCK inhibitor
  3. c Change culture medium daily
  4. d Monitor for neural rosette appearance
Phase 6 · Isolation and Expansion of Neural Rosettes Days 7-10
  1. a Manually dissect neural rosette clusters
  2. b Culture rosette clusters in suspension
  3. c Transfer to low-attachment 96-well plates
Phase 7 · Infection of Early-Stage Organoids from Neural Rosettes Days 10-30
  1. a Wash organoids
  2. b Prepare infection inoculum
  3. c Infect organoids
  4. d Incubate organoids
  5. e Remove inoculum
  6. f Wash organoids twice
  7. g Transfer to culture dishes
  8. h Monitor organoid development
Phase 8 · Generation of Early-Stage Brain Organoids from iPSCs (Alternative Method) Days 1-9
  1. a Generate embryoid bodies
  2. b Culture embryoid bodies
  3. c Switch to neural induction medium
  4. d Observe neuroectoderm formation
  5. e Verify neural rosette presence
  6. f Prepare organoids for infection
Phase 9 · Infection of Early-Stage Brain Organoids from Embryoid Bodies Day 9 onwards
  1. a Infect organoids individually
  2. b Allow viral absorption
  3. c Remove inocula
  4. d Wash organoids
  5. e Culture in neuronal differentiation medium
  6. f Change culture medium regularly
  7. g Monitor organoid status
Phase 10 · Generation and Infection of Adherent 3D (A-3D) Cultures Days 1-35
  1. a Generate monolayer NPC cultures
  2. b Infect NPC monolayers
  3. c Remove inocula and dissociate cells
  4. d Seed cells at high density
  5. e Culture in NPC maintenance medium
  6. f Switch to differentiation medium
  7. g Withdraw small molecules
  8. h Change medium schedule
  9. i Monitor A-3D cultures
Phase 11 · Immunofluorescence Analysis Days 4, 8, 15, 20, 35
  1. a Fix organoids in paraformaldehyde
  2. b Wash organoids
  3. c Cryoprotect organoids
  4. d Embed organoids
  5. e Prepare frozen sections
  6. f Prepare slides
  7. g Apply primary antibodies
  8. h Apply secondary antibodies
  9. i Acquire images
Phase 12 · Quantitative Confocal Analysis Post-immunofluorescence
  1. a Acquire confocal images
  2. b Process images with DenoiseAI
  3. c Perform segmentation
  4. d Count nuclei
  5. e Measure and normalize volumes
  6. f Generate representative images

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Attribution

This SOP was written by Organthis based on the published method in D'Aiuto et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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