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BRAIN Publication-derived

Human Cerebral Organoid Model of Prion Disease

Source Groveman et al. · National Institute of Allergy and Infectious Diseases (NIAID/NIH), Hamilton, MT, USA · 10.1186/s40478-019-0742-2

👤 Groveman BR, Foliaki ST, Orru CD, Zanusso G, Carroll JA, Race B, Bhatt SM, Bhatt AH, Chesebro B ⏱ 169 days 📋 11 phases 🧫 Human iPSC / Prion-Infected Primary Tissue

Abstract

Groveman et al. established the first human cerebral organoid model of prion disease by inoculating organoids with CJD brain homogenate, demonstrating prion propagation, PrPSc accumulation, and spongiform degeneration over 6 months. This BSL-3 model enables anti-prion drug screening in human tissue without animal models.

Cell source
Human iPSC / Prion-Infected Primary Tissue
Application
Prion Disease Modeling

Protocol overview

55 steps across 11 phases

Human induced pluripotent stem cell culture and embryoid body formation Days 0-6
  1. 1 Culture hu-iPSCs
  2. 2 Seed embryoid body formation
  3. 3 Prepare hES medium
  4. 4 Supplement medium on day 0
  5. 5 Add medium at days 2 and 4
  6. 6 Transfer to neural induction medium
Cerebral organoid generation and Matrigel embedding Days 4-8 post-neural induction
  1. 1 Embed organoids in Matrigel
  2. 2 Incubate in cerebral organoid medium (without retinoic acid)
  3. 3 Transition to medium with retinoic acid
Long-term organoid culture and maturation Days 8-140
  1. 1 Establish agitated long-term culture
  2. 2 Perform twice-weekly media changes
  3. 3 Monitor organoid maturation
Prion inoculum preparation Day 140 (before infection)
  1. 1 Prepare prion inoculum from brain homogenate
  2. 2 Sterilize inoculum by filtration
  3. 3 Verify infectious seeds by RT-QuIC
Prion infection of human cerebral organoids Days 140-147 (initial infection phase)
  1. 1 Distribute organoids for infection
  2. 2 Replace media with inoculated media
  3. 3 Add fresh media at 24 hours post-inoculation
  4. 4 Perform half media exchange at 3 days
  5. 5 Perform full media and vessel exchange at 7 days
  6. 6 Maintain routine culture with three media changes per week
Organoid health monitoring Days 0-169 post-infection
  1. 1 Perform weekly Prestoblue assay
  2. 2 Measure Prestoblue fluorescence
  3. 3 Perform LDH assay
  4. 4 Read LDH absorbance
RT-QuIC seeding activity analysis Days 7-169 post-infection
  1. 1 Collect samples for RT-QuIC at specified time points
  2. 2 Prepare organoid homogenates for RT-QuIC
  3. 3 Prepare RT-QuIC reaction mix for media seeding
  4. 4 Prepare RT-QuIC reaction mix for organoid seeding
  5. 5 Set up RT-QuIC reactions
  6. 6 Incubate RT-QuIC plate
  7. 7 Record ThT fluorescence
  8. 8 Analyze RT-QuIC data using Spearman-Kärber method
Proteinase-K digestion and Western blotting Day 169 post-infection (at harvest)
  1. 1 Prepare organoid homogenates
  2. 2 Treat with Proteinase K
  3. 3 Stop Proteinase K reaction
  4. 4 Prepare samples for Western blotting
  5. 5 Run gel electrophoresis
  6. 6 Transfer proteins to PVDF membrane
  7. 7 Detect PrP with 3F4 antibody
  8. 8 Apply secondary antibody and detect signal
Immunofluorescence staining Day 96 and Day 169 post-infection
  1. 1 Fix organoids
  2. 2 Apply primary antibodies
  3. 3 Apply secondary antibodies
  4. 4 Mount organoids
  5. 5 Image by confocal microscopy
Immunohistochemistry and histochemistry Day 96 and Day 169 post-infection
  1. 1 Prepare organoid tissue sections
  2. 2 Perform antigen retrieval
  3. 3 Stain for prion protein with 6H4 antibody
  4. 4 Stain for astrocytes with anti-GFAP
  5. 5 Perform H&E staining
  6. 6 Analyze histopathology slides
Cytokine array analysis Days 14, 35, 68, 95, 127, 159, 169 post-infection
  1. 1 Collect conditioned media
  2. 2 Perform Bio-Plex cytokine assay
  3. 3 Exclude cytokines below detection limit

Full SOP

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Attribution

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

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