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BRAIN Publication-derived

Sporadic Creutzfeldt-Jakob Disease Cerebral Organoids

Full SOP with Academic or a plan

Source Williams et al., 2023 · National Institute of Allergy and Infectious Diseases, National Institutes of Health · 10.1186/s13287-023-03591-2

Authors: Katie Williams, Simote T. Foliaki, Brent Race, Anna Smith, Tina Thomas, Bradley R. Groveman, Cathryn L. Haigh Duration: 180 days 12 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of human cerebral organoids from iPSCs, their infection with sporadic Creutzfeldt-Jakob disease (sCJD) prions, and subsequent seeding with neural progenitor cells (NPCs) to assess therapeutic effects on neuroelectrophysiological function and disease parameters. NPCs successfully integrated into infected organoids and restored impaired neuronal spike rates despite only modest reductions in prion accumulation.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

49 steps across 12 phases

Phase 1 · iPSC culture and maintenance Ongoing
  1. a Routine culture of hu-iPSCs
  2. b Passage iPSC colonies
Phase 2 · Embryoid body (EB) production Days 0-4
  1. a Seed cells for EB formation
  2. b Add Y27632 on day 0
  3. c Feed EBs on days 2 and 4
Phase 3 · Neural induction and Matrigel embedding Days 5-10
  1. a Transfer to neural induction medium
  2. b Embed in Matrigel
  3. c Transfer organoids to maintenance culture
  4. d Switch to B27 plus retinoic acid on day 10
  5. e Establish agitated long-term culture
Phase 4 · NPC differentiation and preparation 11 days prior to CO embedding
  1. a Thaw NPCs and passage
  2. b Collect and count NPCs
  3. c Prepare NPC-Matrigel mixture
Phase 5 · NPC seeding into COs Day 90 post-infection (dpi)
  1. a Prepare organoids for seeding
  2. b Apply NPC-Matrigel mixture
  3. c Coat organoids in Matrigel
  4. d Allow Matrigel to solidify
  5. e Transfer seeded organoids to culture
  6. f Prepare mock-seeded controls
Phase 6 · sCJD prion infection Day 0 of infection
  1. a Prepare inoculum
  2. b Inoculate organoids
  3. c Add fresh media at 24 hours
  4. d Perform full media exchange at day 7
  5. e Maintain infected organoids
Phase 7 · Organoid viability assessment Days 0-180 dpi
  1. a Prestoblue metabolism assay
  2. b LDH cytotoxicity assay
Phase 8 · Prion infection parameter assessment Day 180 dpi
  1. a Prepare organoid homogenates
  2. b RT-QuIC assay
  3. c Proteinase-K digestion and Western blotting
Phase 9 · Neuroelectrophysiology recording Day 180 dpi
  1. a Coat MEA wells with PEI and laminin
  2. b Plate organoids on MEA
  3. c Incubate before recording
  4. d Record local field potential
Phase 10 · Immunohistochemistry and immunofluorescence Day 180 dpi
  1. a Fix organoids for histology
  2. b Routine H&E staining
  3. c Immunohistochemistry for PrP
  4. d Immunofluorescence for NPC characterization
  5. e MAP2 staining of organoids
  6. f General organoid characterization IHC
  7. g Immunofluorescence on frozen sections
Phase 11 · Cell signaling analysis Day 180 dpi
  1. a Prepare samples for BioPlex
  2. b Quantify total protein
  3. c Run BioPlex cell signaling assays
Phase 12 · Western blotting for neuronal and glial markers Day 180 dpi
  1. a Prepare samples (non-PK treated)
  2. b Run gels and transfer
  3. c Probe with primary antibodies
  4. d Probe with secondary antibodies
  5. e Visualize bands
  6. f Quantification

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Attribution

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

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