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BRAIN Publication-derived

Human cerebral organoids as a therapeutic drug screening model for Creutzfeldt–Jakob disease

Source Groveman et al., 2021 · Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health · 10.1038/s41598-021-84689-6

👤 Bradley R. Groveman, Natalia C. Ferreira, Simote T. Foliaki, Ryan O. Walters, Clayton W. Winkler, Brent Race, Andrew G. Hughson, Gianluigi Zanusso, Cathryn L. Haigh ⏱ 120 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation, maintenance, and infection of human cerebral organoids derived from iPSCs with sporadic Creutzfeldt–Jakob disease (sCJD) prions, followed by evaluation of anti-prion drug candidates such as pentosan polysulfate using both prophylactic and therapeutic treatment paradigms. The organoids serve as a human 3D cell culture system for screening therapeutic candidates for prion diseases.

Cell source
Human iPSC
Application
Disease modeling and drug screening

Protocol overview

64 steps across 9 phases

Cerebral Organoid Generation and Culture Day -120 to Day 0 (approximately 5 months prior to infection)
  1. 1 Cell source and preparation
  2. 2 Organoid generation protocol
  3. 3 Organoid culture medium preparation
  4. 4 Maintain organoid culture until day 0
Prophylactic Treatment Protocol with PPS Day -7 to Day 14 (21 days total)
  1. 1 Prepare treatment compounds
  2. 2 Begin prophylactic treatment (Day -7)
  3. 3 Prepare inoculum on day 0
  4. 4 Inoculate organoids (Day 0)
  5. 5 Continue treatment during infection (Day 0–7)
  6. 6 Dilute inoculum (Day 1 and Day 4)
  7. 7 Transfer to fresh vessels with treatment (Day 7)
  8. 8 Remove treatment and perform media change (Day 14)
Sample Collection and Analysis - Prophylactic Arm Day 28, 63, and 120 post-inoculation
  1. 1 Collect samples at 28 dpi (Day 28)
  2. 2 Collect samples at 63 dpi (Day 63)
  3. 3 Collect samples at 120 dpi (Day 120)
Therapeutic Treatment Protocol with PPS Day 0 to Day 63 (infection phase), then Day 63 to Day 91 (treatment phase)
  1. 1 Establish sCJD infection without prophylactic treatment
  2. 2 Begin therapeutic treatment at 63 dpi (Day 63)
  3. 3 Continue therapeutic treatment for 28 days (Day 63–91)
  4. 4 Collect samples at 91 dpi (Day 91, end of treatment)
  5. 5 Remove treatment and enter recovery phase (Day 91)
  6. 6 Collect final samples at 120 dpi (Day 120, 57 days post-treatment)
Real-Time Quaking-Induced Conversion (RT-QuIC) Assay Performed on samples from all collection timepoints
  1. 1 Prepare organoid homogenate
  2. 2 Prepare serial dilutions
  3. 3 Prepare RT-QuIC reaction mix
  4. 4 Load RT-QuIC plates
  5. 5 Perform RT-QuIC in shaking incubator
  6. 6 Determine positive wells and calculate SD50
Proteinase-K Digestion and Western Blotting Performed on organoid tissue collected at 120 dpi (final timepoint)
  1. 1 Prepare organoid homogenate
  2. 2 Split sample for proteinase K digestion
  3. 3 Perform proteinase K digestion
  4. 4 Terminate proteinase K digestion
  5. 5 Prepare samples for gel electrophoresis
  6. 6 Load and run SDS-PAGE gel
  7. 7 Transfer proteins to PVDF membrane
  8. 8 Block and incubate with primary antibody
  9. 9 Wash and incubate with secondary antibody
  10. 10 Detect chemiluminescence
  11. 11 Detect total protein and quantify
Histochemistry and Immunohistochemistry Performed on organoid tissue collected at 120 dpi
  1. 1 Fix organoid tissue
  2. 2 Prepare tissue for sectioning
  3. 3 Embed and freeze tissue
  4. 4 Section tissue on cryostat
  5. 5 Perform hematoxylin and eosin (H&E) staining
  6. 6 Perform immunohistochemistry for total PrP
  7. 7 Analyze histopathology slides
Immunofluorescence Staining and Confocal Microscopy Performed on organoid tissue collected at 120 dpi or other timepoints
  1. 1 Fix organoids for immunofluorescence
  2. 2 Prepare tissue for cryosectioning
  3. 3 Embed and freeze tissue
  4. 4 Block and permeabilize tissue
  5. 5 Incubate with SAF32 primary antibody
  6. 6 Incubate with secondary antibody and phalloidin
  7. 7 Stain cell nuclei with Hoechst
  8. 8 Mount slides with coverslips
  9. 9 Acquire confocal microscopy images
  10. 10 Deconvolve and process images
Metabolic and Viability Monitoring (PrestoBlue and LDH Assays) Performed throughout the culture period (baseline and at collection timepoints)
  1. 1 Prepare PrestoBlue reagent
  2. 2 Add PrestoBlue to organoid cultures
  3. 3 Measure PrestoBlue fluorescence
  4. 4 Normalize PrestoBlue data
  5. 5 Collect media samples for LDH analysis
  6. 6 Prepare LDH assay
  7. 7 Measure LDH in media
  8. 8 Assess cytotoxicity (optional toxicity evaluation)
  9. 9 Normalize and plot LDH data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Groveman et al., 2021. 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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