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BRAIN Publication-derived

Cerebral Organoids for Drug Screening

Full SOP with Academic or a plan

Source Raja et al. · Rutgers University / The Rockefeller University, New York, USA · 10.1371/journal.pone.0161969

Authors: Raja WK, Mungenast AE, Lin YT, Ko T, Bhatt F, Bhatt SM, Bhatt RM, Bhatt KJ, Bhatt NP, Bhatt RS, Bhatt AM, Bhatt AH, Bhatt JE, Bhatt AB, Bhatt MN, Bhatt RN, Tsai LH Duration: 100 days 8 phases Cell source: Patient-Derived iPSC (Familial AD: APP Duplication, PSEN1 A246E)

Abstract

Raja et al. generated cerebral organoids from iPSCs of familial Alzheimer disease patients carrying APP duplication or PSEN1 A246E mutations, demonstrating spontaneous amyloid-β aggregation, tau hyperphosphorylation, and endosomal abnormalities. Treatment with β- and γ-secretase inhibitors reduced pathological phenotypes, validating the platform for AD drug screening.

Cell source
Patient-Derived iPSC (Familial AD: APP Duplication, PSEN1 A246E)
Application
Alzheimer Disease Drug Screening

Protocol overview

47 steps across 8 phases

Phase 1 · iPSC Maintenance and Preparation Days 0–Culture initiation
  1. a Culture iPSCs on MEF feeder layer
  2. b Dissociate iPSCs into single-cell suspension
  3. c Separate iPSCs from MEF feeders
  4. d Verify pluripotency by immunocytochemistry
Phase 2 · Embryoid Body (EB) Formation Days 1-20
  1. a Prepare ultra-low adherence 96-well plates
  2. b Load iPSCs at 12,000 cells per well
  3. c Add Dorsomorphin for neural lineage promotion
  4. d Maintain EBs in 96-well format for 18-20 days
  5. e Transfer organoids to non-adherent culture dishes
Phase 3 · Neuroepithelial Formation and Maturation Days 20-70
  1. a Culture organoids in neuroepithelial differentiation medium
  2. b Add Matrigel and FBS after 15-20 days
  3. c Increase Matrigel concentration and add B27 supplement
  4. d Maintain organoids until endpoint
Phase 4 · Tissue Processing and Immunohistochemistry Days 60 and 90-100
  1. a Fix organoids in 4% paraformaldehyde
  2. b Cryoprotect in 30% sucrose solution
  3. c Embed in OCT compound and freeze
  4. d Section organoids at 30 μm thickness
  5. e Permeabilize tissue sections
  6. f Block non-specific binding
  7. g Incubate with primary antibodies overnight
  8. h Wash sections in PBST with horse serum
  9. i Incubate with secondary antibodies and Hoechst
  10. j Final washes before mounting
  11. k Mount sections and seal
  12. l Image sections on confocal microscope
Phase 5 · Thioflavin-S Staining for Tau Aggregates Days 60 and 90-100
  1. a Pre-treat sections with potassium permanganate
  2. b De-stain and prepare for Thioflavin-S
  3. c Stain with Thioflavin-S
  4. d Develop and wash in darkness
  5. e Mount and image
Phase 6 · Enzyme-Linked Immunosorbent Assay (ELISA) for Aβ Days 60 and 90
  1. a Collect organoid culture supernatant
  2. b Prepare ELISA kit and standards
  3. c Incubate samples with primary antibody
  4. d Wash and incubate with HRP-conjugated secondary
  5. e Develop with HRP substrate and measure
  6. f Calculate Aβ concentration from standard curve
Phase 7 · Transferrin Endocytosis Assay for Endosome Assessment Days 90-100
  1. a Prepare live organoids for transferrin uptake
  2. b Incubate with transferrin
  3. c Wash and fix organoids
  4. d Wash with PBS and stain with Hoechst
  5. e Mount organoids on glass coverslips
  6. f Image and analyze transferrin-positive endosomes
Phase 8 · Drug Treatment with Secretase Inhibitors Days 30-90
  1. a Prepare drug stock solutions
  2. b Begin drug treatment at day 30
  3. c Maintain drug treatment through media changes
  4. d Process organoids for immunohistochemistry
  5. e Quantify drug-induced changes in AD pathology

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Attribution

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

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