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BRAIN Publication-derived

Sporadic Alzheimer's Disease Brain Organoids

Full SOP with Academic or a plan

Source Chen et al., 2021 · Beckman Research Institute of City of Hope · 10.1002/advs.202101462

Authors: Xianwei Chen, Guoqiang Sun, E Tian, Mingzi Zhang, Hayk Davtyan, Thomas G. Beach, Eric M. Reiman, Mathew Blurton-Jones, David M. Holtzman, Yanhong Shi Duration: 110 days 12 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of human iPSC-derived brain organoids and their exposure to human serum to model sporadic Alzheimer's disease (sAD). The serum-exposed organoids recapitulate AD-like pathologies including amyloid-beta aggregation, phosphorylated tau accumulation, synaptic loss, and impaired neural network activity, providing a platform for mechanistic study and therapeutic development.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

47 steps across 12 phases

Phase 1 · hiPSC Derivation and Maintenance Day 0 - Day 5 (routine maintenance)
  1. a Reprogram human fibroblasts to hiPSCs
  2. b Culture hiPSCs in maintenance medium
Phase 2 · Brain Organoid Generation Day 0 - Day 50
  1. a Initiate organoid differentiation
  2. b Screen organoids for quality (Day 20-40)
  3. c Culture organoids to day 50-60
Phase 3 · Serum and Compound Treatment Day 50-60 to Day 90-110 (≈12 days of treatment)
  1. a Prepare serum and treatment groups
  2. b Apply serum and compounds to organoids
  3. c Treat organoids for approximately 12 days
Phase 4 · Immunostaining of Brain Organoids On harvest (Day 90-110)
  1. a Fix and cryopreserve organoids
  2. b Permeabilize and block organoid sections
  3. c Incubate with primary antibodies
  4. d Wash and incubate with secondary antibodies
  5. e Mount and image organoid sections
Phase 5 · Protein Extraction and Western Blotting On harvest (Day 90-110)
  1. a Prepare soluble and insoluble protein fractions
  2. b Prepare protein lysates for Western blot
  3. c Run SDS-PAGE and transfer to PVDF membrane
  4. d Block and incubate with primary antibodies
  5. e Wash and incubate with secondary antibodies
  6. f Develop and image Western blot
Phase 6 · Aβ1-40 ELISA On harvest (Day 90-110)
  1. a Prepare organoid lysates
  2. b Perform Aβ1-40 ELISA
Phase 7 · Transmission Electron Microscopy (TEM) On harvest (Day 90-110)
  1. a Fix organoids for TEM
  2. b Post-fix, dehydrate, and embed
  3. c Prepare ultra-thin sections and image
Phase 8 · Calcium Imaging Day 90 to Day 97 (1 week post-serum treatment)
  1. a Dissociate organoids and seed on Matrigel-coated slides
  2. b Prepare serum or control conditions
  3. c Load calcium indicator and acquire images
  4. d Process and quantify calcium imaging data
Phase 9 · Microelectrode Array (MEA) Recording Day 90 to Day 97 (1 week post-serum treatment)
  1. a Dissociate organoids and seed on MEA plates
  2. b Culture in BrainPhys medium and apply treatments
  3. c Perform MEA recording
  4. d Analyze MEA data
Phase 10 · Single-Cell RNA Sequencing (scRNA-seq) Day 90-110 (on harvest)
  1. a Dissociate organoids to single cells
  2. b Capture cells on 10X Chromium device
  3. c Construct and analyze sequencing libraries
  4. d Process raw sequencing data
  5. e Preprocess and integrate scRNA-seq data
  6. f Perform dimensionality reduction and clustering
  7. g Annotate cell types
  8. h Identify differentially expressed genes (DEGs)
  9. i Perform functional enrichment analysis (GSEA)
Phase 11 · Real-Time PCR Validation On harvest (Day 90-110)
  1. a Extract total RNA
  2. b Synthesize complementary DNA (cDNA)
  3. c Perform real-time PCR
Phase 12 · Immunostaining of Human Brain Tissues On analysis (using post-mortem AD and control tissues)
  1. a Fix and cryopreserve frozen brain tissues
  2. b Perform antigen retrieval and autofluorescence quenching
  3. c Immunostain brain tissue sections

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Attribution

This SOP was written by Organthis based on the published method in Chen et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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