Skip to content
← Back to browse
BRAIN Publication-derived

Modeling Sporadic Alzheimer's Disease in Human Brain Organoids under Serum Exposure

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

👤 Xianwei Chen, Guoqiang Sun, E Tian, Mingzi Zhang, Hayk Davtyan, Thomas G. Beach, Eric M. Reiman, Mathew Blurton-Jones, David M. Holtzman, Yanhong Shi ⏱ 110 days 📋 12 phases 🧫 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

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

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Chen 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.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence