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BRAIN Publication-derived

Alzheimer's Disease Cerebral Organoids for Neuroinflammation Studies

Full SOP with Academic or a plan

Source Park et al. · Department of Biochemistry & Biomedical Sciences, Seoul National University College of Medicine · 10.1073/pnas.1314145110

Authors: Jong-Chan Park, So-Yeong Jang, Dongjoon Lee, Jeongha Lee, Uiryong Kang, Hongjun Chang, Haeng Jun Kim, Sun-Ho Han, Jinsoo Seo, Murim Choi, Dong Young Lee, Min Soo Byun, Dahyun Yi, Kwang-Hyun Cho, Inhee Mook-Jung Duration: 65 days 10 phases Cell source: Human iPSC, Patient-Derived iPSC (Alzheimer's Disease)

Abstract

This protocol describes the generation of human iPSC-derived cerebral organoids (iCOs) from patient peripheral blood mononuclear cells, with characterization of normal and Alzheimer's disease-associated pathological features including amyloid-beta and phospho-tau accumulation. The iCOs are used in a high-throughput drug screening platform to identify therapeutic candidates targeting key AD-associated signaling pathways (ApoE ε4 allele and LPL variants).

Cell source
Human iPSC, Patient-Derived iPSC (Alzheimer's Disease)
Application
Disease modeling and drug screening for Alzheimer's disease

Protocol overview

37 steps across 10 phases

Phase 1 · iPSC Generation and Characterization Days 0-25
  1. a PBMC Isolation and T Cell Activation
  2. b Sendai Virus Reprogramming
  3. c iPSC Colony Derivation and Passage
  4. d iPSC Characterization
Phase 2 · Embryoid Body (EB) Formation Days 0-6
  1. a Embryoid Body Initiation
  2. b Neural Induction with SMAD Inhibitors
Phase 3 · Cerebral Organoid Formation and Expansion Days 6-43+
  1. a EB Collection and Organoid Seeding
  2. b Early Proliferation Phase (Days 6-15)
  3. c Maturation Phase with Reduced EGF/bFGF (Days 16-24)
  4. d Neurotrophic Factor Phase (Days 25-42)
  5. e Maturation Phase without Growth Factors (Days 43+)
Phase 4 · Organoid Quality Control and Selection Days 7, 60, and drug screening period
  1. a Manual Quality Control (Days 7 and 60)
  2. b Automated Quality Control During Drug Screening
Phase 5 · Immunohistochemistry and Pathology Characterization Days 60+
  1. a Organoid Fixation and Cryopreservation
  2. b Cryosectioning and Permeabilization
  3. c Blocking and Primary Antibody Staining
  4. d Secondary Antibody Staining and Mounting
  5. e Confocal Microscopy and Image Acquisition
Phase 6 · 3D Tissue Clearing (Ethyl-Cinnamate ECi Protocol) Days 60+
  1. a Fixation and Blocking/Permeabilization
  2. b Primary Antibody Staining
  3. c Washing and Secondary Antibody Staining
  4. d Post-Staining Fixation and Dehydration
  5. e Index Matching and Imaging
Phase 7 · Drug Treatment and Screening Days 60-65 (or as specified)
  1. a Organoid Preparation for Drug Treatment
  2. b Drug Dose Preparation and Application
  3. c Drug Response Readout via Cell Viability (MTT Assay)
  4. d High-Content Screening with Automated Imaging
Phase 8 · Functional Assays: Calcium Oscillation Analysis Days 60-65
  1. a Organoid Preparation and Dye Loading
  2. b Calcium Flux Detection
Phase 9 · Molecular and Genomic Analyses Days 60+
  1. a RNA Extraction and RT-qPCR
  2. b RNA Sequencing and Library Preparation
  3. c RNA-Seq Analysis: Sequencing and Read Mapping
  4. d Differential Gene Expression (DEG) Analysis
  5. e Pathway Enrichment and Gene Ontology Analysis
Phase 10 · Immunocytochemistry for iPSCs Parallel to iPSC characterization (days 15-25)
  1. a Slide Preparation and Cell Fixation
  2. b Blocking and Primary Antibody Incubation
  3. c Secondary Antibody Staining and Mounting

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Attribution

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

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