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BRAIN Publication-derived

iPSC-derived cerebral organoids reveal mitochondrial, inflammatory and neuronal vulnerabilities in bipolar disorder

Source El Sabbagh et al., 2025 · University of Toronto, Krembil Brain Institute, Toronto Western Hospital, University Health Network · 10.1038/s41398-025-03529-7

👤 Dana El Soufi El Sabbagh, Alencar Kolinski Machado, Lauren Pappis, Erika Leigh Beroncal, Delphine Ji, George Nader, Prathyusha Ravi Chander, Jaehyoung Choi, Angela Duong, Hyunjin Jeong, Bruna Panizzutti, Chiara Cristina Bortolasci, Andrea Szatmari, Peter Carlen, Margaret Hahn, Liliana Attisano, Michael Berk, Ken Walder, Ana Cristina Andreazza ⏱ 180 days 📋 11 phases 🧫 Patient-Derived iPSC (Bipolar Disorder)

Abstract

This protocol describes the generation and characterization of cerebral organoids (COs) from iPSCs derived from bipolar disorder (BD) patients and healthy controls to model disease-specific mitochondrial dysfunction, metabolic dysregulation, and inflammatory responses. BD-derived COs exhibit impaired mitochondrial membrane potential, reduced ATP levels, increased neuronal hyperactivity, enhanced NLRP3 inflammasome activation sensitivity, and elevated markers of oxidative stress and cell-free mitochondrial DNA release.

Cell source
Patient-Derived iPSC (Bipolar Disorder)
Application
Disease modeling

Protocol overview

69 steps across 11 phases

Blood Collection and PBMC Isolation Day 0-1
  1. 1 Venous Blood Collection
  2. 2 Plasma Separation
  3. 3 PBMC Isolation using Density Gradient Centrifugation
  4. 4 PBMC Washing and Red Blood Cell Removal
  5. 5 PBMC Cryopreservation
  6. 6 PBMC Culture Initiation
iPSC Generation and Characterization Day 3-30 (approximately)
  1. 1 PBMC Transfection with Episomal Vectors
  2. 2 iPSC Culture Setup
  3. 3 iPSC Colony Characterization - Immunofluorescence
  4. 4 Epi-Pluri-Score Analysis
  5. 5 Karyotyping
  6. 6 Quality Control Filter
Cerebral Organoid (CO) Generation Day 0-30 (approximately)
  1. 1 iPSC Singularization and Embryonic Body (EB) Formation
  2. 2 Neural Induction
  3. 3 Matrigel Embedding
  4. 4 Organoid Culture on Orbital Shaker
  5. 5 Batch Documentation
CO Characterization at 1 Month: Cellular Composition Day 28-30
  1. 1 Preparation for Immunofluorescence Staining
  2. 2 Cryosectioning
  3. 3 SOX2 Immunostaining for Neural Progenitor Cells
  4. 4 Confocal Microscopy Imaging
  5. 5 Automated Image Quantification
  6. 6 Cellular Composition Comparison
Metabolomic Profiling and ATP Measurements Day 28-30
  1. 1 CO Singularization
  2. 2 Cell Counting
  3. 3 Sample Preparation for Metabolomics
  4. 4 Metabolomic Analysis by LC-MS
  5. 5 Intracellular ATP Measurement - Cell Titer Glo Assay
  6. 6 Luminescence Measurement
Transmission Electron Microscopy (TEM) - Mitochondrial Morphology Day 28-35 (sample preparation time)
  1. 1 Primary Fixation
  2. 2 Phosphate Buffer Washing
  3. 3 Secondary Fixation with Osmium Tetroxide
  4. 4 Dehydration with Graded Ethanol Series
  5. 5 Propylene Oxide Treatment
  6. 6 Epoxy Resin Infiltration
  7. 7 Resin Polymerization
  8. 8 Ultramicrotomy
  9. 9 Staining with Uranyl Acetate and Lead Citrate
  10. 10 TEM Imaging and Mitochondrial Morphology Analysis
CO Characterization at 6 Months: Electrophysiology and Cellular Maturity Day 170-180
  1. 1 CO Slice Preparation
  2. 2 Vibratome Slicing
  3. 3 CO Slice Recovery Culture
  4. 4 Immunofluorescence for Mature Neurons and Astrocytes
  5. 5 Electrophysiology Setup
  6. 6 Local Field Potential (LFP) Electrode Positioning
  7. 7 LFP Recording
  8. 8 Electrophysiology Data Analysis
Mitochondrial Health Assessment - Membrane Potential and Morphology Day 28-30
  1. 1 MitoTracker Green Staining - Mitochondrial Mass
  2. 2 MitoTracker Red CMX ROS Staining - Mitochondrial Membrane Potential
  3. 3 JC-1 Staining for Membrane Potential Assessment
  4. 4 Confocal Microscopy Imaging
  5. 5 Automated Quantification of Mitochondrial Parameters
Extracellular Markers of Inflammation and Mitochondrial Stress Day 28-30
  1. 1 Supernatant Collection
  2. 2 Reactive Oxygen Species (ROS) Measurement
  3. 3 Double-Stranded DNA (dsDNA) Release Measurement
  4. 4 Cell-Free Mitochondrial DNA (ccf-mtDNA) Quantification
  5. 5 IL-1β Inflammatory Marker Measurement
NLRP3 Inflammasome Activation Assessment Day 28-30
  1. 1 Baseline ASC Speck Imaging
  2. 2 NLRP3 Inflammasome Activation with LPS and Nigericin
  3. 3 Activated ASC Speck Imaging
  4. 4 ASC Speck Quantification
  5. 5 Activation Sensitivity Calculation
  6. 6 Acute ccf-mtDNA Release Upon Activation
NLRP3 Inflammasome Inhibition and Therapeutic Rescue Day 28-30
  1. 1 Pre-treatment with MCC950 (NLRP3 Inhibitor)
  2. 2 Pre-treatment with Bioactive Flavonoid Extract (BFE)
  3. 3 Immunofluorescence Imaging After Inhibitor Treatment
  4. 4 ASC Speck Quantification - Treatment Conditions
  5. 5 Rescue Effect Calculation
  6. 6 ccf-mtDNA Release Measurement - Treatment Conditions

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in El Sabbagh et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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