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BRAIN Publication-derived

Schizophrenia and Bipolar Disorder Cerebral Organoids

Full SOP with Academic or a plan

Source Cheng et al., 2025 · Johns Hopkins University · 10.1063/5.0250559

Authors: Kai Cheng, Autumn Williams, Anannya Kshirsagar, Sai Kulkarni, Rakesh Karmacharya, Deok-Ho Kim, Sridevi V. Sarma, Annie Kathuria Duration: 270 days 15 phases Cell source: Patient-Derived iPSC (Schizophrenia, Bipolar Disorder)

Abstract

This protocol describes the generation, maintenance, and electrophysiological characterization of patient-derived cerebral organoids (COs) and two-dimensional cortical interneuron cultures (2DNs) from individuals with schizophrenia and bipolar disorder. Multi-electrode array recordings combined with stimulus–response dynamic network modeling and machine learning enable identification of disease-specific electrophysiological signatures for objective psychiatric biomarker discovery.

Cell source
Patient-Derived iPSC (Schizophrenia, Bipolar Disorder)
Application
Disease modeling and biomarker identification

Protocol overview

53 steps across 15 phases

Phase 1 · iPSC Line Preparation and Validation Passage 25-30
  1. a iPSC Genomic Integrity Testing with KaryoStat Analysis
  2. b Copy Number Variation Analysis
  3. c Mycoplasma Testing
Phase 2 · Cerebral Organoid Differentiation Days 0-7 (EB formation and transition)
  1. a Culture iPSCs in Basal Medium
  2. b Form Embryoid Bodies in U-Bottom Plates
  3. c Transfer EBs to Cerebral Organoid Induction Media
  4. d Embed EBs in Matrigel
Phase 3 · Cerebral Organoid Maturation Days 7-270 (9 months in vitro)
  1. a Culture Organoids on Orbital Shakers
  2. b Add BDNF Starting at Day 30
  3. c Continue Culture for 6-9 Months
Phase 4 · Organoid Integrity Validation Multiple timepoints during culture (e.g., Days 30, 60, 90, 180, 270)
  1. a Immunohistochemical Characterization
  2. b Quantify Cell Populations
  3. c RT-qPCR Validation of Marker Expression
Phase 5 · Two-Dimensional Cortical Neuron Differentiation Days 0-120 (neuronal maturation)
  1. a Culture iPSCs on Geltrex-Coated Plates
  2. b Neural Induction with N2/B27 and SMAD Inhibitors
  3. c Split and Plate NPCs on Day 8
  4. d Forebrain Specification with Purmorphamine
  5. e Plate on ECM-Coated Substrate
  6. f Neuronal Maturation in BrainPhys Media with DAPT
  7. g Continue Culture to Maturation
Phase 6 · Preparation of Organoids for MEA Recording Day 270 (9 months in vitro, after MEA plate preparation)
  1. a Prepare MEA Plates with ECM Coating
  2. b Transfer Organoids to MEA Plates
  3. c Culture Organoids on MEA Plates for 3 Additional Months
Phase 7 · Electrophysiological Recording Setup and Media Preparation Day of recording
  1. a Prepare Recording Media
  2. b Perform 50% Media Exchange 24 Hours Before Recording
  3. c Complete Media Exchange 1 Hour Before Recording
  4. d Set Up MEA Recording System with Temperature and pH Control
  5. e Verify Recording Media Osmolarity
Phase 8 · Baseline and Post-Electrical Stimulation Recording Day of recording (9-month-old organoids or 90-120-day neurons)
  1. a Check Electrode Impedance and Integrity
  2. b Record Baseline Spontaneous Activity
  3. c Apply Electrical Stimulation Protocol
  4. d Record Post-Stimulation Response
Phase 9 · Signal Validation and Spike Detection During and immediately after recording
  1. a Characterize Baseline Firing Properties
  2. b Apply Tetrodotoxin (TTX) to Verify Neuronal Origin
  3. c Verify Signal-to-Noise Ratio
Phase 10 · Electrophysiological Data Preprocessing During data analysis (post-recording)
  1. a Apply Bandpass Filtering
  2. b Apply Notch Filtering at Power Line Harmonics
  3. c Spike Detection and Time Series Binarization
  4. d Downsample Spike Train to 1 kHz
  5. e Rate Code Spike Train with 200 ms Sliding Window
Phase 11 · Stimulus–Response Dynamic Network Modeling (SRDNM) During data analysis
  1. a Develop SRDNM to Capture Stimulus-Response Relationships
  2. b Estimate State Transition Matrix A
Phase 12 · Sink Index Feature Extraction During data analysis
  1. a Define Sink Index for Each Channel
  2. b Generate Comprehensive Sink Index Feature Map
Phase 13 · Feature Selection Using Minimum Redundancy Maximum Relevance (MRMR) During data analysis
  1. a Apply MRMR Feature Selection Framework
  2. b Identify Top-Ranked Features for Classification
Phase 14 · Support Vector Machine Classifier Development and Validation During data analysis
  1. a Implement Nested Cross-Validation Strategy
  2. b Train Support Vector Machine (SVM) Classifier
  3. c Evaluate SVM on Held-Out Test Fold
  4. d Aggregate Performance Metrics Across Outer Folds
Phase 15 · Hyperparameter Tuning and Algorithm Comparison During data analysis
  1. a Optimize SVM Hyperparameters via Bayesian Optimization
  2. b Compare Performance of Multiple Classification Algorithms
  3. c Select SVM as Primary Classifier

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Attribution

This SOP was written by Organthis based on the published method in Cheng et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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