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BRAIN Publication-derived

Semi-Automated Generation and Molecular Characterization of Midbrain Organoids from Bipolar Disorder Patient-Derived hiPSCs

Source Meyer et al. · Department of Genetics, Blavatnik Institute, Harvard Medical School; Wyss Institute for Biologically Inspired Engineering, Harvard University; BD2: Breakthrough Discoveries for thriving with Bipolar Disorder · 10.1101/2025.04.01.646642;

👤 Katharina Meyer, Matthew Woodworth, Maria Carolina Bittencourt Gonçalves, Michelle Yue, Hoor AlJandal, Shad Morton, Michael Lewandowski, Ninning Liu, Eric Zigon, Patrick Fortuna, Mariana Garcia-Corral, Bogdan Budnik, George M. Church, Jenny M. Tam ⏱ 56 days 📋 9 phases 🧫 Patient-Derived iPSC (Bipolar Disorder); Healthy Control iPSC

Abstract

This protocol describes the semi-automated generation of region-specific midbrain organoids from human induced pluripotent stem cells (hiPSCs) derived from bipolar disorder (BD) patients and healthy controls. The protocol incorporates automated liquid handling and high-content imaging to improve reproducibility and throughput. Organoids are characterized via electrophysiology (MEA recordings), transcriptomics, proteomics, and phosphoproteomics to identify molecular alterations associated with BD, including dysregulation of calcium signaling and kinase pathways that are partially normalized by lithium treatment.

Cell source
Patient-Derived iPSC (Bipolar Disorder); Healthy Control iPSC
Application
Disease modeling; Drug screening

Protocol overview

48 steps across 9 phases

hiPSC Maintenance and Embryoid Body Formation DIV -∞ to DIV 0
  1. 1 hiPSC Culture Preparation
  2. 2 Cell Dissociation with ROCK Inhibitor
  3. 3 Cell Quenching and Centrifugation
  4. 4 Cell Resuspension and Counting
  5. 5 Embryoid Body Seeding
Embryoid Body to Midbrain Organoid Differentiation (Days 1–13) DIV 1–DIV 13
  1. 1 Initial Media Changes (Days 1–2)
  2. 2 Day 3 Half-Media Change
  3. 3 Day 4 Full Media Change with Midbrain 1st Media
  4. 4 Days 6–7 Midbrain 2nd Media Changes
  5. 5 Days 8, 10, 12 Midbrain 3rd Media Changes
Maturation and Neuronal Differentiation (Days 14–35) DIV 14–DIV 35
  1. 1 Transition to Midbrain 4th Media (Every Other Day from Day 14)
  2. 2 Automated Media Changes Using Tecan Fluent 780
  3. 3 High-Content Imaging for Quality Control (DIV 7–DIV 35)
  4. 4 Quality Control Outlier Analysis (DIV 7/8 and DIV 35)
Electrophysiological Assessment (DIV 36–56) DIV 36–DIV 56
  1. 1 MEA Plate Surface Sterilization and Pre-conditioning
  2. 2 MEA Plate Coating: PEI Primary Coating
  3. 3 MEA Plate Coating: Laminin Secondary Coating
  4. 4 Organoid Plating on MEA (DIV 36)
  5. 5 Transition to BrainPhys Media (DIV 41–46)
  6. 6 Baseline Electrophysiology Recordings (DIV 42, 44, 46)
  7. 7 Axon Tracking Assay (DIV 49–56)
  8. 8 Media Maintenance During Recording Period (DIV 48–88)
Cell Dissociation and Immunocytochemistry (DIV 35 and onwards) DIV 35–DIV 46
  1. 1 Organoid Dissociation for Flow Cytometry
  2. 2 Cell Fixation
  3. 3 Immunocytochemistry Blocking
  4. 4 Primary Antibody Staining
  5. 5 Primary Antibody Wash
  6. 6 Secondary Antibody Staining
  7. 7 Final Wash and Suspension
  8. 8 Immunofluorescence on Intact Organoids (Optional)
Lithium Treatment (DIV 46–53) DIV 46–DIV 53
  1. 1 Lithium Carbonate Treatment Initiation
  2. 2 Media Replacement with Lithium
  3. 3 Sample Collection After Treatment
RNA Sequencing and Transcriptomics DIV 35–DIV 53 (harvest and analysis)
  1. 1 Organoid Lysis for RNA Extraction
  2. 2 RNA Sequencing Library Preparation and Sequencing
  3. 3 RNA-Seq Read Alignment and Gene Expression Quantification
  4. 4 Gene Annotation and Ontology Analysis
Proteomics and Phosphoproteomics DIV 35–DIV 53 (harvest and analysis)
  1. 1 Sample Preparation Using S-Trap Protocol
  2. 2 Protein Digestion
  3. 3 Peptide Resuspension and LC-MS/MS Analysis
  4. 4 Phosphoproteomics Enrichment
  5. 5 Mass Spectrometry Analysis
  6. 6 Mass Spectrometry Data Analysis
  7. 7 Kinase Enrichment Analysis (KEA3 and PhosD)
Flow Cytometry and Cell Type Analysis DIV 35–DIV 46 (harvest and analysis)
  1. 1 Flow Cytometry Setup on Sony SH800
  2. 2 Cell Gating and Sorting on Sony SH800
  3. 3 Subsequent Analysis on FACSymphony A3
  4. 4 Cell Type Quantification and Data Analysis

Full SOP

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Attribution

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

This wording is awaiting legal review.

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