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BRAIN Publication-derived

Familial Bipolar Disorder Cortical Organoids

Full SOP with Academic or a plan

Source Phalnikar et al., 2024 · Institute for Stem Cell Science and Regenerative Medicine (inStem), Bengaluru, India · 10.1093/oons/kvae007

Authors: Kruttika Phalnikar, M Srividya, SV Mythri, NS Vasavi, Archisha Ganguly, Aparajita Kumar, Padmaja S, Kishan Kalia, Srishti S Mishra, Sreeja Kumari Dhanya, Pradip Paul, Bharath Holla, Suhas Ganesh, Puli Chandramouli Reddy, Reeteka Sud, Biju Viswanath, Bhavana Muralidharan Duration: 50 days 10 phases Cell source: Patient-Derived iPSC (Familial Bipolar Disorder)

Abstract

This protocol generates 3D cortical organoids and 2D neural stem cells from patient-derived iPSCs to model neurodevelopmental deficits in familial bipolar disorder. The organoids recapitulate early cortical development and reveal reduced progenitor proliferation, defective neuroepithelial bud organization, and abnormal neuronal migration in BD patients compared to healthy controls.

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

Protocol overview

52 steps across 10 phases

Phase 1 · iPSC Culture and Maintenance Ongoing (passages maintained up to 10)
  1. a Culture hiPSCs in StemFlex Medium
  2. b Passage iPSC Colonies
  3. c Verify Pluripotency and Karyotype
Phase 2 · Cortical Organoid Generation – Induction Phase Day 0-5
  1. a Dissociate iPSCs to Single Cells
  2. b Centrifuge and Resuspend in Rock Inhibitor
  3. c Seed Cells in Ultra-Low Attachment 96-well Plate
  4. d Transfer Spheroids to 6-well Ultra-Low Attachment Plate
  5. e Daily Media Change During Induction Phase
Phase 3 · Cortical Organoid Development – Neural Differentiation Phase Day 6-25
  1. a Transition to Neural Differentiation Medium with FGF and EGF
  2. b Daily Media Change (Days 6-10)
  3. c Alternate Day Media Change (Days 11-25)
  4. d Maintain Orbital Shaker Conditions
Phase 4 · Cortical Organoid Maturation – BDNF/NT3 Phase Day 25-43
  1. a Replace Growth Factors with BDNF and NT3
  2. b Media Change Every 1-2 Days
  3. c Continue Orbital Shaker Cultivation
Phase 5 · Long-Term Organoid Maintenance Day 43 onwards
  1. a Transition to Maintenance Medium Without Growth Factors
  2. b Media Change Every 2-3 Days
  3. c Maintain Shaker and Incubator Conditions
Phase 6 · Neural Stem Cell Generation and Maintenance (2D) Varies (typically 3 passages before use)
  1. a Dissociate iPSCs and Culture in Neural Induction Medium
  2. b Culture Through Three Passages of Neural Induction
  3. c Transition to Neural Progenitor Medium
  4. d Characterize NSCs for Marker Expression
  5. e Prepare NSCs for Migration Assay
Phase 7 · Immunohistochemistry of Organoids Variable (typically day 40-50 of organoid culture)
  1. a Harvest and Fix Organoids
  2. b Equilibrate in Sucrose Solution
  3. c Embed and Snap-Freeze Organoids
  4. d Cryosection Organoids
  5. e Dry Slides and Fix Sections
  6. f Quench and Permeabilize Sections
  7. g Block and Incubate with Primary Antibodies
  8. h Secondary Antibody and DAPI Staining
  9. i Mount Slides and Store
Phase 8 · EdU Proliferation Assay for Organoids DIV40 (typically)
  1. a Pulse Organoids with EdU
  2. b Fix Organoids
  3. c Process Through Immunohistochemistry
  4. d Co-stain with SOX2 and Ki67 Antibodies
  5. e Quantify Proliferation Markers
Phase 9 · 2D Neural Stem Cell Migration Assay Variable (P2-P6 NSCs, 20 hours of live imaging)
  1. a Prepare NSCs in Culture-Insert 2-well Dish
  2. b Remove Culture-Insert and Image Setup
  3. c Acquire Time-Lapse Images
  4. d Track Individual Cell Trajectories
  5. e Calculate Migration Parameters
  6. f Perform Statistical Analysis
Phase 10 · RNA Sequencing and Transcriptome Analysis DIV43 organoids (harvest to analysis: ~3-4 weeks)
  1. a Harvest Organoids and Preserve in TRIzol
  2. b Perform RNA Extraction and Quantification
  3. c Isolate Poly(A) mRNA
  4. d Prepare cDNA Library
  5. e Perform Sequencing
  6. f Quality Control and Alignment
  7. g Count Features and Normalize Expression
  8. h Identify Differentially Expressed Genes
  9. i Cell-Type Deconvolution

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Attribution

This SOP was written by Organthis based on the published method in Phalnikar et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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