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BRAIN Publication-derived

Stroke Brain Organoids for Drug Screening

Full SOP with Academic or a plan

Source Kawano et al., 2025 · Kitasato University School of Medicine · 10.3791/69303

Authors: Shihori Kawano, Chika Saegusa, Yusuke Masano, Franziska Becker, Mari Nakamura, Seiji Shiozawa, Junji Fujikura, Takafumi Toyohara, Takaaki Abe, Hideyuki Okano, Masato Fujioka Duration: 60 days 8 phases Cell source: Patient-Derived iPSC (MELAS with m.3243A>G variant)

Abstract

This protocol generates brain organoids from patient-derived iPSCs carrying the m.3243A>G mitochondrial variant associated with MELAS. The method produces 3D cerebral organoids containing FOXG1-positive forebrain neurons within 30 days, which can be dissociated into 2D neural cultures for phenotypic evaluation and drug screening. The protocol enables investigation of MELAS pathophysiology and heteroplasmy-dependent phenotypes.

Cell source
Patient-Derived iPSC (MELAS with m.3243A>G variant)
Application
Disease modeling

Protocol overview

50 steps across 8 phases

Phase 1 · Cell Culture Preparation Day -1 to Day 1
  1. a Coat 6-well plates with laminin
  2. b Prepare prewarmed culture medium with ROCK inhibitor
  3. c Retrieve and rapidly thaw frozen iPSC vial
  4. d Dilute and centrifuge thawed cells
  5. e Resuspend and seed cells into laminin-coated plates
  6. f Medium change on Days 1-2
Phase 2 · Generation of Cerebral Organoids Day 0 to Day 30
  1. a Dissociate iPSCs and prepare for organoid initiation
  2. b Resuspend cells in differentiation medium with inhibitors
  3. c Initial organoid culture
  4. d Medium change at Day 6
  5. e Wash organoids
  6. f Transfer organoids to 96-well plate
  7. g Culture organoids for 3 days
  8. h Medium change at Day 9
  9. i Transfer organoids to 6-well plates
  10. j Medium change at Day 15
  11. k Continue medium changes until Day 30
Phase 3 · Dissociation of Cerebral Organoids for 2D Culture Day 30
  1. a Coat plates with poly-L-ornithine (PO)
  2. b Coat plates with laminin
  3. c Prepare dissociation enzymes
  4. d Rinse and dissociate organoids
  5. e Centrifuge dissociated cells
  6. f Layer isolation solution and centrifuge
  7. g Resuspend cells and filter
  8. h Count cells and seed onto coated plates
Phase 4 · Sample Preparation for Analysis Day 30 and Day 60
  1. a Collect organoids for fixation (Day 30)
  2. b Fix organoids with paraformaldehyde
  3. c Store fixed organoids
  4. d Collect organoids for RNA extraction (Day 30)
  5. e Fix 2D-cultured neurons (Day 60)
Phase 5 · RT-qPCR Analysis Day 30 onwards
  1. a Extract total RNA from organoids
  2. b Synthesize complementary DNA (cDNA)
  3. c Set up quantitative PCR reactions
  4. d Run qPCR program
  5. e Normalize and analyze data
Phase 6 · Immunofluorescence Staining Day 30 and Day 60
  1. a Prepare organoid cryosections
  2. b Remove embedding medium
  3. c Permeabilize tissue
  4. d Block non-specific binding
  5. e Incubate with primary antibodies
  6. f Incubate with secondary antibodies and counterstain
  7. g Mount and image
Phase 7 · Heteroplasmy Analysis Day 0 (on iPSC lines)
  1. a Harvest iPSCs for DNA extraction
  2. b Extract total DNA
  3. c Set up heteroplasmy-specific qPCR
  4. d Run heteroplasmy-detection qPCR
  5. e Calculate heteroplasmy levels
Phase 8 · Organoid Morphology Analysis Day 13-19
  1. a Capture organoid images
  2. b Analyze organoid size and circularity
  3. c Perform statistical analysis

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Attribution

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

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