BRAIN Publication-derived

Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS)

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

👤 Shihori Kawano, Chika Saegusa, Yusuke Masano, Franziska Becker, Mari Nakamura, Seiji Shiozawa, Junji Fujikura, Takafumi Toyohara, Takaaki Abe, Hideyuki Okano, Masato Fujioka ⏱ 60 days 📋 8 phases 🧫 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

Cell Culture Preparation Day -1 to Day 1

1 Coat 6-well plates with laminin
2 Prepare prewarmed culture medium with ROCK inhibitor
3 Retrieve and rapidly thaw frozen iPSC vial
4 Dilute and centrifuge thawed cells
5 Resuspend and seed cells into laminin-coated plates
6 Medium change on Days 1-2

Generation of Cerebral Organoids Day 0 to Day 30

1 Dissociate iPSCs and prepare for organoid initiation
2 Resuspend cells in differentiation medium with inhibitors
3 Initial organoid culture
4 Medium change at Day 6
5 Wash organoids
6 Transfer organoids to 96-well plate
7 Culture organoids for 3 days
8 Medium change at Day 9
9 Transfer organoids to 6-well plates
10 Medium change at Day 15
11 Continue medium changes until Day 30

Dissociation of Cerebral Organoids for 2D Culture Day 30

1 Coat plates with poly-L-ornithine (PO)
2 Coat plates with laminin
3 Prepare dissociation enzymes
4 Rinse and dissociate organoids
5 Centrifuge dissociated cells
6 Layer isolation solution and centrifuge
7 Resuspend cells and filter
8 Count cells and seed onto coated plates

Sample Preparation for Analysis Day 30 and Day 60

1 Collect organoids for fixation (Day 30)
2 Fix organoids with paraformaldehyde
3 Store fixed organoids
4 Collect organoids for RNA extraction (Day 30)
5 Fix 2D-cultured neurons (Day 60)

RT-qPCR Analysis Day 30 onwards

1 Extract total RNA from organoids
2 Synthesize complementary DNA (cDNA)
3 Set up quantitative PCR reactions
4 Run qPCR program
5 Normalize and analyze data

Immunofluorescence Staining Day 30 and Day 60

1 Prepare organoid cryosections
2 Remove embedding medium
3 Permeabilize tissue
4 Block non-specific binding
5 Incubate with primary antibodies
6 Incubate with secondary antibodies and counterstain
7 Mount and image

Heteroplasmy Analysis Day 0 (on iPSC lines)

1 Harvest iPSCs for DNA extraction
2 Extract total DNA
3 Set up heteroplasmy-specific qPCR
4 Run heteroplasmy-detection qPCR
5 Calculate heteroplasmy levels

Organoid Morphology Analysis Day 13-19

1 Capture organoid images
2 Analyze organoid size and circularity
3 Perform statistical analysis

Full SOP

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Attribution

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

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