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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Lee et al., 2022 · Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea · 10.3389/fnagi.2022.925227

Authors: Youngsun Lee, Ji Su Kang, On-Ju Ham, Mi-Young Son, Mi-Ok Lee Duration: 30 days 5 phases Cell source: Human ESC (H9) and Human iPSC (LRRK2 G2019S mutant and gene-corrected)

Abstract

This protocol generates midbrain organoids from human pluripotent stem cells and treats them with trimethylamine N-oxide (TMAO) to induce aging-associated phenotypes. The TMAO-treated midbrain organoids exhibit increased cellular senescence, epigenetic alterations, loss of dopaminergic neurons, astrocyte activation, and pathological protein phosphorylation (α-synuclein and Tau), providing an in vitro model for studying brain aging and Parkinson's disease pathogenesis without genetic modification.

Cell source
Human ESC (H9) and Human iPSC (LRRK2 G2019S mutant and gene-corrected)
Application
Disease modeling; Aging model development for late-onset neurodegenerative diseases

Protocol overview

14 steps across 5 phases

Phase 1 · Cell Culture and Maintenance Ongoing until organoid generation
  1. a Culture pluripotent stem cells in TeSR-E8 medium
  2. b Passage cells using ReLeSR
Phase 2 · Embryoid Body Formation and Early Patterning Day 0-2
  1. a Dissociate cells to single-cell suspension
  2. b Culture in EBM medium (Day 0-1)
  3. c Switch to BGM medium (Day 1-2)
Phase 3 · Midbrain Patterning and Matrigel Embedding Day 2-9
  1. a Add mesencephalon patterning factors to BGM (Day 2)
  2. b Embed organoids in growth factor-reduced matrigel
  3. c Transfer to ultralow attachment 6-well plate and culture on orbital shaker (Day 9)
Phase 4 · Midbrain Maturation (Weeks 4-30) Day 9 onwards (4WM to 30WM)
  1. a Culture in BMM medium with daily medium changes
  2. b Begin TMAO treatment at 4 weeks of maturation (4WM)
  3. c Maintain organoids until endpoint (30WM total maturation)
Phase 5 · Sample Preparation for Analysis At designated timepoints (8WM, 30WM)
  1. a Harvest organoids for RNA extraction
  2. b Prepare organoids for immunostaining
  3. c Prepare conditioned media for dopamine and biochemical assays

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Attribution

This SOP was written by Organthis based on the published method in Lee et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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