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BRAIN Publication-derived

Reproducible generation of human midbrain organoids for in vitro modeling of Parkinson's disease

Source Nickels et al., 2020 · Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg · 10.1016/j.scr.2020.101870

👤 Sarah Louise Nickels, Jennifer Modamio, Bárbara Mendes-Pinheiro, Anna Sophia Monzel, Fay Betsou, Jens Christian Schwamborn ⏱ 38 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes an optimized and standardized approach for generating human midbrain organoids from midbrain floor plate neuronal progenitor cells (mfNPCs) derived from iPSCs. The optimized protocol addresses key limitations including necrotic core formation and batch-to-batch variability while maintaining dopaminergic neuron differentiation and astrocyte development. The organoids are suitable for in vitro modeling of Parkinson's disease and toxin-induced neurodegeneration.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

27 steps across 6 phases

mfNPC Maintenance and Colony Formation Day 0–8 (Condition A), Day 0–2 (Condition B), Day 0–5 (Condition C)
  1. 1 Prepare mfNPC maintenance medium
  2. 2 Split mfNPCs and seed into ultralow adhesion plates
  3. 3 Maintain mfNPC colonies in 96-well plates
Pre-Patterning and Patterning Day 8–17 (Condition A), Day 2–8 (Condition B), Day 5–14 (Condition C)
  1. 1 Prepare Patterning I medium
  2. 2 Apply Patterning I medium
  3. 3 Prepare Patterning II medium
  4. 4 Apply Patterning II medium
Embedding and Maturation Initiation Day 8 (Condition A), Day 2 (Condition B), Day 5 (Condition C)
  1. 1 Prepare geltrex droplets
  2. 2 Embed colonies into geltrex droplets
  3. 3 Initiate shaking culture
Differentiation Day 17–38 (Condition A: 21 days), Day 8–32 (Condition B: 24 days), Day 14–35 (Condition C: 21 days)
  1. 1 Prepare differentiation medium
  2. 2 Replace with differentiation medium
  3. 3 Continue differentiation under shaking conditions
  4. 4 Harvest organoids at day 30
Immunostaining and Analysis Day 30 and beyond
  1. 1 Fix organoids for immunostaining
  2. 2 Whole mount staining protocol (optional acetone treatment)
  3. 3 Block organoids
  4. 4 Incubate with primary antibodies
  5. 5 Wash organoids
  6. 6 Incubate with secondary antibodies
  7. 7 Final wash and mount
  8. 8 Image organoids using confocal microscopy
6-OHDA Treatment (Optional, for Parkinson's Disease Modeling) Day 30–36
  1. 1 Prepare 6-OHDA treatment solutions
  2. 2 Replace medium with 6-OHDA treatment solution
  3. 3 Incubate with 6-OHDA for two consecutive days
  4. 4 Replace with recovery medium
  5. 5 Continue recovery culture

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Nickels et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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