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BRAIN Publication-derived

Midbrain Organoid Model of Parkinson Disease with α-Synuclein

Source Smits et al. · University of Luxembourg, Luxembourg · 10.1038/s41531-019-0078-4

👤 Smits LM, Reinhardt L, Reinhardt P, Glatza M, Monzel AS, Stanber N, Delber MS, Reinber RM, Sterneckert J, Schwamborn JC ⏱ 70 days 📋 6 phases 🧫 Patient-Derived iPSC (SNCA Triplication)

Abstract

Smits et al. generated midbrain organoids from iPSCs of patients carrying SNCA triplication, which develop endogenous α-synuclein aggregation and selective dopaminergic neuron vulnerability over 60 days of culture. This model recapitulates key Parkinson disease pathology and provides a platform for testing neuroprotective compounds.

Cell source
Patient-Derived iPSC (SNCA Triplication)
Application
Parkinson Disease Modeling

Protocol overview

34 steps across 6 phases

iPSC Preparation and Derivation of mfNPCs (2D Protocol) Day 0–8
  1. 1 iPSC Dissociation and Colony Preparation
  2. 2 Neural Induction Medium Preparation and Culture
  3. 3 Medium Exchanges (Days 2 and 4)
  4. 4 Embryoid Body (EB) Collection and Replating
  5. 5 Transition to mfNPC Expansion Medium
  6. 6 First Passage and High-Density Culture
  7. 7 Subsequent Passages (P2–P5)
Derivation of mfNPCs Under 3D Conditions (Alternative Protocol for Organoid Starting Material) Day 0–8+ (up to Passage 4–5)
  1. 1 iPSC Dissociation and EB Formation in AggreWell
  2. 2 EB Culture and Transfer
  3. 3 N2B27 Medium Transition (Day 2)
  4. 4 Ascorbic Acid Addition (Days 4 and 6)
  5. 5 EB Trituration and Transfer to Ultra-Low Attachment Plates (Day 8)
  6. 6 Subsequent Passages (from Passage 1 onward)
  7. 7 Expansion to Passages 4–5
Expansion of mfNPCs in 2D Culture Ongoing (Passages 5–15)
  1. 1 mfNPC Culture Setup
  2. 2 mfNPC Expansion Medium Preparation and Exchange
  3. 3 Cell Dissociation and Passage
  4. 4 Passage Range and Cell Identity Maintenance
Expansion of 3D-cultured mfNPCs Ongoing (from Passage 1–5 onward, every 7–14 days)
  1. 1 Spherical Colony Collection and Dissociation
  2. 2 Re-seeding and Aggregation
  3. 3 ROCK Inhibitor Addition and Medium Management
2D Differentiation of mfNPCs into mDANs Day 0–25 (14 days minimum for analysis)
  1. 1 mfNPC Seeding for Differentiation
  2. 2 Dopaminergic Patterning Medium (Day 2)
  3. 3 Neuronal Maturation Medium Initiation (Day 6)
  4. 4 Optional DAPT Supplementation for Maturation Enhancement
  5. 5 Cell Density Management During Maturation
  6. 6 Analysis Timepoint
3D Differentiation of mfNPCs into Midbrain-Specific Organoids (hMOs) Day 0–70 (or longer)
  1. 1 Seeding and Maintenance Phase (Days 0–7)
  2. 2 Pre-patterning Phase (Days 7–10)
  3. 3 Dopaminergic Differentiation Initiation (Day 9–10)
  4. 4 Maturation Phase Initiation (Day 12)
  5. 5 Optional DAPT Addition (Day 12 onward)
  6. 6 Static Culture with Regular Medium Changes
  7. 7 Analysis Timepoints

Full SOP

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Attribution

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

This wording is awaiting legal review.

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