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BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids

Full SOP with Academic or a plan

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

Authors: Smits LM, Reinhardt L, Reinhardt P, Glatza M, Monzel AS, Stanber N, Delber MS, Reinber RM, Sterneckert J, Schwamborn JC Duration: 70 days 6 phases Cell source: 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

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

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Attribution

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

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