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BRAIN Publication-derived

3D Cultures of Parkinson's Disease-Specific Dopaminergic Neurons for High Content Phenotyping and Drug Testing

Source Bolognin et al., 2019 · Luxembourg Centre for Systems Biomedicine, University of Luxembourg · 10.1002/advs.201800927

👤 Silvia Bolognin, Marie Fossépré, Xiaobing Qing, Javier Jarazo, Janez Šcancˇar, Edinson Lucumi Moreno, Sarah L. Nickels, Kobi Wasner, Nassima Ouzren, Jonas Walter, Anne Grünewald, Enrico Glaab, Luis Salamanca, Ronan M. T. Fleming, Paul M. A. Antony, Jens C. Schwamborn ⏱ 42 days 📋 8 phases 🧫 Patient-Derived iPSC (Parkinson's disease, LRRK2-G2019S)

Abstract

This protocol describes the generation and culture of 3D patient-derived dopaminergic neurons from induced pluripotent stem cells carrying the LRRK2-G2019S mutation associated with Parkinson's disease. The 3D microfluidic system reveals robust disease-relevant phenotypes including decreased dopaminergic differentiation, mitochondrial abnormalities, and increased cell death that are undetectable in standard 2D cultures, enabling high-content phenotyping and testing of therapeutic compounds such as LRRK2 inhibitors.

Cell source
Patient-Derived iPSC (Parkinson's disease, LRRK2-G2019S)
Application
Disease modeling and drug screening

Protocol overview

24 steps across 8 phases

Cell Culture Preparation and hNESC Generation Day 0–14
  1. 1 Prepare N2B27 Culture Medium
  2. 2 Culture hNESC Lines in Matrigel-Coated Plates
  3. 3 Passage hNESCs Using Accutase
3D Microfluidic Culture Setup and Differentiation Initiation Day 1–6
  1. 1 Load Cells into OrganoPlates
  2. 2 Initiate Neuronal Differentiation Medium (Days 1–6)
Continued Neuronal Differentiation and Maturation Day 7–42
  1. 1 Switch to PMA-Free Differentiation Medium (Day 7 onwards)
  2. 2 Optional Drug Treatment: Add LRRK2 Inhibitor (if testing)
  3. 3 Maintain Gravity-Driven Perfusion Throughout Culture
Sample Collection and Immunofluorescence Staining (2, 3, and 6 weeks) Day 14, 21, 42
  1. 1 Fix Cells for Immunofluorescence
  2. 2 Permeabilize Cells
  3. 3 Block Non-Specific Antibody Binding
  4. 4 Primary Antibody Incubation
  5. 5 Secondary Antibody Incubation
  6. 6 Mount and Image Samples
Live-Cell Imaging for Mitochondrial Assays (2 and 3 weeks) Day 14, 21
  1. 1 Stain Mitochondrial Membrane Potential with TMRM
  2. 2 Acquire Live Confocal Images for Mitochondrial Analysis
Cell Viability Assays (2 and 3 weeks) Day 14, 21
  1. 1 Live/Dead Staining with Calcein and Ethidium Homodimer
  2. 2 Fix Samples and Stain for Cleaved-Caspase 3
Supplementary Assays: LDH Release and mtDNA Analysis Day 21 (LDH); Day 21 (mtDNA)
  1. 1 Quantify LDH Release (Cell Death Marker)
  2. 2 Analyze Mitochondrial DNA (mtDNA) Copy Number
Image Analysis and Data Extraction After imaging completion
  1. 1 Automated Image Analysis: Morphometric Features
  2. 2 Automated Image Analysis: Mitochondrial Features
  3. 3 Automated Image Analysis: Cell Death Features
  4. 4 Statistical Analysis and Multivariate Classification

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Bolognin et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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