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BRAIN Publication-derived

Integrated, Automated Maintenance, Expansion, and Differentiation of Patient-Derived Cellular Models for High-Throughput Drug Screening

Source Boussaad et al., 2021 · Luxembourg Centre for Systems Biomedicine, University of Luxembourg & Luxembourg Institute of Health · 10.1038/s41598-021-81129-3

👤 Ibrahim Boussaad, Gérald Cruciani, Silvia Bolognin, Paul Antony, Claire M. Dording, Yong-Jun Kwon, Peter Heutink, Eugenio Fava, Jens C. Schwamborn, Rejko Krüger ⏱ 30 days 📋 7 phases 🧫 Human iPSC, Human Fibroblasts, Small Molecule-Derived Neural Progenitor Cells (smNPC)

Abstract

This protocol describes an integrated automated platform for maintaining, expanding, and differentiating patient-derived cellular models including fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC). The system enables automated cultivation, splitting, expansion, and differentiation of these cell types into 2D dopaminergic neurons and 3D midbrain organoids suitable for high-throughput and high-content screening (HTS/HCS) applications in translational neurodegenerative disease research.

Cell source
Human iPSC, Human Fibroblasts, Small Molecule-Derived Neural Progenitor Cells (smNPC)
Application
High-throughput drug screening, Disease modeling, Phenotypic screening

Protocol overview

41 steps across 7 phases

Fibroblast Maintenance and Splitting Passage 1 to continuous maintenance
  1. 1 Prepare plates and trypsin treatment
  2. 2 Tilt and collect detached cells
  3. 3 Wash source well and pool cells
  4. 4 Divide cell suspension into deepwell plate wells
  5. 5 Seed into destination wells
  6. 6 Incubate seeded cells
iPSC Maintenance and Splitting Passage 1 to continuous maintenance
  1. 1 Prepare matrix-coated plates and wash iPSC
  2. 2 Detach iPSC with EDTA solution
  3. 3 Collect and centrifuge iPSC clumps
  4. 4 Resuspend and distribute iPSC clumps
  5. 5 Seed into destination wells
  6. 6 Incubate and monitor colony formation
Small Molecule-Derived Neural Progenitor Cell (smNPC) Maintenance and Splitting Passage 1 to continuous maintenance
  1. 1 Prepare matrix-coated plates and wash smNPC
  2. 2 Detach smNPC with protease solution
  3. 3 Collect and wash detached smNPC
  4. 4 Distribute cell suspension
  5. 5 Seed into destination plates
  6. 6 Incubate and feed smNPC
Cell Expansion to Large-Scale Culture Days 1-3 post-seeding
  1. 1 First expansion: 6-well to 1-well plate (fibroblasts)
  2. 2 Second expansion: 1-well to 1-well plates (fibroblasts)
  3. 3 First expansion: 6-well to 1-well plate (iPSC)
  4. 4 Second expansion: 1-well to 1-well plates (iPSC)
  5. 5 First expansion: 6-well to 1-well plate (smNPC)
  6. 6 Second expansion: 1-well to 1-well plates (smNPC)
Automated 2D Midbrain Dopaminergic Neuronal Differentiation Days 0-21+
  1. 1 Initiate differentiation: Seed smNPC in differentiation matrix
  2. 2 First splitting during differentiation (Day 3-4)
  3. 3 Second splitting during differentiation (Day 8)
  4. 4 Switch to maturation medium (Day 10)
  5. 5 Neuronal maturation (Days 10-21+)
  6. 6 Harvest neurons for assay plates (Day 17 or later)
Automated Generation of Midbrain-Specific Organoids Days 0-30
  1. 1 Prepare cell suspension and ultra-low adherence plates
  2. 2 Dispense cells into 96-well organoid plate
  3. 3 Centrifuge plate for cell settling
  4. 4 Spheroid formation phase (Days 0-7)
  5. 5 Transition to differentiation medium (Day 8)
  6. 6 Maturation phase (Days 14-30)
  7. 7 Assess organoid size and homogeneity (Day 30)
Quality Control: Confluency and Cell Distribution Assessment 24 hours post-seeding
  1. 1 Acquire confluence images with multimode plate reader
  2. 2 Calculate confluency percentage
  3. 3 Calculate coefficient of variance (CV) for cell distribution
  4. 4 Compare inter-well confluence differences

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Boussaad et al., 2021. 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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