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BRAIN Publication-derived

Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids

Source Renner et al., 2021 · Department for Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany · 10.3389/fnmol.2021.715054

👤 Henrik Renner, Katharina J. Becker, Theresa E. Kagermeier, Martha Grabos, Farsam Eliat, Patrick Günther, Hans R. Schöler, Jan M. Bruder ⏱ 56 days 📋 7 phases 🧫 Human iPSC-derived small molecule neural precursor cells (smNPCs)

Abstract

This protocol describes the generation and toxicity screening of automated midbrain organoids (AMOs) derived from human iPSCs to evaluate general and dopaminergic neuron-specific toxicity of compounds at the single-cell level. The standardized workflow enables high throughput assessment of compound effects on complex human neural tissue in vitro, combining ATP-based viability assays with high-content imaging of dopaminergic and general neuronal populations.

Cell source
Human iPSC-derived small molecule neural precursor cells (smNPCs)
Application
High throughput toxicity screening and cell-type-specific toxicity assessment

Protocol overview

41 steps across 7 phases

Small Molecule Neural Precursor Cell (smNPC) Culture and Maintenance Ongoing maintenance, split every 5-7 days
  1. 1 Coat culture plates with Matrigel
  2. 2 Culture smNPCs in N2B27 medium with small molecule supplements
  3. 3 Split smNPCs every 5-7 days
Generation of Automated Midbrain Organoids (AMOs) Day 0 to Day 50
  1. 1 Prepare single-cell suspension and seed into conical 96-well plates
  2. 2 Initiate ventral patterning (Days 2-6)
  3. 3 Add maturation factors and activin A (Day 6)
  4. 4 Continue maintenance feeding (Days 6-50)
Primary Organoid Viability Screen Day 47-50
  1. 1 Switch to toxicity medium (Day 47)
  2. 2 Treat organoids with compound library (Day 48)
  3. 3 Measure organoid viability with CellTiter-Glo 3D assay
  4. 4 Record luminescence
  5. 5 Normalize and analyze viability data
Whole-Mount Staining and Clearing of Organoids Day 50 (after primary screen compound incubation and recovery)
  1. 1 Fix organoids with 4% paraformaldehyde
  2. 2 Block and permeabilize organoids
  3. 3 Incubate with primary antibodies (6 days)
  4. 4 Wash after primary antibodies
  5. 5 Incubate with secondary antibodies and DAPI (6 days)
  6. 6 Wash after secondary antibodies
  7. 7 Dehydrate organoids via methanol series
  8. 8 Transfer to organic solvent-resistant plates
  9. 9 Clear organoids with BABB solvent
Primary Cell-Type-Specific Toxicity Screen and High Content Image Analysis Day 50-56
  1. 1 Prepare and treat organoids with compound library (Day 50)
  2. 2 Recovery period in standard medium (6 days)
  3. 3 Fix, stain, and clear organoids
  4. 4 Position organoids for imaging
  5. 5 Acquire high-content confocal images
  6. 6 Analyze images and quantify cell populations
  7. 7 Normalize and analyze cell-type-specific toxicity data
Dose-Response Analysis of Selected Compounds Day 47-56
  1. 1 Prepare organoids and switch to toxicity medium (Day 47)
  2. 2 Treat organoids with compound dose series (Day 48)
  3. 3 Recovery period and medium exchange (Days 50-56)
  4. 4 Measure viability with CellTiter-Glo 3D assay
  5. 5 Fix, stain, and clear organoids for cell-type-specific analysis
  6. 6 Acquire images and analyze cell populations
  7. 7 Normalize and fit dose-response curves
Comparison of Toxicity in 2D and 3D Cultures (Optional) Day 0-50
  1. 1 Generate 2D dopaminergic neurons in parallel with 3D organoids
  2. 2 Reseed 2D neurons to 96-well plates (Day 48)
  3. 3 Switch both 2D and 3D to toxicity medium (Day 49)
  4. 4 Treat 2D and 3D with dose series (Day 50)
  5. 5 Measure viability in both 2D and 3D (48 h post-treatment)
  6. 6 Normalize and compare 2D vs. 3D toxicity

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Renner et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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