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BRAIN Publication-derived

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

Source Renner et al., 2020 · Max Planck Institute for Molecular Biomedicine, Münster, Germany · 10.7554/elife.52904

👤 Henrik Renner, Martha Grabos, Katharina J Becker, Theresa E Kagermeier, Jie Wu, Mandy Otto, Stefan Peischard, Dagmar Zeuschner, Yaroslav TsyTsyura, Paul Disse, Jürgen Klingauf, Sebastian A Leidel, Guiscard Seebohm, Hans R Schöler, Jan M Bruder ⏱ 59 days 📋 11 phases 🧫 Patient-Derived iPSC (from same line as smNPCs, human midbrain-specific neural precursor cells)

Abstract

This protocol describes a fully automated workflow for the generation, maintenance, and optical analysis of homogeneous human midbrain organoids in standard 96-well plates. The resulting organoids exhibit dopaminergic differentiation, spontaneous synchronized neural activity, and reproducible morphology, enabling high-throughput screening of compounds for midbrain-specific disorders such as Parkinson's disease.

Cell source
Patient-Derived iPSC (from same line as smNPCs, human midbrain-specific neural precursor cells)
Application
Drug screening, Disease modeling, High-throughput toxicity testing

Protocol overview

48 steps across 11 phases

smNPC Maintenance and Preparation Days 0-5 (prior to organoid generation)
  1. 1 Coat 6-well plates with Matrigel
  2. 2 Culture smNPCs in N2B27 medium with small molecules
  3. 3 Passage smNPCs
Organoid Generation and Initial Aggregation Days 0-2 (organoid days 0-2)
  1. 1 Prepare smNPCs for seeding
  2. 2 Seed 9000 smNPCs per well in U-bottom 96-well plates
  3. 3 Add polyvinyl alcohol (PVA) to promote cell adhesion
Ventral Neural Patterning Days 2-6 (organoid days 2-6)
  1. 1 Initiate ventral patterning by removing CHIR99021
  2. 2 Add BDNF and GDNF during ventralization
  3. 3 Feed organoids every other day
Midbrain Differentiation and Maturation Days 6-30+ (organoid days 6-30 and beyond)
  1. 1 Remove SAG and add TGF-β3 on day 6
  2. 2 Add dibutyryl cyclic AMP (dbcAMP) on day 6
  3. 3 Increase BDNF and GDNF concentration on day 6
  4. 4 Add Activin A on day 6 (single dose)
  5. 5 Continue automated feeding every other day
  6. 6 Culture organoids for minimum 30 days; extend as needed
Organoid Fixation At desired timepoint (typically day 25-30)
  1. 1 Fix organoids with 4% paraformaldehyde (PFA)
  2. 2 Wash fixed organoids with PBS
  3. 3 Store fixed organoids or proceed to whole mount staining
Whole Mount Immunostaining Days 1-14 after fixation (6 days primary, 6 days secondary, 2 days washing/recovery)
  1. 1 Prepare blocking and permeabilization solution
  2. 2 Incubate with primary antibodies for 6 days
  3. 3 Wash after primary antibody incubation
  4. 4 Incubate with secondary antibodies for 6 days
  5. 5 Final wash after secondary antibody incubation
Tissue Clearing (BABB Method) 1 day (approximately 2-3 hours active incubation)
  1. 1 Dehydrate organoids through methanol gradient
  2. 2 Transfer organoids to BABB-compatible 96-well plates
  3. 3 Pre-clearing in 1:1 methanol/BABB mixture
  4. 4 Complete clearing in 100% BABB
Organoid Positioning and High-Content Imaging 1 day (imaging day)
  1. 1 Uniform aggregate positioning by tilting
  2. 2 Acquire overview images at 2× magnification
  3. 3 Acquire tiled 3D confocal stacks at 10× magnification
  4. 4 Quantitative image analysis using Harmony and Columbus software
  5. 5 Data export and statistical analysis
Functional Analysis: Calcium Imaging (Optional) 1 day (performed at desired developmental timepoint, e.g., day 35)
  1. 1 Load organoids with Fluo-4 AM calcium indicator
  2. 2 Image calcium dynamics using spinning-disk or widefield confocal microscopy
  3. 3 Analyze calcium transient dynamics
  4. 4 Optional: Measure calcium dynamics on plate reader
  5. 5 Optional: Test inhibitors to validate calcium signal
Functional Analysis: Electrophysiology (Optional) 1 day per experiment (performed at desired developmental timepoint, e.g., day 35)
  1. 1 Prepare MEA substrate
  2. 2 Transfer organoids to MEA substrate and allow attachment
  3. 3 Record basal electrical activity
  4. 4 Add pharmacological modulators and record activity
  5. 5 Analyze electrical field potentials
Toxicity Screening: Dopaminergic Neuron-Selective Toxins (Optional) Days 47-59 (example protocol shown; timing adjustable based on organoid maturity)
  1. 1 Replace antioxidant-containing medium with Tox medium
  2. 2 Treat organoids with dopaminergic toxins
  3. 3 Refresh toxin medium at 24 hours
  4. 4 Recovery period and fixation
  5. 5 Fix organoids and perform whole mount immunostaining
  6. 6 High-content imaging and quantification of TH+ cell loss

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Renner et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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