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BRAIN Publication-derived

Midbrain Organoids for Drug Screening

Full SOP with Academic or a plan

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

Authors: 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 Duration: 59 days 11 phases Cell source: 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

Phase 1 · smNPC Maintenance and Preparation Days 0-5 (prior to organoid generation)
  1. a Coat 6-well plates with Matrigel
  2. b Culture smNPCs in N2B27 medium with small molecules
  3. c Passage smNPCs
Phase 2 · Organoid Generation and Initial Aggregation Days 0-2 (organoid days 0-2)
  1. a Prepare smNPCs for seeding
  2. b Seed 9000 smNPCs per well in U-bottom 96-well plates
  3. c Add polyvinyl alcohol (PVA) to promote cell adhesion
Phase 3 · Ventral Neural Patterning Days 2-6 (organoid days 2-6)
  1. a Initiate ventral patterning by removing CHIR99021
  2. b Add BDNF and GDNF during ventralization
  3. c Feed organoids every other day
Phase 4 · Midbrain Differentiation and Maturation Days 6-30+ (organoid days 6-30 and beyond)
  1. a Remove SAG and add TGF-β3 on day 6
  2. b Add dibutyryl cyclic AMP (dbcAMP) on day 6
  3. c Increase BDNF and GDNF concentration on day 6
  4. d Add Activin A on day 6 (single dose)
  5. e Continue automated feeding every other day
  6. f Culture organoids for minimum 30 days; extend as needed
Phase 5 · Organoid Fixation At desired timepoint (typically day 25-30)
  1. a Fix organoids with 4% paraformaldehyde (PFA)
  2. b Wash fixed organoids with PBS
  3. c Store fixed organoids or proceed to whole mount staining
Phase 6 · Whole Mount Immunostaining Days 1-14 after fixation (6 days primary, 6 days secondary, 2 days washing/recovery)
  1. a Prepare blocking and permeabilization solution
  2. b Incubate with primary antibodies for 6 days
  3. c Wash after primary antibody incubation
  4. d Incubate with secondary antibodies for 6 days
  5. e Final wash after secondary antibody incubation
Phase 7 · Tissue Clearing (BABB Method) 1 day (approximately 2-3 hours active incubation)
  1. a Dehydrate organoids through methanol gradient
  2. b Transfer organoids to BABB-compatible 96-well plates
  3. c Pre-clearing in 1:1 methanol/BABB mixture
  4. d Complete clearing in 100% BABB
Phase 8 · Organoid Positioning and High-Content Imaging 1 day (imaging day)
  1. a Uniform aggregate positioning by tilting
  2. b Acquire overview images at 2× magnification
  3. c Acquire tiled 3D confocal stacks at 10× magnification
  4. d Quantitative image analysis using Harmony and Columbus software
  5. e Data export and statistical analysis
Phase 9 · Functional Analysis: Calcium Imaging (Optional) 1 day (performed at desired developmental timepoint, e.g., day 35)
  1. a Load organoids with Fluo-4 AM calcium indicator
  2. b Image calcium dynamics using spinning-disk or widefield confocal microscopy
  3. c Analyze calcium transient dynamics
  4. d Optional: Measure calcium dynamics on plate reader
  5. e Optional: Test inhibitors to validate calcium signal
Phase 10 · Functional Analysis: Electrophysiology (Optional) 1 day per experiment (performed at desired developmental timepoint, e.g., day 35)
  1. a Prepare MEA substrate
  2. b Transfer organoids to MEA substrate and allow attachment
  3. c Record basal electrical activity
  4. d Add pharmacological modulators and record activity
  5. e Analyze electrical field potentials
Phase 11 · Toxicity Screening: Dopaminergic Neuron-Selective Toxins (Optional) Days 47-59 (example protocol shown; timing adjustable based on organoid maturity)
  1. a Replace antioxidant-containing medium with Tox medium
  2. b Treat organoids with dopaminergic toxins
  3. c Refresh toxin medium at 24 hours
  4. d Recovery period and fixation
  5. e Fix organoids and perform whole mount immunostaining
  6. f High-content imaging and quantification of TH+ cell loss

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Attribution

This SOP was written by Organthis based on the published method in Renner et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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