Skip to content
Back to browse
BRAIN Publication-derived

Parkinson's Disease Midbrain Organoids for Drug Screening (2021)

Full SOP with Academic or a plan

Source Zanetti et al., 2021 · Vienna University of Technology (TUW), Austria · 10.1039/d0an02206c

Authors: Cristian Zanetti, Sarah Spitz, Emanuel Berger, Silvia Bolognin, Lisa M. Smits, Philipp Crepaz, Mario Rothbauer, Julie M. Rosser, Martina Marchetti-Deschmann, Jens C. Schwamborn, Peter Ertl 6 phases Cell source: Human iPSC

Abstract

This protocol describes a non-invasive electrochemical sensing approach using redox cycling and 3-mercaptopropionic acid (MPA) self-assembled monolayers to detect dopamine release from human midbrain organoids (hMOs). The method enables label-free, time-resolved monitoring of dopamine levels in complex cell culture matrices and demonstrates the ability to identify differences between healthy and Parkinson's disease-specific hMOs, including phenotypic rescue upon LRRK2 inhibitor II treatment.

Cell source
Human iPSC
Application
Disease modeling and drug screening

Protocol overview

21 steps across 6 phases

Phase 1 · Electrode preparation and MPA modification Day -1 to Day 0
  1. a Electrode pre-treatment and cleaning
  2. b MPA self-assembled monolayer modification
  3. c Confirmation of MPA modification by electrochemical impedance spectroscopy (EIS)
Phase 2 · hNESC maintenance and hMO generation Day -10 to Day 9
  1. a hNESC seeding for hMO generation
  2. b Organoid maintenance phase (Days 0-7)
  3. c Pre-patterning phase (Days 7-10)
  4. d Transition to neuronal maturation medium (Day 9)
Phase 3 · Long-term organoid cultivation Day 10 to Day 70
  1. a Static culture and maintenance
  2. b Optional drug treatment with LRRK2 inhibitor II
Phase 4 · Electrochemical measurement setup and dopamine detection Day 23 onwards
  1. a Electrochemical cell assembly
  2. b Sample preparation and calibration
  3. c Electrochemical measurement
Phase 5 · LC-MRM-MS verification analysis Day 60 (sample collection and processing)
  1. a Sample preservation and storage
  2. b Sample preparation for LC-MRM-MS
  3. c LC-MRM-MS analysis
Phase 6 · Immunofluorescence characterization Day 60-70
  1. a Organoid fixation
  2. b Agarose embedding and sectioning
  3. c Permeabilization
  4. d Blocking and primary antibody incubation
  5. e Secondary antibody and nuclear staining
  6. f Mounting and imaging

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Zanetti et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence