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BRAIN Publication-derived

Midbrain Organoids for Drug Screening and Disease Modelling

Full SOP with Academic or a plan

Source Parrotta et al., 2025 · University 'Magna Graecia', Catanzaro, Italy · 10.1002/ana.27172

Authors: Elvira Immacolata Parrotta, Valeria Lucchino, Clara Zannino, Desirèe Valente, Stefania Scalise, Davide Bressan, Giorgia Lucia Benedetto, Maria Roberta Iazzetta, Mariagrazia Talarico, Monica Gagliardi, Francesco Conforti, Silvia Di Agostino, Alessandro Fiorenzano, Aldo Quattrone, Giovanni Cuda, Andrea Quattrone Duration: 120 days 5 phases Cell source: Patient-Derived iPSC (Sporadic Progressive Supranuclear Palsy-Richardson's syndrome)

Abstract

This protocol describes the generation and comprehensive characterization of multi-donor midbrain organoids (MOs) derived from induced pluripotent stem cells (iPSCs) of patients with sporadic progressive supranuclear palsy-Richardson's syndrome (PSP-RS) over a 120-day maturation period. The organoids recapitulate key pathological hallmarks of PSP including accumulation of hyperphosphorylated tau, neurofibrillary tangles, tufted astrocytes, and dopaminergic neuron degeneration, providing a robust in vitro model for disease pathophysiology studies and drug discovery.

Cell source
Patient-Derived iPSC (Sporadic Progressive Supranuclear Palsy-Richardson's syndrome)
Application
Disease modeling

Protocol overview

19 steps across 5 phases

Phase 1 · Induced Pluripotent Stem Cell (iPSC) Generation and Characterization Days 1-30
  1. a Blood Sample Collection and Processing
  2. b iPSC Reprogramming using Sendai Virus Kit
  3. c MAPT Gene Sequencing and H1/H2 Haplotype Detection
Phase 2 · Multi-Donor Midbrain Organoid (MO) Generation and Early Development Days -3 to 30
  1. a iPSC Preparation and Pooling for Organoid Generation
  2. b Embryoid Body (EB) Formation in Ultra-Low Adhesion Plates
  3. c Midbrain Organoid Differentiation Initiation
  4. d Early Midbrain Marker Expression Analysis (Day 20)
Phase 3 · Mid-Stage Organoid Maturation and Dopaminergic Neuron Development Days 30-60
  1. a Dopaminergic Neuron Marker Expression (Day 30)
  2. b Late Midbrain Marker Analysis (Day 60)
  3. c Organoid Size Monitoring
Phase 4 · Late Maturation, Tau Pathology Development, and Comprehensive Characterization Days 60-120
  1. a Organoid Cryopreservation Preparation (Day 60-90)
  2. b Cryosectioning for Immunofluorescence and Immunochemistry
  3. c Immunofluorescence Analysis of Hyperphosphorylated Tau Forms (Days 60, 90, 120)
  4. d Immunochemistry (IHC) for Histological Confirmation (Day 90)
  5. e Western Blot Analysis of Tau and Dopaminergic Markers
  6. f RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
  7. g Neuromelanin Pigmentation Assessment (Day 120)
Phase 5 · iPSC Proliferation and Cell Cycle Analysis (Supporting Studies) Days 1-30 (parallel to organoid generation)
  1. a iPSC Proliferation Assay
  2. b Cell Cycle Profile Analysis by Flow Cytometry

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Attribution

This SOP was written by Organthis based on the published method in Parrotta et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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