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OTHER Publication-derived

Patient-Derived Organoid (PDO) Establishment and Characterization for Neuroblastoma

Source Fusco et al., 2019 · Fondazione Istituto di Ricerca Pediatrica Città della Speranza (IRP) - Neuroblastoma Laboratory, Padova, Italy · 10.1186/s12885-019-6149-4

👤 P. Fusco, B. Parisatto, E. Rampazzo, L. Persano, C. Frasson, A. Di Meglio, A. Leslz, L. Santoro, B. Cafferata, A. Zin, E. Cimetta, G. Basso, M. R. Esposito, G. P. Tonini ⏱ 90 days 📋 10 phases 🧫 Patient-Derived iPSC (Neuroblastoma)

Abstract

This protocol describes the establishment and characterization of patient-derived organoids (PDOs) from neuroblastoma tumor biopsies. PDOs are cultured in Matrigel-based systems and maintain the histological, genomic, and phenotypic features of primary tumors, including MYCN amplification, 1p deletion, and 17q gain. PDOs enable personalized drug screening and disease pathogenesis studies.

Cell source
Patient-Derived iPSC (Neuroblastoma)
Application
Disease modeling

Protocol overview

54 steps across 10 phases

PDO Establishment and Initial Culture Days 0-7
  1. 1 Prepare Single-Cell Suspension
  2. 2 Embed Cells in Matrigel
  3. 3 Incubate Matrigel Droplets
  4. 4 Transfer to Culture Plates
  5. 5 Monitor Initial Growth
PDO Culture and Maintenance Days 7-40
  1. 6 Prepare Complete PDO Culture Medium
  2. 7 Culture PDOs in Complete Medium
  3. 8 Monitor Growth and Viability
PDO Dissociation and Viability Assessment Days 30, 60
  1. 9 Dissociate PDOs to Single Cells
  2. 10 Assess Cell Viability
PDO Cryopreservation and Recovery Days 21-90
  1. 11 Prepare PDOs for Cryopreservation (Method I: Single Cells)
  2. 12 Freeze Single-Cell PDOs
  3. 13 Thaw and Recover Single-Cell PDOs
  4. 14 Re-establish PDOs from Thawed Cells
  5. 15 Prepare PDOs for Cryopreservation (Method II: Whole Organoids)
  6. 16 Freeze Whole Organoids
  7. 17 Thaw and Culture Whole Organoids
Histological Characterization Days 40+
  1. 18 Collect PDOs for Histology
  2. 19 Prepare Paraffin-Embedded Sections
  3. 20 Perform H&E Staining
Immunohistochemical Analysis Days 40+
  1. 21 Perform Automated Immunostaining
  2. 22 Stain for NB84a Antigen
  3. 23 Stain for Synaptophysin (SYP)
  4. 24 Stain for Chromogranin A (CHGA)
  5. 25 Stain for Mitotic Index (pHH3)
  6. 26 Image Acquisition
Genomic Characterization (aCGH Analysis) Days 40+
  1. 27 Extract and Prepare Genomic DNA
  2. 28 Prepare aCGH Samples
  3. 29 Purify Labeled DNA
  4. 30 Perform aCGH Hybridization
  5. 31 Scan Microarray Chips
  6. 32 Perform Digital Analysis
Protein Expression Analysis (Western Blot) Days 40+
  1. 33 Collect and Dissociate PDOs
  2. 34 Prepare Cell Lysates
  3. 35 Measure Protein Concentration
  4. 36 Prepare and Load Protein Samples
  5. 37 Perform SDS-PAGE Electrophoresis
  6. 38 Transfer Proteins to Membrane
  7. 39 Block Non-specific Binding
  8. 40 Incubate with Primary Antibodies
  9. 41 Wash Membrane
  10. 42 Incubate with Secondary Antibody
  11. 43 Wash and Detect
In Vitro Limiting Dilution Assay (Stemness Assessment) Days 60+
  1. 44 Dissociate PDOs to Single-Cell Suspension
  2. 45 Prepare Serial Dilutions
  3. 46 Culture for Colony Formation
  4. 47 Score Wells for Colony Presence
  5. 48 Analyze Data Using ELDA Software
Flow Cytometry Analysis Days 60+
  1. 49 Dissociate PDOs and Prepare Single-Cell Suspension
  2. 50 Prepare Cells for Staining
  3. 51 Perform Multi-Parameter Staining
  4. 52 Wash and Resuspend Cells
  5. 53 Perform Flow Cytometry Analysis
  6. 54 Analyze Flow Cytometry Data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fusco et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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