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BRAIN Publication-derived

Paediatric Brain Tumour Organoids

Full SOP with Academic or a plan

Source Lago et al., 2023 · Armenise-Harvard Laboratory of Brain Disorders and Cancer, CIBIO, Trento, Italy · 10.15252/emmm.202318199

Authors: Chiara Lago, Aniello Federico, Gloria Leva, Norman L Mack, Benjamin Schwalm, Claudio Ballabio, Matteo Gianesello, Luana Abballe, Isabella Giovannoni, Sofia Reddel, Sabrina Rossi, Nicolas Leone, Andrea Carai, Angela Mastronuzzi, Alessandra Bisio, Alessia Soldano, Concetta Quintarelli, Franco Locatelli, Marcel Kool, Evelina Miele, Luca Tiberi Duration: 61 days 7 phases Cell source: Patient-Derived Tumor Biopsies (Ependymoma, Medulloblastoma, Low-Grade Glioma)

Abstract

This protocol enables efficient generation, expansion, and biobanking of patient-derived organoids (PDOs) from pediatric brain tumor biopsies and patient-derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the original tumors and demonstrate therapeutic responses comparable to patient treatment outcomes, enabling personalized medicine applications.

Cell source
Patient-Derived Tumor Biopsies (Ependymoma, Medulloblastoma, Low-Grade Glioma)
Application
Disease modeling and personalized drug screening for pediatric brain tumors

Protocol overview

34 steps across 7 phases

Phase 1 · Tumor Sample Collection and Initial Processing Day 0-1
  1. a Collect fresh surgically resected tumor samples
  2. b Transfer tissue to sterile culture hood
  3. c Mechanical dissection into tumor pieces (preferred method)
  4. d Remove red blood cells and debris (if applicable)
Phase 2 · PDO Culture Initiation and Early Growth Day 1-7
  1. a Prepare PDOs culture medium
  2. b Seed tumor pieces into low-adhesion 96-well plates
  3. c Incubate tumor pieces in culture
  4. d Perform first medium change
Phase 3 · PDO Expansion and Maintenance (Weeks 2-12+) Day 8 onwards
  1. a Monitor organoid growth and morphology
  2. b Split organoids for amplification (enzymatic method for PDXOs)
  3. c Split organoids for amplification (mechanical method for PDOs)
  4. d Continue routine medium changes
  5. e Test for Mycoplasma contamination
Phase 4 · Cryopreservation and Recovery of PDOs/PDXOs As needed
  1. a Prepare organoids for freezing
  2. b Load into cryovials and freeze slowly
  3. c Thaw organoids for recovery
  4. d Monitor post-thaw recovery
  5. e Confirm Mycoplasma status post-recovery
Phase 5 · In Vivo Xenografting of PDOs (Optional) Day 20-35 post-establishment
  1. a Prepare PDOs for orthotopic engraftment
  2. b Anesthetize and position mice for injection
  3. c Perform stereotactic injection
  4. d Monitor animal health and tumor development
  5. e Harvest and analyze xenograft tumors
Phase 6 · Quality Control and Characterization Throughout culture; Days 14-61 typical endpoints
  1. a Collect samples for DNA methylation analysis
  2. b Collect samples for histological analysis
  3. c Perform immunofluorescence staining
  4. d Perform single-cell RNA sequencing (optional, for comprehensive characterization)
  5. e Perform copy number variation (CNV) and mutational profiling
Phase 7 · Drug Sensitivity Testing (Optional Translational Application) As needed for clinical translation
  1. a Plate organoids for drug testing
  2. b Prepare drug combinations at specified concentrations
  3. c Perform medium changes during drug treatment
  4. d Live-stain organoids with Calcein-AM and image
  5. e Fix and immunostain for proliferation and apoptosis markers
  6. f Analyze drug response and compare to patient outcome

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Attribution

This SOP was written by Organthis based on the published method in Lago et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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