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LIVER Publication-derived

Hepatoblastoma Liver Tumouroids for Drug Screening

Full SOP with Academic or a plan

Source Kluiver et al., 2024 · Princess Máxima Center for Pediatric Oncology · 10.1038/s41467-024-52757-w

Authors: Thomas A. Kluiver, Yuyan Lu, Stephanie A. Schubert, Lianne J. Kraaier, Femke Ringnalda, Philip Lijnzaad, Jeff DeMartino, Wouter L. Megchelenbrink, Vicky Amo-Addae, Selma Eising, Flavia W. de Faria, Daniel Münter, Marc van de Wetering, Kornelius Kerl, Evelien Duiker, Marius C. van den Heuvel, Vincent E. de Meijer, Ruben H. de Kleine, Jan J. Molenaar, Thanasis Margaritis, Hendrik G. Stunnenberg, Ronald R. de Krijger, József Zsiros, Hans Clevers, Weng Chuan Peng Duration: 10 days 5 phases Cell source: Patient-Derived Hepatoblastoma

Abstract

This protocol describes the establishment and molecular characterization of patient-derived hepatoblastoma tumor organoids (HBTOs) from biopsies and surgical resections. The organoids recapitulate the molecular heterogeneity of hepatoblastomas (fetal and embryonal subtypes) and enable high-throughput drug screening to identify therapeutic vulnerabilities, including sensitivity to HDAC, FGFR, and EGFR inhibitors.

Cell source
Patient-Derived Hepatoblastoma
Application
Disease modeling and drug screening

Protocol overview

32 steps across 5 phases

Phase 1 · Tissue Dissociation and Single Cell Preparation Day 0
  1. a Tissue Mincing
  2. b Liver Perfusion Medium Incubation
  3. c Wash and Centrifuge
  4. d Enzymatic Digestion
  5. e Assess Dissociation Progress
  6. f Mechanical Dissociation
  7. g Red Blood Cell Lysis (Optional)
  8. h Enzyme Wash-off
Phase 2 · Organoid Plating and Initial Culture Day 0-Day 7
  1. a Basement Membrane Extract (BME) Preparation
  2. b Culture Plate Preparation
  3. c Droplet Plating
  4. d BME Gelation
  5. e Culture Medium Addition
  6. f Initial Incubation
Phase 3 · Ongoing Organoid Culture and Maintenance Day 7 onwards
  1. a Medium Replacement Schedule
  2. b Monitor Organoid Morphology
  3. c Passage Organoids (Every 1-3 weeks)
  4. d Dispase-mediated BME Dissociation
  5. e PBS Wash
  6. f Single-Cell Dissociation with TryPLE
  7. g Final PBS Wash
  8. h Replating in Fresh BME
Phase 4 · Full Culture Medium Composition and Optimization Ongoing
  1. a Full Medium Preparation
  2. b Add Basal Supplements
  3. c Add Growth Factors
  4. d Add Small Molecule Regulators
  5. e Reduced Medium Alternative
Phase 5 · Validation and Quality Control Upon establishment and at passages P2, P5, P10
  1. a CTNNB1 Mutation Verification
  2. b Western Blot for β-Catenin
  3. c Copy Number Variation (CNV) Profiling
  4. d Single-Cell RNA-Seq (scRNA-seq) Characterization
  5. e Immunofluorescence (IF) Staining for HNF4A and LEF1

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Attribution

This SOP was written by Organthis based on the published method in Kluiver et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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