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LIVER Publication-derived

Patient-Derived Hepatoblastoma Tumoroid Culture and Functional Characterization

Source Wu et al., 2024 · Stanford University School of Medicine · 10.1038/s41467-024-53802-4

👤 Peng V. Wu, Matt Fish, Florette K. Hazard, Chunfang Zhu, Sujay Vennam, Hannah Walton, Dhananjay Wagh, John Coller, Joanna Przybyl, Maurizio Morri, Norma Neff, Robert B. West, Roel Nusse ⏱ 3 days 📋 11 phases 🧫 Patient-Derived Tumor (Hepatoblastoma)

Abstract

This protocol describes the establishment and culture of patient-derived hepatoblastoma tumoroids in three-dimensional Matrigel-based systems. The tumoroids are propagated in defined media containing specific growth factors (EGF, FGF10, HGF) to generate functional 3D tumor models that recapitulate primary tumor heterogeneity, including differential expression of Wnt pathway components, biliary lineage markers (SOX4, KRT19), and FGF19 expression patterns. These systems enable functional studies of growth factor dependencies and signaling pathway requirements.

Cell source
Patient-Derived Tumor (Hepatoblastoma)
Application
Disease modeling and growth factor dependency testing

Protocol overview

55 steps across 11 phases

Tumor Tissue Procurement and Initial Processing Day 0
  1. 1 Tissue collection and transport
  2. 2 Tissue mincing
  3. 3 Tissue washing
Matrigel Embedding and Media Preparation Day 0–1
  1. 1 Prepare tumoroid media
  2. 2 Embed cell clusters in Matrigel
  3. 3 Overlay media on Matrigel
Tumoroid Propagation and Maintenance Day 2–onwards
  1. 1 Monitor tumoroid growth
  2. 2 Perform media changes
  3. 3 Passage tumoroids (every 1–2 weeks)
Single-Cell Isolation and Colony Formation Assay Day of assay
  1. 1 Dissociate tumoroids to single cells
  2. 2 Count and seed single cells for colony assay
  3. 3 Incubate colonies and perform media changes
  4. 4 Quantify colonies
RNA Isolation and qRT-PCR Analysis Day of assay
  1. 1 Release tumoroids and harvest for RNA
  2. 2 Extract total RNA
  3. 3 Perform reverse transcription
  4. 4 Perform quantitative RT-PCR (qRT-PCR)
  5. 5 Calculate relative gene expression
FGF19 Protein Quantification by ELISA Day of assay
  1. 1 Embed tumoroids and collect conditioned media
  2. 2 Perform sandwich ELISA
  3. 3 Normalize FGF19 concentration
Lentiviral Transduction (Gene Knockdown/Overexpression) Day 0–3
  1. 1 Transfect 293T cells with lentiviral plasmids
  2. 2 Collect lentiviral supernatant
  3. 3 Infect tumoroids with lentivirus
  4. 4 Perform antibiotic selection
  5. 5 Harvest selected cells and validate
Immunofluorescence of Fixed Tumoroids Day of assay
  1. 1 Seed and grow tumoroids for staining
  2. 2 Fix tumoroids
  3. 3 Permeabilize and block tumoroids
  4. 4 Incubate with primary antibodies
  5. 5 Incubate with secondary antibodies and mount
  6. 6 Image tumoroids
Cell Cycle Analysis by Flow Cytometry Day of assay
  1. 1 Synchronize tumoroids
  2. 2 Release into test conditions
  3. 3 Label with EdU
  4. 4 Dissociate tumoroids to single cells
  5. 5 Fix cells
  6. 6 Stain cells with EdU and propidium iodide
  7. 7 Acquire and analyze flow cytometry data
Protein Analysis by Western Blotting Day of assay
  1. 1 Harvest cells and prepare lysates
  2. 2 Clarify lysates
  3. 3 Prepare samples for SDS-PAGE
  4. 4 Perform gel electrophoresis and transfer
  5. 5 Block and incubate with primary antibodies
  6. 6 Wash and incubate with secondary antibodies
  7. 7 Detect and analyze
Single-Cell RNA Sequencing (scRNA-seq) Day of library preparation
  1. 1 Dissociate tumoroids to single cells
  2. 2 Count and prepare cells for 10x loading
  3. 3 Load cells onto 10x Chromium
  4. 4 Prepare sequencing libraries
  5. 5 Sequence libraries
  6. 6 Process sequencing data
  7. 7 Perform clustering and UMAP visualization
  8. 8 Identify marker genes and gene signatures
  9. 9 Perform RNA velocity and pseudotime analyses

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wu et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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