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LIVER Publication-derived

Patient-Derived Hepatocellular Carcinoma with Neuroendocrine Differentiation (HCC-NED) Organoid Generation and Drug Screening

Source Meier et al., 2022 · University Hospital and University of Basel, Switzerland · 10.1038/s43856-022-00150-3

👤 Marie-Anne Meier, Sandro Nuciforo, Mairene Coto-Llerena, John Gallon, Matthias S. Matter, Caner Ercan, Jürg Vosbeck, Luigi M. Terracciano, Savas D. Soysal, Daniel Boll, Otto Kollmar, Raphaël Delaloye, Salvatore Piscuoglio, Markus H. Heim ⏱ 28 days 📋 7 phases 🧫 Patient-Derived Tumor Tissue (HCC-NED)

Abstract

This protocol describes the generation of three-dimensional organoids from patient-derived hepatocellular carcinoma tissue with neuroendocrine differentiation (HCC-NED), including tissue dissociation, basement membrane culture, and subsequent characterization by histology, immunohistochemistry, and drug sensitivity testing. The HCC-NED organoids retain key histological and molecular features of the primary tumor and enable in vitro drug screening to guide personalized treatment decisions in this rare liver cancer entity.

Cell source
Patient-Derived Tumor Tissue (HCC-NED)
Application
Disease modeling and personalized drug screening for rare hepatocellular carcinoma subtype

Protocol overview

41 steps across 7 phases

Tissue Dissociation and Organoid Seeding Day 0–1
  1. 1 Tissue Collection and Preparation
  2. 2 Dissociation to Small Cell Clusters
  3. 3 Seed Cell Clusters in Basement Membrane Extract Type 2 (BME2) Domes
Organoid Expansion Culture Day 1–7 (Weekly cycles)
  1. 1 Overlay BME2 Domes with Expansion Medium
  2. 2 Incubate at 37°C in Humidified CO₂ Incubator
  3. 3 Weekly Medium Replacement
  4. 4 Monitor Organoid Growth
Organoid Passaging and Cryopreservation Day 7 and onwards
  1. 1 Dissociate Organoids for Passage
  2. 2 Reseed Dissociated Cells in Fresh BME2 Domes
  3. 3 Prepare Frozen Stocks
  4. 4 Test for Mycoplasma Contamination
Histology and Immunohistochemistry Day 21+ (after organoid expansion)
  1. 1 Release Organoids from BME2
  2. 2 Fix Organoids in 4% Phosphate-Buffered Formalin
  3. 3 Encapsulation in HistoGel
  4. 4 Dehydration and Paraffin Embedding
  5. 5 Immunohistochemical Staining
  6. 6 Histopathological Evaluation
In Vivo Tumorigenicity Assessment via Xenograft Day 21+ (parallel to organoid expansion)
  1. 1 Obtain NSG Mice and Ethics Approval
  2. 2 Release Organoids from BME2 and Prepare Injection Suspension
  3. 3 Subcutaneous Injection into Flank
  4. 4 House Mice Under Standard Conditions
  5. 5 Monitor Tumor Growth
  6. 6 Harvest Tumor at Target Size
  7. 7 Fix and Process Xenograft Tumor
In Vitro Drug Sensitivity Screening Day 21–28 (after organoid expansion)
  1. 1 Prepare Drug Stock Solutions
  2. 2 Dissociate Organoids and Seed in 384-Well Plates
  3. 3 Add Drug Compounds in 2-Fold Dilution Series
  4. 4 Incubate Plates for 6 Days
  5. 5 Measure Cell Viability
  6. 6 Normalize Results and Calculate Dose-Response Curves
  7. 7 Interpret Results
DNA Extraction and Whole-Exome Sequencing Day 21+ (parallel to organoid expansion)
  1. 1 Collect Tumor, Adjacent Non-Tumoral Liver, and Organoid Samples
  2. 2 Extract DNA Using Qiagen DNeasy Kit
  3. 3 Perform Whole-Exome Capture
  4. 4 Sequence on Illumina NovaSeq 6000
  5. 5 Align Reads to Reference Genome
  6. 6 Post-Alignment Processing
  7. 7 Detect Somatic Variants
  8. 8 Apply Variant Filtering Criteria
  9. 9 Detect Copy Number Alterations
  10. 10 Identify Loss of Heterozygosity (LOH)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Meier et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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