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LIVER Publication-derived

Liver Organoids for Drug Screening (2022)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 28 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Meier et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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