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LIVER Publication-derived

Patient-derived HCC organoid establishment and drug screening protocol

Source Wong et al., 2025 · The Chinese University of Hong Kong, Department of Surgery · 10.1016/j.jhepr.2025.101426

👤 Alissa Michelle Wong, Hao Huang, Aikha Melissa Wong, Howard Ho Wai Leung, Winnie Wing Yan Cheung, Yin Kau Lam, Xiaofan Ding, Luyao Zhang, Clarice Chu, Yue Liu, Cecilia Ka Wing Chan, Anthony Wing Hung Chan, Kelvin Kwok Chai Ng, Xin Wang, Stephen Lam Chan, Nathalie Wong ⏱ 24 days 📋 10 phases 🧫 Patient-Derived iPSC (Hepatocellular Carcinoma)

Abstract

This protocol describes the establishment of patient-derived tumor organoids (PDTOs) from hepatocellular carcinoma tissues using growth factor-reduced media. The PDTOs are characterized for genomic, transcriptomic, and histological features, and subjected to high-throughput drug screening (100-drug panel) to identify pharmacogenomic vulnerabilities and inform precision medicine approaches for HCC patients.

Cell source
Patient-Derived iPSC (Hepatocellular Carcinoma)
Application
Disease modeling, Drug screening, Pharmacogenomic analysis

Protocol overview

58 steps across 10 phases

Tissue collection and dissociation Day 0
  1. 1 Tissue collection and transport
  2. 2 Mechanical mincing
  3. 3 Enzymatic digestion
  4. 4 Cell cluster filtration
Organoid seeding and initial culture establishment Day 1–7
  1. 5 Matrigel preparation and embedding
  2. 6 Culture media addition
  3. 7 Initial incubation
  4. 8 Media change schedule
Organoid expansion and passage Week 1–8+
  1. 9 Media retention and ROCK inhibitor addition
  2. 10 TrypLE enzymatic dissociation
  3. 11 Organoid recovery and resuspension
  4. 12 Re-embedding in fresh Matrigel
  5. 13 Post-passage media addition
Cryopreservation and storage Week 2–8
  1. 14 Organoid harvest and cell recovery
  2. 15 Freezing medium preparation
  3. 16 Cryopreservation
  4. 17 Long-term storage
  5. 18 Thawing and recovery
Genomic and transcriptomic characterization Week 1–4
  1. 19 DNA/RNA extraction
  2. 20 Whole exome sequencing (WES)
  3. 21 RNA-Seq transcriptomic profiling
  4. 22 Bioinformatic analysis
Histological and immunological characterization Week 1–3
  1. 23 Organoid fixation and paraffin embedding
  2. 24 Microtome sectioning
  3. 25 H&E histological staining
  4. 26 Immunofluorescence (IF) staining
  5. 27 Immunofluorescence imaging and analysis
High-throughput drug screening and pharmacogenomic analysis Week 3–8
  1. 28 Organoid preparation for drug screening
  2. 29 Multi-well plate seeding
  3. 30 Drug library preparation and serial dilution
  4. 31 Drug addition and incubation
  5. 32 Cell viability assessment
  6. 33 Data normalization and IC50 calculation
  7. 34 Elastic net regression analysis
  8. 35 Tumor specificity assessment
Validation studies and functional experiments Week 4–12
  1. 36 Live/dead cell imaging
  2. 37 Gene knockdown via shRNA
  3. 38 Gene overexpression via lentiviral transduction
  4. 39 Quantitative PCR (qPCR) validation
  5. 40 Immunofluorescence quantification
  6. 41 Chromatin immunoprecipitation-qPCR (ChIP-qPCR)
  7. 42 Dual-luciferase promoter reporter assay
In vivo xenotransplantation studies Week 4–16
  1. 43 Animal preparation and housing
  2. 44 Organoid preparation for implantation
  3. 45 Subcutaneous xenotransplantation
  4. 46 Orthotopic xenotransplantation
  5. 47 Tumor growth monitoring
  6. 48 Drug treatment initiation
  7. 49 Tumor harvest and histological analysis
Clinical translation and proof-of-concept studies Week 8–24
  1. 50 Patient selection and informed consent
  2. 51 Tumor tissue biopsy acquisition
  3. 52 Whole-exome sequencing and variant annotation
  4. 53 Rapid PDTO establishment for drug screening
  5. 54 Candidate drug selection based on genomic and pharmacogenomic data
  6. 55 High-throughput drug screening on patient PDTOs
  7. 56 Clinician consultation and treatment recommendations
  8. 57 Off-label drug administration and clinical monitoring
  9. 58 Clinical outcome assessment and retrospective analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wong et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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