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LIVER Publication-derived

Hepatoblastoma Liver Tumouroids with Single-Cell Profiling

Full SOP with Academic or a plan

Source Kluiver et al., 2023 · Princess Máxima Center for Pediatric Oncology · 10.1101/2023.08.28.554783

Authors: Thomas A. Kluiver, Yuyan Lu, Stephanie A. Schubert, Lianne J. Kraaier, Femke Ringnalda, Philip Lijnzaad, Jeff DeMartino, Wout L. Megchelenbrink, Vicky Amo-Addae, Selma Eising, Flavia W. de Faria, Daniel J. Münter, Marc van de Wetering, Kornelius Kerl, Evelien Duiker, Marius van den Heuvel, Vincent E. de Meijer, Ruben H. de Kleine, Ronald R. de Krijger, Jan J. Molenaar, Thanasis Margaritis, Henk Stunnenberg, József Zsiros, Hans Clevers, Weng Chuan Peng 7 phases Cell source: Patient-Derived Tumor Tissue (Hepatoblastoma)

Abstract

This protocol describes the establishment and comprehensive characterization of patient-derived hepatoblastoma organoids from primary tumor material. The organoids are cultured in 3D basement membrane extract and subjected to single-cell RNA-seq, spatial transcriptomics, single-cell ATAC-seq, and high-throughput drug screening to identify tumor subpopulations and therapeutic targets.

Cell source
Patient-Derived Tumor Tissue (Hepatoblastoma)
Application
Disease modeling and drug screening

Protocol overview

45 steps across 7 phases

Phase 1 · Tissue Dissociation and Organoid Establishment Day 0-3
  1. a Tissue mincing
  2. b Liver perfusion medium incubation
  3. c Tissue washing and centrifugation
  4. d Enzymatic digestion
  5. e Mechanical dissociation
  6. f Red blood cell lysis (if necessary)
  7. g Enzyme wash-off
  8. h BME preparation and plating
Phase 2 · Organoid Culture and Expansion Day 3-ongoing
  1. a Initial culture medium preparation
  2. b Alternative reduced medium for difficult-to-expand lines
  3. c Organoid culture and expansion
  4. d Organoid passaging
Phase 3 · Single-Cell RNA Sequencing Day prior to sequencing
  1. a Tissue preparation for scRNA-seq
  2. b Cell cleanup
  3. c Organoid preparation for scRNA-seq
  4. d Single-cell capture using 10x Chromium
  5. e cDNA library preparation and sequencing
Phase 4 · Spatial Transcriptomics Day of tissue processing
  1. a Tissue freezing
  2. b Cryosectioning
  3. c Tissue fixation and H&E staining
  4. d Tissue permeabilization optimization
  5. e cDNA library preparation and sequencing
Phase 5 · Immunofluorescence Staining for Biomarker Validation Day of staining
  1. a Tissue/organoid fixation
  2. b Tissue processing and embedding
  3. c Section preparation
  4. d Deparaffinization and rehydration
  5. e Antigen retrieval
  6. f Permeabilization and blocking
  7. g Primary antibody incubation
  8. h Secondary antibody incubation
  9. i Counterstaining and mounting
  10. j Imaging
Phase 6 · High-Throughput Drug Screening Days of drug testing
  1. a Organoid recovery and cell viability baseline
  2. b Drug addition
  3. c Incubation period
  4. d Cell viability measurement
  5. e Data analysis and IC50 calculation
Phase 7 · Molecular Characterization: CTNNB1 Sequencing and qRT-PCR Day of molecular analysis
  1. a Genomic DNA extraction
  2. b CTNNB1 exon 3 PCR amplification
  3. c Sanger sequencing
  4. d RNA extraction
  5. e Reverse transcription
  6. f qRT-PCR setup
  7. g Real-time PCR amplification
  8. h Data analysis and relative expression calculation

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Attribution

This SOP was written by Organthis based on the published method in Kluiver et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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