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LIVER Publication-derived

Hepatocellular Carcinoma Liver Organoids for Biobanking

Full SOP with Academic or a plan

Source Nuciforo et al., 2018 · University Hospital Basel, University of Basel · 10.1016/j.celrep.2018.07.001

Authors: Sandro Nuciforo, Isabel Fofana, Matthias S. Matter, Tanja Blumer, Diego Calabrese, Tujana Boldanova, Salvatore Piscuoglio, Stefan Wieland, Femke Ringnalda, Gerald Schwank, Luigi M. Terracciano, Charlotte K.Y. Ng, Markus H. Heim Duration: 28 days 8 phases Cell source: Patient-Derived Hepatocellular Carcinoma (HCC) and Intrahepatic Cholangiocarcinoma (CCC) cells from needle biopsies

Abstract

This protocol describes the generation of long-term organoid cultures from tumor needle biopsies of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCC) patients. The derived organoids retain morphology, genetic heterogeneity, and tumor marker expression of the originating tumors, enabling phenotypic and genotypic characterization and drug sensitivity testing such as sorafenib response.

Cell source
Patient-Derived Hepatocellular Carcinoma (HCC) and Intrahepatic Cholangiocarcinoma (CCC) cells from needle biopsies
Application
Disease modeling, Drug screening, Patient-derived tumor organoid biobank development

Protocol overview

30 steps across 8 phases

Phase 1 · Biopsy Collection and Tissue Preparation Day 0
  1. a Perform ultrasound-guided coaxial needle biopsy
  2. b Immediately process biopsy samples
  3. c Transport tumor biopsy fragments to laboratory
Phase 2 · Tissue Digestion and Organoid Initiation Day 0
  1. a Place biopsy pieces in advanced DMEM/F-12 medium
  2. b Perform limited enzymatic digestion
  3. c Seed cell clusters into reduced growth factor BME
Phase 3 · Organoid Culture Initiation and Expansion (Weeks 1-4) Days 1-28
  1. a Add expansion medium to polymerized BME
  2. b Culture organoids in standard incubation conditions
  3. c Perform initial medium changes
Phase 4 · Organoid Passaging and Long-Term Culture (Weeks 4 onwards) Day 28 onwards
  1. a Passage tumor organoids via enzymatic dissociation
  2. b Collect and re-seed dissociated organoids
  3. c Continue regular passaging and maintenance
  4. d Test for Mycoplasma contamination
Phase 5 · Organoid Cryopreservation Any time from passage 1 onwards
  1. a Prepare organoids for freezing
  2. b Resuspend in cryopreservation medium
  3. c Freeze organoids
Phase 6 · Organoid Characterization via Histology Any culture period
  1. a Release organoids from BME matrix
  2. b Fix organoids for paraffin embedding
  3. c Perform H&E and immunohistochemical staining
  4. d Histopathological evaluation
Phase 7 · Sorafenib Drug Sensitivity Testing Any culture period (typical: weeks 4-12)
  1. a Prepare sorafenib solutions
  2. b Plate organoids for drug treatment
  3. c Add sorafenib to organoid cultures
  4. d Measure cell viability using CellTiter-Glo 3D assay
  5. e Calculate IC50 values
Phase 8 · Organoid Xenotransplantation Weeks 4-12 (timing flexible; not critical path)
  1. a Prepare organoid cell suspension for transplantation
  2. b Prepare injection mixture
  3. c Inject organoids subcutaneously into immunodeficient mice
  4. d Monitor xenograft tumor growth
  5. e Harvest xenograft tumors for histological analysis

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Attribution

This SOP was written by Organthis based on the published method in Nuciforo et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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