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Biliary Tract Carcinoma Tumouroids

Full SOP with Academic or a plan

Source Saito et al., 2019 · Keio University Faculty of Pharmacy, Division of Pharmacotherapeutics · 10.1016/j.celrep.2019.03.088

Authors: Yoshimasa Saito, Toshihide Muramatsu, Yae Kanai, Hidenori Ojima, Aoi Sukeda, Nobuyoshi Hiraoka, Eri Arai, Yuko Sugiyama, Juntaro Matsuzaki, Ryoei Uchida, Nao Yoshikawa, Ryo Furukawa, Hidetsugu Saito Duration: 365 days 6 phases Cell source: Patient-Derived Surgical Tissue (Intrahepatic Cholangiocarcinoma, Gallbladder Cancer, Neuroendocrine Carcinoma)

Abstract

This protocol describes the establishment and long-term in vitro culture of organoids derived from biliary tract carcinoma (BTC) tissues, including intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of Vater. The organoids recapitulate histopathology, gene expression, and genetic alterations of primary tumors and enable identification of prognostic biomarkers and therapeutic agents through drug screening.

Cell source
Patient-Derived Surgical Tissue (Intrahepatic Cholangiocarcinoma, Gallbladder Cancer, Neuroendocrine Carcinoma)
Application
Disease modeling and drug screening

Protocol overview

22 steps across 6 phases

Phase 1 · Tissue Preparation and Cell Isolation Day 0
  1. a Tissue sectioning
  2. b Enzymatic digestion
  3. c Cell collection and washing
Phase 2 · Organoid Plating and Initial Culture Day 0-1
  1. a Matrigel embedding
  2. b Allow Matrigel to solidify
  3. c Add culture medium
Phase 3 · Organoid Expansion and Maintenance Day 1-365
  1. a Monitor organoid formation
  2. b Medium change
  3. c Organoid passaging
  4. d Long-term culture maintenance
Phase 4 · Drug Screening and Sensitivity Testing Drug treatment: 6 days per compound
  1. a Organoid plating for drug screening
  2. b Compound addition
  3. c Cell viability assessment
  4. d Data analysis and interpretation
Phase 5 · Molecular Characterization and Analysis Parallel to culture phases
  1. a RNA extraction for gene expression profiling
  2. b Microarray analysis
  3. c DNA extraction for whole-exome sequencing
  4. d Whole-exome sequencing
Phase 6 · Histopathological Validation Parallel to culture phases
  1. a Tissue fixation and embedding
  2. b H&E and PAS staining
  3. c Immunohistochemistry
  4. d Microscopic examination and documentation

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Attribution

This SOP was written by Organthis based on the published method in Saito et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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