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PANCREAS Publication-derived

Human Pancreatic Tumor Organoid Establishment and Functional Characterization

Source Seino et al., 2018 · Keio University School of Medicine, Department of Gastroenterology, Tokyo · 10.1016/j.stem.2017.12.009

👤 Takashi Seino, Shintaro Kawasaki, Mariko Shimokawa, Hiroki Tamagawa, Kohta Toshimitsu, Masayuki Fujii, Yuki Ohta, Mami Matano, Kosaku Nanki, Kenta Kawasaki, Sirirat Takahashi, Shinya Sugimoto, Eisuke Iwasaki, Junichi Takagi, Takao Itoi, Minoru Kitago, Yuko Kitagawa, Takanori Kanai, Toshiro Sato ⏱ 24 days 📋 8 phases 🧫 Patient-Derived PDAC

Abstract

This protocol describes the establishment and culture of patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids from surgical specimens, fine-needle aspirates, or ascites samples, followed by functional characterization of Wnt/R-spondin niche dependencies. The method enables generation of a comprehensive tumor organoid library for investigating niche factor requirements and transcriptional programs underlying PDAC heterogeneity.

Cell source
Patient-Derived PDAC
Application
Disease modeling; Drug screening

Protocol overview

34 steps across 8 phases

Sample Processing and Tissue Dissociation Day 1
  1. 1 Tissue Washing and Mincing
  2. 2 Enzymatic Digestion with Liberase TH
  3. 3 Secondary Digestion with TrypLE Express
  4. 4 Enzyme Inactivation
Organoid Culture Initiation Day 1–14
  1. 5 Matrigel Embedding
  2. 6 Basal Culture Medium Preparation
  3. 7 Initial Culture Establishment
Enrichment of PDAC Organoids Week 2–4
  1. 8 EGF Removal Selection
  2. 9 Sequential Niche Factor Removal Selection
Functional Characterization of Niche Dependencies Week 4–12
  1. 10 Systematic Niche Factor Withdrawal
  2. 11 Classification into Wnt Niche Subtypes
  3. 12 Porcupine Inhibitor Treatment to Validate Wnt Production
  4. 13 ICG001 Treatment to Assess Wnt-Signal Dependency
Molecular Analysis and Quality Control Week 4–24
  1. 14 Whole-Exome Sequencing
  2. 15 Copy-Number Analysis by CGH/SNP Microarray
  3. 16 Gene-Expression Microarray Analysis
  4. 17 DNA Methylation Analysis
  5. 18 Real-Time Quantitative PCR Validation
CAF Co-culture and In Vivo Validation Week 4–16
  1. 19 Cancer-Associated Fibroblast (CAF) Establishment
  2. 20 PDAC Organoid–CAF Aggregation
  3. 21 Transfer to Collagen–Matrigel Embedding
  4. 22 Assessment of CAF-Mediated Wnt Support
  5. 23 Xenotransplantation of Organoids with or without CAFs
  6. 24 In Vivo Porcupine Inhibitor Treatment
GATA6 Functional Analysis Week 8–20
  1. 25 GATA6 Knockdown and Knockout Generation
  2. 26 Assessment of Wnt Niche Adaptation upon GATA6 Loss
  3. 27 GATA6 Overexpression in W+ Organoids
  4. 28 Clinical Validation: GATA6 Expression Correlation with Epithelial Wnt
CRISPR-Cas9 Engineering of Pancreatic Organoids Week 8–24
  1. 29 Generation of Engineered Organoids with Driver Mutations
  2. 30 Transcriptome Validation of Engineered Organoids
  3. 31 Subcutaneous Xenotransplantation of Engineered Organoids
  4. 32 Assessment of Wnt-Niche Independency Acquisition during Tumorigenesis
  5. 33 Gene-Expression and Epigenetic Analysis of Adapted Organoids
  6. 34 Validation of Porcupine-Inhibitor Sensitivity in Adapted Organoids

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Seino et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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