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PANCREAS Publication-derived

Human Pancreatic Tumor Organoid Establishment and Characterization

Source Seino et al., 2018 · Keio University School of Medicine, 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 ⏱ 365 days 📋 12 phases 🧫 Patient-Derived Pancreatic Tumor Tissue and Patient-Derived iPSC

Abstract

This protocol establishes patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids from surgical, fine-needle aspiration, and ascites specimens, and characterizes their functional Wnt niche dependencies to model tumor heterogeneity and disease progression. The resulting organoid library enables investigation of tumor-stroma interactions and therapeutic targeting strategies.

Cell source
Patient-Derived Pancreatic Tumor Tissue and Patient-Derived iPSC
Application
Disease modeling, Drug screening

Protocol overview

62 steps across 12 phases

Tissue Sample Preparation and Enzymatic Digestion Day 0–1
  1. 1 Tissue Collection and Initial Processing
  2. 2 Enzymatic Digestion with Liberase
  3. 3 Secondary Enzymatic Digestion with TrypLE
  4. 4 Enzyme Inactivation and Cell Recovery
  5. 5 Red Blood Cell Lysis (for ascites samples only)
Organoid Embedding and Initial Culture Day 1–7
  1. 1 Matrigel Embedding
  2. 2 Basal Culture Medium Overlay
  3. 3 Initial Niche Factor Supplementation
  4. 4 Culture Incubation
  5. 5 Organoid Morphology Assessment
PDAC Organoid Enrichment via Niche Selection Day 7–30
  1. 1 EGF Removal for KRAS Mutant Selection
  2. 2 TP53 Mutant Selection (Alternative to EGF Removal)
  3. 3 SMAD4 Mutant Selection via Noggin Removal/BMP4 Addition
  4. 4 Assessment of PDAC Origin
  5. 5 Extended Culture and Cryopreservation
Functional Wnt Niche Dependency Characterization Day 30–60
  1. 1 Successive Niche Factor Removal
  2. 2 Wnt3A Removal Test
  3. 3 R-spondin Removal Test
  4. 4 Porcupine Inhibitor (Porcn-i) Treatment Validation
  5. 5 Wnt Pathway Inhibitor (ICG001) Treatment
Cancer-Associated Fibroblast (CAF) Co-Culture Day 60–90
  1. 1 CAF Isolation and Culture
  2. 2 Single-Cell Dissociation of PDAC Organoids and CAFs
  3. 3 Cell Aggregation in Ultra-Low Attachment Plates
  4. 4 Transfer to Collagen/Matrigel Mixture
  5. 5 Growth Assessment in Wnt-Free Conditions
  6. 6 Porcn-i Treatment of Co-Cultures
CRISPR-Cas9-Mediated Genetic Engineering Day 90–150
  1. 1 sgRNA Design and Cloning
  2. 2 Electroporation of px330 Plasmids and GFP-Puro PiggyBac Vector
  3. 3 Antibiotic Selection and Enrichment
  4. 4 Generation of Multi-Mutant Organoid Lines (KCT, KCTS)
  5. 5 Genomic Confirmation of Mutations
Molecular Characterization: Whole-Exome Sequencing and Mutation Analysis Day 150–180
  1. 1 Genomic DNA Extraction
  2. 2 Library Preparation for Whole-Exome Sequencing
  3. 3 Read Mapping and Variant Detection
  4. 4 Variant Filtering and Annotation
  5. 5 Hotspot Mutation Confirmation (Optional)
Molecular Characterization: Copy Number Analysis and Methylation Day 180–210
  1. 1 Copy Number Alteration (CNA) Analysis
  2. 2 DNA Methylation Analysis
  3. 3 Identification of Differentially Methylated Regions
Molecular Characterization: Gene Expression and qPCR Validation Day 210–240
  1. 1 RNA Extraction
  2. 2 Microarray Gene Expression Profiling
  3. 3 Real-Time Quantitative PCR (qPCR)
  4. 4 Validation of Wnt Target Gene Expression
  5. 5 Validation of Epithelial Wnt Ligand Expression
Molecular Characterization: GATA6 Genetic Perturbation Day 240–300
  1. 1 GATA6 Knockdown via shRNA
  2. 2 GATA6 Knockout via CRISPR-Cas9
  3. 3 Validation of GATA6 Loss of Function
  4. 4 Assessment of Wnt Self-Activation in GATA6-Depleted Organoids
  5. 5 GATA6 Overexpression in W[+] Organoids
  6. 6 Assessment of Wnt Dependency Induction by GATA6 Overexpression
Xenotransplantation and In Vivo Tumor Formation Day 300–365 (up to 3 months post-transplant)
  1. 1 Organoid Preparation and GFP Labeling
  2. 2 Subcutaneous Xenotransplantation with or without CAFs
  3. 3 Orthotopic Xenotransplantation (Optional)
  4. 4 Tumor Monitoring and Measurement
  5. 5 Porcn-i Treatment (Optional)
  6. 6 Tissue Harvest and Histological Analysis
Adaptive Wnt-Free Culture and Phenotypic Transition Day 300–365
  1. 1 Passage 0 (P0): Establishment of Control Cultures
  2. 2 Passage 1 (P1): Initiation of Wnt-Free Culture
  3. 3 Passage 2–4 (P2–P4): Continued Adaptation to Wnt-Free Conditions
  4. 4 Assessment of Wnt Niche Adaptation
  5. 5 Molecular Analysis of Adapted Organoids
  6. 6 Gene Set Enrichment Analysis (GSEA)

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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