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PANCREAS Publication-derived

Pancreatic Ductal Adenocarcinoma Organoids for Transplantation

Full SOP with Academic or a plan

Source Boj et al., 2015 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht; Cold Spring Harbor Laboratory, USA · 10.1016/j.cell.2014.12.021

Authors: Sylvia F. Boj, Chang-Il Hwang, Lindsey A. Baker, Hans Clevers, David A. Tuveson Duration: 20 days 8 phases Cell source: Human pancreatic ductal cells from resected tissue and endoscopic biopsies; Mouse pancreatic ductal cells from wild-type and transgenic strains (KC, KPC mice)

Abstract

This protocol describes the establishment and propagation of three-dimensional pancreatic ductal organoids from normal and neoplastic murine and human pancreatic tissue. The organoids recapitulate pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDA) progression in vitro and in vivo, enabling molecular profiling and identification of disease-stage-specific biomarkers and therapeutic targets.

Cell source
Human pancreatic ductal cells from resected tissue and endoscopic biopsies; Mouse pancreatic ductal cells from wild-type and transgenic strains (KC, KPC mice)
Application
Disease modeling; Pancreatic cancer progression modeling and therapeutic target discovery

Protocol overview

31 steps across 8 phases

Phase 1 · Tissue Harvest and Preparation Day 0
  1. a Collect pancreatic tissue
  2. b Tissue mincing (for tumors and metastases)
Phase 2 · Enzymatic Digestion Day 0-1
  1. a Digest mouse normal and preneoplastic ductal tissue
  2. b Digest murine tumor and metastatic tissue
  3. c Digest human normal pancreatic tissue
  4. d Digest human tumor tissue
Phase 3 · Organoid Establishment in Matrigel Day 0-1
  1. a Prepare Matrigel suspension for murine ducts
  2. b Prepare Matrigel suspension for human tissue
  3. c Plate in culture wells
Phase 4 · Culture Medium Preparation and Application Day 0
  1. a Prepare murine organoid culture medium
  2. b Prepare human normal (hN) organoid culture medium
  3. c Prepare human tumor (hT) organoid culture medium
  4. d Add culture medium to wells
Phase 5 · Organoid Outgrowth and Passaging Day 1-20+
  1. a Observe organoid outgrowth
  2. b Change medium every 3-4 days
  3. c Passage murine organoids
  4. d Passage human normal organoids
  5. e Passage human tumor organoids
Phase 6 · Cryopreservation Any passage
  1. a Prepare organoids for cryopreservation
  2. b Freeze organoids gradually
  3. c Thaw organoids for use
Phase 7 · Genetic Manipulation (Optional - Adenoviral Cre Infection for Kras Activation) Passage 3 onward
  1. a Prepare organoids for adenoviral infection
  2. b Infect organoids via spinoculation
  3. c Culture infected organoids
  4. d Assess Cre-mediated recombination
Phase 8 · Orthotopic Transplantation (Optional) Passage 1-3+
  1. a Prepare organoids for transplantation
  2. b Prepare recipient mice for surgery
  3. c Perform orthotopic injection into pancreas
  4. d Close surgical incision
  5. e Post-operative care
  6. f Monitor for tumor development

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Attribution

This SOP was written by Organthis based on the published method in Boj et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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