A Reproducible 2d Monolayer Culture System for Murine Primary Pancreatic Ductal Cells

Document Type

Conference Proceeding

Publication Date

2-20-2026

Publication Title

Ann Surg Oncol

Keywords

amylase, collagen, collagen type 1, fibroblast growth factor 10, lectin, noggin, tamoxifen, acinar cell, adult, animal cell, animal experiment, animal tissue, carcinogenesis, colony formation, conditioned medium, conference abstract, controlled study, female, human, human cell, monolayer culture, mouse, neoplastic cell transformation, nonhuman, organoid, pancreas cancer, pancreatic duct, treatment response

Abstract

Patient-derived organoids (PDOs) are now widely used to model pancreatic cancer but lack matched "normal" epithelial controls, a critical limitation in studying tumorigenesis, adaptation and therapeutic response. Existing "normal" pancreatic cell lines are immortalized or partially transformed, confounding interpretation. Existing methods in mice are inconsistent and biased towards large or small ducts; we therefore aimed to optimize a method for culturing primary murine ductal cells that could then lay the groundwork for future human-derived primary cell lines. METHODS: Duct fragments were isolated from genetically normal murine pancreata using a combination of mechanical and enzymatic digestion, and plated on stiff (2.31 mg/mL) collagen matrices. We varied acinar cell contamination, matrix stiffness, and media composition. Colony formation was quantified, and lineage tracing was performed by injecting Sox9CreER;KrasG12D;Ptenflox/flox;R26RYFP mice with tamoxifen at eight weeks of age. RESULTS: Colonies arise from duct fragments and not single cells. The presence of acinar cells reduces colony formation by approximately 3-4 fold, and colony formation is almost completely suppressed when ducts are plated in acinar cell conditioned media. Changing matrix density (within a range of 1.00-2.31mg type I collagen/mL) ormedia supplementation with FGF-10, RSPOl and Noggin (factors important for long-term pancreatic duct organoid growth) did not significantly affect initial colony formation. Colonies formed from lineage traced ductal cells were DBA-lectin+, amylase- and YFP+, indicating ductal origin and identity. CONCLUSIONS: We describe a robust, reproducible method for culturing primary murine pancreatic ductal cells as 2D monolayers. While optimized in mice, this approach provides a framework for future development of patient-derived "normal" ductal cultures, which could be done in parallel with patient-derived organoids and provide a valuable matched-control system for mechanistic and therapeutic studies.

Volume

33

Issue

1

First Page

S145

Last Page

S146

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