Purified Human Pancreatic Duct Cell Culture Conditions Defined by Serum-Free High-Content Growth Factor Screening

Corinne A Hoesli,James D Johnson,James M Piret,Kathrin Maedler

doi:10.1371/journal.pone.0033999

Corinne A Hoesli, James D Johnson + Show 2 more

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https://doi.org/10.1371/journal.pone.0033999

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Abstract

The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.

Highlights

The pancreas is a complex organ containing multiple interspersed cell types with diverse exocrine and endocrine functions
Most pancreatic duct cell culture protocols require the use of serumcontaining medium, which is undesirable for research or therapeutic cell culture because serum is undefined, suffers from high batch-to-batch variation and may contain dangerous contaminants that are problematic for transplantation applications [21,22]
We present a novel method to deplete fibroblast-like cells from pancreatic tissue using CD90-based selection, as well as a serumfree high content screening platform to identify pancreatic duct mitogens

Summary

Introduction

The pancreas is a complex organ containing multiple interspersed cell types with diverse exocrine and endocrine functions. Established protocols exist for isolating, studying and transplanting relatively pure cultures of endocrine islet cells [1,2,3], but the culture of enriched CK19-positive ductal cells has proven challenging. Human CK19-positive cells are an interesting cell population [4] as they have been shown to proliferate in several disease states, including pancreatic ductal adenocarcinoma [5] and diabetes [6]. Most pancreatic duct cell culture protocols require the use of serumcontaining medium, which is undesirable for research or therapeutic cell culture because serum is undefined, suffers from high batch-to-batch variation and may contain dangerous contaminants that are problematic for transplantation applications [21,22]. Improved methods to separate exocrine cell populations and to culture them in defined optimized media are needed to enable studies of duct cell biology

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