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

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.

胰腺导管样CK19+细胞的增殖与胰腺癌、糖尿病等多种疾病状态存在密切关联。这类重要细胞的体外研究长期受限于其增殖能力不足：相较之下，成纤维细胞样波形蛋白阳性（vimentin+）细胞会过度增殖并覆盖原代培养体系。本研究旨在构建一套导管细胞有丝分裂原筛选平台，首先完成vimentin+细胞群的清除。
我们采用磁激活细胞分选（magnetic-activated cell sorting）技术，通过细胞表面标记物CD90，从去除胰岛的人胰腺细胞培养物中分离清除vimentin+细胞。经分选后，培养物中的CD90+细胞污染率从34±20%降至1.3±0.6%，最终获得具有上皮细胞形态的纯化CK19+细胞培养物。
随后采用全因子实验设计，测试碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）、肝细胞生长因子（HGF）、角质形成细胞生长因子（KGF）以及血管内皮生长因子（VEGF）的促有丝分裂活性。在测试条件下培养6天后，使用溴脱氧尿苷（BrdU）对细胞进行标记，随后染色并通过高通量成像完成分析。
本次筛选实验验证了bFGF、EGF、HGF及KGF对CK19+细胞的预期促有丝分裂效果，同时还揭示了上述因子与VEGF之间的相互作用。添加bFGF、EGF、HGF与KGF的无血清培养基，可实现与添加10%血清相当的CK19+细胞增殖水平。
本研究开发的方法可提供高效的细胞培养与高通量筛选平台，用于纯化的原代胰腺CK19+细胞的相关研究，从而推动胰腺癌与糖尿病领域的研究进展。