An improved method for the isolation and culture of rat pancreatic ductal epithelial cells.

BACKGROUND

This aim of this study was to explore a novel method that can be used to isolate and culture rat pancreatic ductal epithelial cells.

METHODS

Retrograde injection of indigo carmine solution into the bile duct of rats revealed the main pancreatic duct, which was isolated using the naked eye (without using a stereomicroscope). The main pancreatic duct was sequentially digested with three enzymes, and the digested cells were cultured in RPMI-1640 medium containing 10-15% fetal bovine serum. After 72 h, the primary pancreatic ductal epithelial cells started to adhere to the wall. The cells reached 70-80% confluence after approximately 7 days and were subsequently digested with 0.25% trypsin and subcultured. Cells of the second and fourth passages were harvested. Cytokeratin (CK)-7 and CK-19 protein expressions in the cells and pancreatic tissue were detected by Western blot analysis. q-PCR was employed to examine CK-7, CK-19, somatostatin, amylase, insulin, and glucagon mRNA expression in the cells and pancreatic tissue after the main pancreatic duct was removed.

RESULTS

The rats had one or two main pancreatic ducts meeting the bile ducts at a right or an acute angle. Rat pancreatic ductal epithelial cells isolated by this method grew well and showed a cobblestone-like appearance via microscopy. Western blot analysis showed that both the second and fourth passages of pancreatic ductal epithelial cells expressed CK-7 and CK-19 protein. The q-PCR results showed the expression of CK-7 and CK-19 genes in the second and fourth passages of pancreatic ductal epithelial cells, while the somatostatin, amylase, insulin, and glucagon genes were not expressed. The pancreatic tissue harvested after the removal of the main pancreatic duct did not express CK-7 or CK-19, while the somatostatin, amylase, insulin, and glucagon genes were expressed.

CONCLUSIONS

The preliminary results show that this method can be applied to successfully isolate and culture rat pancreatic ductal epithelial cells.