Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas

Primary cultures of pancreatic stellate cells (PSCs) remain an important basis for in vitro study. However, effective methods for isolating abundant PSCs are currently lacking. We report on a novel approach to isolating PSCs from normal rat pancreases and human pancreatic ductal adenocarcinoma (PDAC) tissue. After anaesthesia and laparotomy of the rat, a blunt cannula was inserted into the pancreatic duct through the anti-mesentery side of the duodenum, and the pancreas was slowly infused with an enzyme solution until all lobules were fully dispersed. The pancreas was then pre-incubated, finely minced and incubated to procure a cell suspension. PSCs were obtained after the cell suspension was filtered, washed and subject to gradient centrifugation with Nycodenz solution. Fresh human PDAC tissue was finely minced into 1×1×1 mm3 cubes with sharp blades. Tissue blocks were placed at the bottom of a culture plate with fresh plasma (EDTA-anti-coagulated plasma from the same patient, mixed with CaCl2) sprinkled around the sample. After culture for 5-10 days under appropriate conditions, activated PSCs were harvested. An intraductal perfusion of an enzyme solution simplified the procedure of isolation of rat PSCs, as compared with the multiple injections technique, and a modified outgrowth method significantly shortened the outgrowth time of the activated cells. Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period, thus facilitating future PSC-related research.