Primary cultures for pancreatic stellate cells (PSCs)

Abstract

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. This purpose of this chapter is to report our novel approach to isolating PSCs from normal rat pancreas and human pancreatic ductal adenocarcinoma (PDAC) tissue. Normal PSCs were isolated with enzyme digestion and ladder centrifugation with Nycodenz solution. Isolated PSCs were cultured in DMEM/F12 containing 10% fetal bovine serum. Cancer-associated PSCs were obtained by an outgrow method from fresh human PDAC tissues. Isolated activated PSCs were cultured in DMEM/F12 containing 20% fetal bovine serum. With our modification, normal pancreas tissue yields an adequate number of PSCs (approximately 0.5–5 million/g pancreas) for in vitro studies, and the cell viability was about 90%. And a modified outgrowth method made tissue blocks attached more tightly and 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.