Abstract: Objective To investigate the methods of isolation, purification and identification of human intrahepatic cholangiocarcinoma-associated fibroblasts (iCAFs), and to explore its impact on cholangiocarcinoma cells.Methods The primary iCAFs were isolated using digestive methods and purified through density gradient centrifugation. Immunocytochemistry, immunofluorescence staining and flow cytometry were performed to confirm iCAFs. Cell counting kit-8 (CCK8) and transwell experiments were performed to assess the effect of iCAFs on cholangiocarcinoma cells.Results The typical morphology of obtained iCAFs were shaped spindle-like, which formed the pattern of net or whirlpool. Typical iCAFs showed positive staining for α-smooth muscle actin and fibroblast-specific protein 1 (FSP-1), with 98.2% positive rate of FSP-1. iCAFs conditioned media significantly promoted the proliferation and migration of the cholangiocarcinoma cells.Conclusion Human iCAFs were successfully isolated and identified, which could promote proliferation and migration of the cholangiocarcinoma cells. It provides the basis for further research of iCAFs in the development and progression of intrahepatic cholangiocarcinoma.

Key words: Intrahepatic cholangiocarcinoma, Cancer-associated fibroblasts, Isolation, Purification, Identification, Proliferation, Migration