A SIMPLIFIED METHOD FOR SUSTAINED PURE CULTURES OF HUMAN PERITONEAL MACROPHAGES (HPM)

Abstract

Peritoneal fluid is a readily available source of human macrophages and monocyte/macrophage colony stimulating factor (M-CSF). We describe a simple means of developing pure macrophage cultures, utilizing dialysate from patients undergoing peritoneal dialysis as a source of both macrophages and M-CSF. Sterile saline was infused and allowed to dwell intraperitoneally for 45 minutes. The fluid was drained and centrifuged at 400G for 20 minutes; the cellular fraction was resuspended in McCoy's modified medium (MMM) with 10% FCS and incubated at 37°C in 5% CO2 for 2 hours. Adherent cells were rinsed with Hanks buffer and overlayed with MMM containing 5% of the supernatant dialysate fluid. Half of the culture medium was replaced twice a week. Cells reached near confluence by 14 days in culture and were characterized histologically and immunologically. The cells stained uniformly for nonspecific esterase and were >98% positive for the monoclonal macrophage markers DR and LeuM5. Active phagocytosis was demonstrated with opsonized fluorescent latex beads. A hemolytic complement assay revealed significant C2 production by cells cultured over 7 days. Arachidonate (AA) metabolism was evaluated following incubation of macrophages with 14C-AA (20μM) and separation by TLC and HPLC. Adherent cells produced the AA metabolites PGD2, TXB2, PGE2, PGF2 α and 6KPGF1 α in the approximate ratio of 4:4:4:4:1. Cells also produced HHT and 15-HETE in small amounts. Macrophage cultures remained viable for >60 days. This method describes a readily available source of cells and M-CSF for developing pure, differentiated, and functional long term cultures of HPM in serum-free medium.