Isolation and Culture of Arterial Smooth Muscle Cells From Human Placenta

Abstract

A simple and economical technique was developed to isolate and culture human arterial smooth muscle cells from chorionic plate vessels. Placentas from healthy women were collected at the time of term delivery. Chorionic plate arteries were identified, excised, and cut into small pieces. An explant technique was used to grow cultures of placental arterial smooth muscle (PASM) cells. Small pieces of vessel with lumens down were placed in 100-mm culture plates and grown in Dulbecco modified eagle medium and 10% fetal bovine serum. Cells appeared from explants within 1 week and grew to confluence in approximately 4 weeks. At confluence, PASM cell cultures had a uniform cell morphology that was characterized by elongated cells in parallel rows, typical of smooth muscle cells. Smooth muscle cell phenotype was evaluated by morphology and by immunoblotting and immunofluorescence of smooth muscle myofilament proteins. All PASM cell cultures expressed α-smooth muscle actin, β-tropomyosin, and h-caldesmon. Expression was similar to that of human aortic smooth muscle cells, but not to endothelial cells or fibroblasts. PASM cells stained uniformly for α-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-α, and interleukin-1β by measurement of interleukin-8 production. Cells cultured for 18 hours showed a progressive increase in interleukin-8 production with time. Treatment with inflammatory mediators increased interleukin-8 production by 3-fold as compared with media control. This technique provides a simple method to obtain normal human arterial smooth muscle cells for in vitro studies of physiology and pathophysiology.