Establishment of a pure vascular endothelial cell line from human placenta

Endothelial cells (EC) from various sectors of the circulatory system have distinct characteristics, some of which have only been identified in cultur es upon their isolation from specific organs or tissues. Cultured vascular EC, derived from the human placenta (HPEC), may be helpful for studying the ir specific function in the fetoplacental unit, such as in the control of m aternofetal traffic. In this paper we report an improved method for isolati on, purification and culture of HPEC, that implies an enzymatic perfusion o f the term placenta, followed by separation of resulting cells on a Percoll density gradient. The inoculated starting suspension was purified by a two -step selection procedure, based on differential trypsinization, leading to a pure population of about 8 x 10(7) cells/placenta, with 2.7-3.4 populati on doublings. The average population doubling time during eight passages wa s 60-65 h and the life span of HPEC was approximately 45-50 population doub lings. The cell morphology at optical and electron microscopical level reve aled a good differentiation of HPEC, which were endowed with numerous plasm alemmal vesicles (caveolae) and Weibel-Palade bodies. The transendothelial electrical resistance of the HPEC monolayer varied between 22 and 52 Ohm/cm (2) The cultures were mycoplasma free, as revealed by fluorescence microsco py using DNA dyes and the polymerase chain reaction (PCR). The negative imm unofluorescent reaction for keratin confirmed that the HPEC were not contam inated with either type of placenta cells, as syncytiotrophoblast. Cultured HPEC demonstrated a strong reaction for von Willebrand factor antigen (by fluorescence microscopy), took up AcLDL-DiI and expressed active angiotensi n converting enzyme. These characteristics substantiate the endothelial nat ure of cultured cells. The interactions with different lectins (BS-I, SEA, RCA, UEA and WGA) assessed by fluorescence microscopy and blotting reveal a strong reaction of HPEC with UEA and a negligible reaction with BS-I lecti n. WGA lectin displayed a marked fluorescence staining in subconfluent HPEC , and at the level of intracellular clefts in post-confluent cultures. In c onclusion: (i) we have obtained a pure line of cultured EC originating from the human placental venous side of the circulatory tree; (ii) the cells ha ve the general characteristics and markers ascribed to EC; (iii) as opposed to large human placental vessels, HPEC do not react to BS-I lectin and, un like human umbilical vein EC, have a much higher proliferation rate and a l ong lifespan; (iv) HPEC expressed a characteristic glycosylated coat partic ularly rich in alpha-L-fucose and beta-GlcNAc containing glycocompounds. (C ) 2000 Harcourt Publishers Ltd.