Culture of Human Mesangial Cells

Human mesangial cells (HMCs) were isolated using the protocol initially described by Harper et al.24 The cortexes of human kidneys surgically removed (from nephrectomies performed for malignancies) from areas away from the neoplasms were used. HMCs were isolated by sieving the cortex through stainless steel screens and a series of different pore size nylon sieves. HMCs were cultured in RPMI 1640 medium (Fisher Scientific, Suwanee, GA), supplemented with 20% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO₂ and treated with a penicillin (100 U/ml)/streptomycin (100 μg/ml) solution (Sigma-Aldrich). HMCs overgrew other cells and became confluent 3 to 4 weeks after plating. In addition, HMCs were purchased from Fisher Scientific and maintained in culture conditions similar to described previously. For both HMC cultures, a homogeneous population of MCs was confirmed by ultrastructural examination showing smooth muscle features with intracytoplasmic myofilaments and attachment plaques at the cell surfaces, as well as immunohistochemical staining showing positivity for HHF-35 (muscle-specific antigen) and vimentin and lack of staining for keratins and factor VIII. On reaching confluence, cells were seeded into 100-mm tissue culture dishes. Fifth to sixth passages of MCs were used for the experiments. The MCs were grown on RPMI 1640 medium containing 15% fetal bovine serum until confluence. Three days before the beginning of the experiments, the fetal bovine serum concentration of the media was decreased to 0.5% with daily exchange of medium to create a quiescent cellular environment prior to the incubation with LCs.