Renal papillary cells grown in cell culture. (A) Isolated renal papillary cells grown in standard cell culture conditions formed cell aggregates within about 24 hours of their isolation and expressed ZO-1 in their tight junctions. (B and C) Phase-contrast (Phase; B) and fluorescence (C) microphotographs of a group of renal papillary cells growing in cell culture conditions 4 days after cell isolation; about 40% of the papillary cells isolated from BrdU-loaded animals were BrdU-retaining cells. (D and E) During the first several days of culture, most cells had an epithelial phenotype expressing ZO-1 in their tight junctions (D), but some cells, in addition to ZO-1, also expressed mesenchymal proteins such as α-smooth muscle actin (αSMA) (E). (F) After more than a week in culture under control conditions with 5% CO₂ and 95% room air, most cells were spindle-shaped and stained strongly for α-smooth muscle actin. (G) However, when cells were grown under hypoxic conditions (5% CO₂, 1.5% O₂, and 93.5% N₂), most cells retained an epithelial phenotype, with prominent ZO-1 expression. (H) When grown in standard control cell culture media on plastic, most cells adhered to the culture dish but frequently formed cell aggregates that resembled neurospheres. (I) The tendency for the cells to aggregate was markedly enhanced by growth of the cells in the absence of sera. The picture shows a 3-week-old culture with many cellular aggregates. (J) Many of the cells inside the aggregates were positive for nestin. Scale bars: 50 μm.