In Vitro Model of the Glomerular Capillary Wall Figure 5. Immunofluorescence staining of (A and B) GEnC and (C and D) podocytes demonstrating monolayer formation when cells are cultured on collagen/PCL electrospun membranes for 1 week at 336C. GEnC are stained for PECAM-1 and podocytes for podocin. Scale bars bar 250 mM (A and C) and 50 mm (B and D). doi:10.1371/journal.pone.0020802.g005 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20802 Figure 6. A WST-1 assay demonstrating proliferation of (A) GEnC and (B) podocytes on collagen/PCL electrospun mem- branes at 336C. Cell number is proportional to absorbance and increases over time in each a case (p,0.05, n = 3). doi:10.1371/journal.pone.0020802.g006 area. These models will be useful for studying cross-talk between glomerular cells via soluble mediators and the mechanisms of GFB function and how this is disturbed in disease states. Materials and Methods Unless otherwise stated all materials were purchased from Sigma-Aldrich (Poole, UK). Cell culture Human conditionally immortalised GEnC and podocytes were used, as described in detail previously [11,12]. These cell lines represent the best recognised human culture model of the respective cell types. At the permissive temperature of 33uC, the tsSV40LT transgene is activated, causing cell proliferation, whereas at 37uC, the transgene is inactivated, rendering cells non-proliferative, quiescent and able to differentiate. GEnC and podocyte behaviour was investigated at both 33uC and 37uC. GEnC were cultured in endothelial growth medium 2 microvas- cular (EGM2-MV, Cambrex, Wokingham, UK) containing FCS (5%) and growth factors as supplied with the exception of VEGF. Podocytes were cultured in RPMI 1640 (Lonza, Basel, Switzer- land) as described previously [11]. When cells were grown in co- culture, EGM2-MV media was used. Co-culture on tissue culture Inserts A GEnC or podocyte suspension of 150,000cells/cm2 was prepared and 100 ml was pipetted into tissue-culture inserts (1 cm diameter, Nunc Int., Rochester, NY) containing polycarbonate supports (0.4 mm pore size, 0.5 cm2 surface area) in 24-well plates. In some cases dual monolayers of the same cell type, or co-cultures of different cell types, were seeded on opposite sides of the membrane. To achieve this inserts were placed upside-down in 24- well plates and 100 ml of cell suspension pipetted onto the support.