In Vitro Model of the Glomerular Capillary Wall Figure 7. Illustrative images of the micro-PEF nickel mesh and the mesh coated with electrospun nanofibres to form a bioartificial composite membrane. (A) Light microscopy of micro-PEF nickel mesh (scale bar 25 mm). The black lines are the bars of the mesh, white squares are the open area. (B) Scanning EM of the micro-PEF nickel mesh coated with electrospun collagen/PCL nanofibres, scale bar 10 mm. (C) Collagen I/PCL/ nickel mesh bioartificial composite membrane secured in 10 mm diameter Cell Crowns. (D,E and F) Light microscopy of the collagen/PCL/mesh composite after 1, 3 and 5 days incubation in tissue culture media at 33uC (scale bar 25 mm). doi:10.1371/journal.pone.0020802.g007 The cells were then cultured at 33uC for at least four hours to allow cell attachment. The inserts were then turned the correct way up and other cells were pipetted into the insert as above. In all cases total media volume inside and outside the insert was made up to 500 ml. Cells were either cultured for 1 week at 33uC only, or for 1 week at 33uC, then switched to 37uC to differentiate. Figure 8. Immunofluorescence demonstrating appearance of GEnC (A–C) and podocyte (D–F) cell layers after one week at 336C on the collagen/PCL/mesh bioartificial composite membrane. PECAM-1 on GEnC labelled red, podocin on podocytes green and nuclei blue (F only). (A and B) GEnC cultured in a single layer or (C) with podocytes co-cultured on the opposite side of the membrane. (D and E) podocytes cultured in a single layer or (C) with GEnC co-cultured on the opposite side of the membrane. Scale bars 250 mm (A and D) and 50 mm (B,C, E and F). doi:10.1371/journal.pone.0020802.g008 PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20802