In Vitro Model of the Glomerular Capillary Wall Figure 2. Scanning EM demonstrating morphology of GEnC (A–C) and podocytes (D–F) when co-cultured on opposite sides of a tissue culture insert porous support. Cells were cultured for 1 week at 33uC (A and D) or a week at 33uC followed by a week at 37uC (B,C,E and F). Scale bars 100 mm (A,B,D and E) or 10 mm (C and F). Images acquired at accelerating voltage of 15 kV. GEnC and podocytes each form a monolayers under co-culture conditions. doi:10.1371/journal.pone.0020802.g002 22.8 V compared to 54+/27 V, p,0.005). There was no significant difference in resistance when dual layers of GEnC and podocytes were compared. However in comparison to dual layers of GEnC or podocytes, the GEnC and podocyte mixed cell type co-culture had a significantly lower resistance (31+/23 V, p,0.003 and p,0.002 respectively). At 37uC (Fig. 3B) dual GEnC layers again showed more than a doubling of resistance compared to a single layer of GEnC (13.8+/21.8 V compared to 40.2+/ 24 V, p,0.001). Dual layers of podocytes showed an almost four- fold increase of resistance values compared to podocyte monolay- ers (2.4+/20.4 V compared to 11.4+/23 V), however this change was not significant. When these resistances were compared to the GEnC and podocyte co-culture resistance (18.6+/21 V), the mixed cell-type co-culture had a significantly lower resistance than the GEnC dual layers (p,0.005), but no difference when compared to the podocyte dual layers. Also, podocyte dual layers had a significantly lower resistance than GEnC co-culture (p,0.005). GEnC resistances in general are comparable between 33 and 37uC [11,12] whereas podocyte resistances are lower at 37uC as we have previously observed [32]. GEnC behaviour on biological matrices GEnC cultured on reduced growth factor (RGF) Matrigel formed tubes, whilst on RGF Matrigel coated with fibronectin or gelatin, clumps of cells were formed (Fig. 4A–C). GEnC also failed to form a monolayer on a peptide hydrogel, collagen type I gel, and collagen type I gels coated with fibronectin or gelatin (Fig. 4D–G). GEnC only formed a confluent monolayer similar to that seen on tissue culture plastic (Fig. 4H) when seeded onto Cellagen (Fig. 4I). Electrospinning of collagen/PCL Cellagen is available only as a thick (35 mm) layer which is impermeable to molecules larger than 3000–4000 Daltons and therefore unsuitable for a model of the GFB. However, it did serve to demonstrate that GEnC will form a confluent monolayer on a collagen membrane provided it is in a suitable form. As an alternative we electrospun a solution of type I collagen and PCL to form a nanofibre membrane, which was stabilised by cross-linking. Scanning electron microscopy demonstrated the fibrous nature of the membranes produced (images of membrane alone not shown, see below for membrane electrospun onto nickel mesh). Cell monolayer formation on electrospun membrane IF staining using cell-specific antibodies demonstrate that GEnC and podocytes formed confluent monolayers on the collagen/PCL membrane when incubated at 33uC for one week (Fig. 5). GEnC and podocyte growth on the collagen/PCL membrane was studied using a WST-1 assay performed 1 and 5 days after seeding (Fig. 6). There was a significant increase in viable cells for both cell types (GEnC p,0.003, podocytes p,0.01) over this time period confirming their proliferation, in addition to attachment, on the collagen/PCL membrane. Formation of the composite bioartificial GBM A micro-PEF nickel mesh (Fig. 7A) was identified as a suitable structural support in view of its physical characteristics and lack of toxicity to cells as assessed by adherence and growth directly on this mesh (data not shown). Next, the collagen/PCL solution was successfully electrospun directly onto this mesh (Fig. 7B) and the resulting biocomposite membrane was then placed in a Cell Crown to form a culture insert (Fig. 7C). Incubation of the membrane/ mesh composite in cell culture media at 33uC for 1, 3 and 5 days did not affect membrane fibre appearance over time (Fig. 7C, D and E). Characterisation of cell growth on the composite bioartificial GBM GEnC and podocytes were cultured on the electrospun collagen/PCL nickel mesh composite in single layers and in co- PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20802