Figure 3. Barrier properties of the cells cultured in tissue culture inserts in single layers and co-culture was measured by TEER. Cells were cultured for 1 week at 33uC (A) or a week at 33uC followed by a week at 37uC (B). Bars show mean +/2 SEM, n = 20 (mean result of 4 experiments, n = 5 in each). At both temperatures resistance increased when cells in co-culture were compared to cells in a single layer. * p,0.05 by ANOVA with Bonferroni post hoc tests. doi:10.1371/journal.pone.0020802.g003 culture. After one week at 33uC GEnC formed a confluent monolayer on the collagen/PCL composite (Fig. 8A and B), whilst podocytes formed a sub-confluent layer (Fig. 8D and E). When the cells were grown in co-culture, GEnC still formed a monolayer and no podocin staining could be seen on the endothelial side (Fig. 8C). Podocytes again formed a sub-confluent monolayer and PECAM-1 staining on GEnC on the opposite side of the bioartificial membrane could be faintly observed (Fig. 8F). When the cells were cultured at 37uC, both GEnC and podocytes formed a confluent monolayer when grow in either a single layer (Fig. 9A– E) or co-culture (Fig. 9C and F). There was no apparent difference in the expression level or distribution of PECAM-1 or podocin between cells cultured on inserts and the bioartificial membrane or between cells in monoculture and in coculture. Scanning electron microscopy of GEnC and podocytes co- cultured on opposite sides of the collagen/PCL/mesh composite demonstrated that GEnC grow in a uniform monolayer (Fig. 10A In Vitro Model of the Glomerular Capillary Wall and B), similar to those seen when these cells were co-cultured on the polycarbonate insert (Fig. 2A–C), whilst podocytes are less densely packed. Discussion We have previously shown that tissue culture inserts can be used to culture human GEnC and podocyte monolayers [6,7,32]. Here we show for the first time that these cultures can effectively be combined either side of the porous support to form a tri-layer model. Cell morphology was preserved when grown on the underside of the support and when in the presence of the other cell type on the other side of the support (Figs. 1 & 2). For both cell types in monoculture the TEER measurements (Fig. 3) were in agreement with previous values [12,32]. The resistance of dual layers of the same cell type at either 33uC or 37uC, was at least double that a single monolayer of either GEnC or podocytes as expected. When GEnC and podocytes were co- cultured at 33uC on opposite sides of the insert, the combined resistance was higher than that of a single layer of either GEnC or podocytes. However, from the resistances of dual layers of the same cell type, the resistance would be expected to be at least the sum of the resistances of single GEnC and podocyte layers. The fact that the resistance was significantly lower than this suggests that one of the cell layers has reduced the resistance of the other, presumably via soluble mediators. A possible candidate for this is VEGF, as this growth factor is produced by podocytes [4], GEnC have receptors for VEGF [5], and VEGF is known to affect GEnC resistance [7]. For differentiated cells (at 37uC) in co-culture the combined resistance of layers of GEnC and podocytes was greater than the ‘expected resistance’. Therefore this does not suggest the same effect of cell-cell communication as seen in undifferentiated cells. The fact that there is a splicing switch in differentiation of podocytes to produce VEGF-A165b [33], which does not reduce GEnC resistance, could potentially explain this observation [34]. These results confirm that tri-layer models of the GFB can successfully be established in vitro and provide evidence that the cell-cell communication which occurs in vivo can be studied in vitro. Given the dissimilarity between polycarbonate porous supports in inserts and the GBM in vivo, we sought to identify a suitable preparation of ECM components with which to replace the porous support as a necessary step in testing our second hypothesis. The aim here was not an unrealistic one of exactly reproducing the GBM in all respects but to produce a predominantly biological membrane more closely analogous to the in vivo GBM and potentially amenable to remodelling by cultured cells. Glomerular cells failed to form monolayers suitable for the proposed model on any of the ECM gels tested. Only when cells were cultured on a rigid collagen membrane (Cellagen) would the cells form a confluent monolayer. This data suggests that not only the composition of the matrix protein, but also its structure and presentation is important in cell monolayer formation, consistent with previous reports [18,35]. GEnC and podocytes grew in a monolayer (similar to that produced when cells were cultured in tissue culture inserts) on an electrospun collagen I/PCL membrane (Fig. 5). A WST-1 proliferation assay confirmed proliferation of both cell types over 5 days culture on the electrospun collagen I/PCL membrane (Fig. 6). Collagen I/PCL membranes have previously been demonstrated as a suitable material for the culture of endothelial cells to form vascular grafts [36]. In order to allow a thin electrospun nanofibre membrane to be used in the model, it was necessary to provide structural support. Metal scaffolds are regularly used in tissue engineering [37] and PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20802