scaffold providing support: it also regulates cellular behaviour by influencing proliferation, survival, shape, migration and differen- tiation [18]. Therefore ideally an artificial GBM should closely mimic the structure and function of the native matrix proteins. Collagen type I has a high degree of biocompatibility and is biodegradable and has therefore been used to mimic the basement membrane in various models, for example of the blood brain barrier [19]. Other biocompatible gels have been used in similar co-culture models, such as self-assembled peptide hydrogel [20] and Matrigel [21]. Usually these components are prepared as thick gels and at least in this form they are not suitable to represent the GBM. Electrospinning has recently come to the fore as a relatively simple technique which can produce fibres on a nanometre scale in random or aligned patterns forming a structure that can mimic the ECM [22]. Biocompatible and biodegradable nanofibres of various synthetic (such as polyglycolic acid [23] and polycapro- lactone, PCL [14]) or naturally occurring (such as collagen type I [22] and chitosan [24]) polymers have been produced, the later being of most relevance to the synthesis of a GBM-like structure. A number of different cell types have been successfully cultured onto collagen type I electrospun membranes including coronary endothelial cells [25], fibroblasts [26] and keratinocytes [27], therefore it is used extensively as an ECM component in tissue engineering. The incorporation of PCL in electrospun collagen nanofibres is attractive to increase their tensile strength whilst also being biocompatible and biodegradable [14,28]. Previously, a few co-culture models have examined interactions between glomerular cells [17,29,30,31], but no model of the glomerular capillary wall including glomerular endothelial cells, basement membrane and podocytes yet exists. Here we describe the sequential construction of two in vitro models of the glomerular capillary wall fulfilling these desirable attributes. We tested two In Vitro Model of the Glomerular Capillary Wall hypotheses, firstly that it would be possible to established a tri-layer model based on tissue culture inserts, with a porous artificial membrane acting as the GBM. Secondly, we hypothesised that we could develop a composite bioartificial membrane consisting of electrospun collagen type I and a supporting micro photo- electroformed (micro-PEF) nickel mesh to replace the porous membrane in the first model. Results Co-culture of cells on insert membranes GEnC and podocytes grew in uniform layers on either side of the polycarbonate support to form a representative model of the glomerular capillary wall (Fig. 1). The density of both cell types appeared to increase when co-cultured opposite the other cell type. Similar results were obtained at 33uC (Fig. 1) and 37uC (data not shown). Scanning EM further confirmed growth of GEnC and podocytes on the polycarbonate support. At both 33uC (Fig. 2A) and 37uC (Fig. 2B and C) GEnC grew in uniform layers when co- cultured with podocytes. Podocytes in co-culture with GEnC at 33uC (Fig. 2D) displayed a more rounded morphology, however when co-cultured at 37uC these cells became flatter and formed a single layer (Fig. 2E and F). The barrier properties of this co-culture model were assessed by measuring trans-endothelial electrical resistance (TEER). TEER is inversely related to the fractional area of pathways across a cell monolayer open to water and small molecules and as such can be used an index of permeability, a higher TEER corresponding to reduced permeability. At 33uC (Fig. 3A) resistance of inserts with GEnC layers both sides was significantly higher than those with a single layer of GEnC (20+/21.7 V compared to 53+/24.7 V, p,0.005). Likewise for podocyte single and dual layers (22+/ Figure 1. IF staining for actin (green) and nuclei (blue) showing GEnC and podocyte monolayer formation in tissue culture inserts after a week at 336C. (A) GEnC cultured alone in a single monolayer. (B) GEnC when podocytes co-cultured on the opposite side of the insert. (C) Podocytes cultured alone in a single monolayer. (D) Podocytes when GEnC co-cultured on the opposite side of the insert. The number of cells, and the confluence of the monolayer, appears to increase when the cells are co-cultured. Scale bar: 250 mm. doi:10.1371/journal.pone.0020802.g001 PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20802