In Vitro Model of the Glomerular Capillary Wall Figure 4. Appearance of GEnC cultured for 1 week at 336C on various matrices to identify a suitable matrix to support growth of GEnC in a uniform layer by light (A–H) or immunofluorescence (I) microscopy. (A) GEnC formed capillary tube-like structures on reduced growth factor (RGF) Matrigel, (B) clumps on RGF Matrigel coated with fibronectin or (C) gelatin, (D) rounded up on peptide hydrogel, (E) formed clumps on collagen type I and on (F) collagen type I coated with 20 mg/ml fibronectin or (G) gelatin. (H) GEnC formed a confluent monolayer on tissue culture plastic and on (I) Cellagen (visualised using IF staining for VE-cadherin, as Cellagen is opaque). All cells were seeded at the same density. Original magnification 610. doi:10.1371/journal.pone.0020802.g004 nickel is widely used in clinical prostheses because of its inert, non- toxic characteristics. We found that a micro-PEF nickel mesh would be suitable for this application. The thickness of this mesh (and the percentage open area) can be further optimised. We devised and successfully applied a novel approach of electrospin- ning collagen/PCL nanofibres directly onto the nickel mesh, forming a supported GBM predominantly composed of a biologically remodelable substrate (Fig. 7). Finally we demonstrate the completed model, composed of collagen type I/PCL electrospun onto a nickel mesh, with GEnC growing across this structure in a confluent monolayer, and podocytes growing in a semi-confluent monolayer. The majority of these cells in co-culture are only separated by the electrospun GBM, allowing close contact of the cells and cellular crosstalk. The composition of the collagen matrix we have developed could be further optimised so that it mimics that of the in vivo GBM to a greater extent. For example, one group has already demonstrated that electrospun collagen membranes can be additionally modified by addition of glycosaminoglycan chains [26]. Collagen IV was not studied here because of the lack of availability of the glomerulo-specific isoforms as well as its non- fibrillary nature which does not lend itself to electrospinning. So far there are no publications detailing electrospinning of collagen type IV or laminin-11 and our approach in future development of the model will be to incorporate these proteins into the electrospun collagen type I/PCL nanofibre membranes. This model could also be used to study the effect of different substrates and coculture on cell differentiation. Although we did not see effects on the endothelial and podocyte markers used in this study (PECAM-1 and podocin respectively), study of a panel of such markers including specific matrix component isoforms would be revealing. In conclusion, we have generated the first tri-layer in vitro models of the human glomerular capillary wall containing the relevant cell types, GEnC and podocytes, separated by a membrane and in so doing have successfully confirmed both experimental hypotheses. In the first model, in which this membrane is the porous support of a tissue culture insert, we provide evidence of the cell-cell communication which is important in regulation of the GFB in vivo. In the second model, the porous support is replaced with a novel composite bioartificial membrane containing collagen type I, successfully establishing a model where the two cell layers are separated only by a biocompatible membrane over the major of its PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20802