An In Vitro Model of the Glomerular Capillary Wall Using Electrospun Collagen Nanofibres in a Bioartificial Composite Basement Membrane Sadie C. Slater1, Vince Beachley2, Thomas Hayes3, Daming Zhang 3 , Gavin I. Welsh1, Moin A. Saleem1, Peter W. Mathieson 1 , Xuejun Wen 2 , Bo Su3, Simon C. Satchell1* 1 Academic Renal Unit, University of Bristol, Bristol, United Kingdom, 2 Department of Bioengineering, Clemson University, Charleston, South Carolina, United States of America, 3 Department of Oral and Dental Science, University of Bristol, Bristol, United Kingdom Abstract The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri- layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease. Citation: Slater SC, Beachley V, Hayes T, Zhang D, Welsh GI, et al. (2011) An In Vitro Model of the Glomerular Capillary Wall Using Electrospun Collagen Nanofibres in a Bioartificial Composite Basement Membrane. PLoS ONE 6(6): e20802. doi:10.1371/journal.pone.0020802 Editor: Markus J. Buehler, Massachusetts Institute of Technology, United States of America Received November 1, 2010; Accepted May 13, 2011; Published June 24, 2011 Copyright: 2011 Slater et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a Wellcome Trust Fellowship to Dr. SC Satchell (075731), the Medical Research Council (G0500053) and the Biotechnology and Biological Sciences Research Council (BB/G000077/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: S.C.Satchell@bristol.ac.uk Introduction The glomerular capillary wall consists of three specialised layers: glomerular endothelial cells (GEnC) on the inside of the capillary, a glomerular basement membrane (GBM) and a layer of podocytes (glomerular epithelial cells) on the outside. The cell layers are separated by a relatively thick basement membrane (300–350 nm), the GBM, a specialised form of extracellular matrix (ECM). It is predominantly composed of particular isoforms of matrix proteins: collagen type IV a3, a4 and a5 chains, and laminin-11 [1,2]. The three layers of this system function as a whole, the glomerular filtration barrier (GFB), to selectively filter the blood [3]. Paracrine communication via soluble mediators between GEnC and podocytes is important in maintenance of the structure and function of the GFB. For example we and others have shown that podocytes secrete the endothelial factors VEGF- A, VEGF-C and angiopoietin 1, whilst GEnC express the cognate receptors for these mediators [4,5,6,7]. Podocyte-specific alter- ations of VEGF-A or angiopoietin expression results in GFB dysfunction including GEnC abnormalities and proteinuria [8,9]. Despite the definition of this structure of the glomerular capillary wall by electron microscopy as far back as 1957 [10], understanding of its filtration function remains incomplete. In vitro work historically has been limited by difficulties with culturing cells of the filtration barrier. Recently, improved culture techniques, and in particular generation of unique conditionally immortalised cell lines of both human GEnC and podocytes in our laboratory, have enabled detailed study of these cells in vitro [11,12]. However, because the components of the glomerular capillary wall function as an inter-related whole, there is a need to develop three- dimensional co-culture models to maximise the relevance of in vitro studies to the intact glomerulus. Ideally such a model would be comprised of a layer of GEnC and a layer of podocytes, separated by a biological membrane acting as the GBM. This membrane should be thin, permeable, able to support the structure, biological, biodegradable, composed of proteins found in the GBM and accessible on both sides (to enable measurement of fluid and molecular movement across the structure). Co-culture techniques have been successfully used to generate functional in vitro models of other tissues, such as the blood-brain barrier [13], skin [14] and the lung alveolar capillary barrier [15]. One of the main challenges in co-culture models is generation of an ECM or basement membrane most appropriate for the tissue in question. In some cases basement membrane has been represented by the synthetic porous support of a tissue culture insert [13,15,16,17]. However basement membrane is more than just a PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20802