Immunofluorescence GEnC or podocytes were fixed in 2% paraformaldehyde, blocked and permeabilised with 0.1% Saponin and 3% BSA in PBS and then incubated with an Alexa Fluor 488-phalloidin conjugate (Invitrogen, Paisley, UK) to label the actin cytoskeleton and with DAPI to label nuclei. The polycarbonate supports were removed from their inserts and were mounted on glass slides using VectashieldH aqueous mounting solution (Vector Laboratories, CA). Samples were examined using a cell imaging system (Leica AF6000LX, Leica Microsystems, Wetzlar, Germany). Scanning electron microscopy Samples were fixed in 2.5% glutaraldehyde, dehydrated in graded ethanols. The samples were then dried using a Samdri 780 critical point dryer (Electron Microscopy Sciences, Pennsylvania, USA), then sputter coated with gold using a sputter coater designed at Bristol University. Images were obtained using a Quanta 400FEI scanning electron microscope (FEI, Eindhoven, Netherlands) operated at an accelerating voltage of 15 kV. Measurement of transendothelial electrical resistance (TEER) Cells were seeded in co-culture onto tissue culture inserts as described above. TEER was measured using an Endohm 12 electrode chamber (World Precision Instruments, Sarasota, FL) connected to an EVOMx voltmeter (World Precision Instruments) as previously described [7]. Tissue culture inserts were placed sequentially into the Endohm chamber and resistance recorded after 10 seconds. Identification of suitable matrix material To develop a bioartificial composite membrane we first sought to identify a suitable matrix material. Twelve-well plates were coated with thick matrix gels, before seeding of GEnC at a density of 20,000cells/cm2. Gels were made of Collagen type I (Millipore, Billerica, MA), reduced growth factor (RGF) Matrigel (BD Biosciences, San Jose, CA) and Puramatrix peptide hydrogel (BD Biosciences), according to the manufacturers’ instructions. In some cases the collagen type I and Matrigel were additionally coated with 20 mg/ml fibronectin or a 2% gelatin solution to enhance cell adhesion. Briefly, 200 ml of solution was pipetted onto the gel and incubated at room temperature for 1 hour. Any excess solution was then removed before cell seeding. Cells were also seeded onto a Cellagen membrane (composed of cross-linked collagen type1, MP Biomedicals, Solon, OH). Cells were cultured for a week at 33uC, then monolayer formation was assessed by light microscopy and images were taken using a CoolPix 4500 camera (Nikon, UK). Cells on Cellagen membranes were visualised by immunofluorescent staining with a VE-cadherin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Primary antibody binding was detected as above. Electrospinning of collagen nanofibres A 10% collagen solution was prepared by dissolving collagen type 1 from calf skin and 2% PCL (Mn = 80,000) in 1,1,1,3,3,3- hexafluoro-2-propanol (Oakwood, West Columbia, SC, USA). PCL was included as it possesses a high tensile strength, yet is biocompatible and has cell binding properties. The solution was stirred overnight at 37uC. Electrospinning was performed in a custom-made chamber, where the spinneret was made from a 25 G tip-ground-to-flat needle (Terumo, Hatagaya, Japan), mounted on an electrically insulated stand. The spinneret needle was maintained at a voltage In Vitro Model of the Glomerular Capillary Wall of 12 kV by a high voltage power supply (PS/EL30R01.5-22, Glassman High Voltage Inc., Hampshire UK), and a glass plate covered with a piece of aluminium foil (14614 cm2) under the needle at a distance of 13 cm was grounded and used as the collector. Collagen solutions were spun directly onto the aluminium foil or alternatively across the open lower end of a cylindrical Cell Crown insert (see below) by placing the Cell Crown top down on the foil prior to electrospinning. The capillary needle spinneret was connected through PTFE tubing to a plastic syringe filled with 3 ml of collagen type I spinning solution. A constant volume flow rate of 3 mL/min was maintained using a syringe pump (KD Scientific, MA). Ambient conditions for the current electrospinning process were 49.8% humidity and 21.0uC (room temperature). Collagen/PCL membranes were cross-linked by immersion in 10%(v/v) HMDI,1,6-diisocyanatohexane (HMDI, Alfa Aesar, MA) in isopropyl alcohol and gently agitated for 2 hours. Membranes were then immersed in isopropyl alcohol for 10 minutes to rinse off HDMI, followed by 1 hour in water. Membranes were placed into Cell Crowns (Scaffdex, Tampere, Finland) to form an assembly similar to the tissue culture inserts used previously. Cell Crowns consist of 2 polycarbonate rings, one fitting inside the other. The electrospun membrane was placed between the two rings, then one ring was pushed over the other, holding the membrane in place to form an insert. This insert system was then sterilised by exposure to UV light for 30 minutes. The insert was then placed in a 24 well plate. For all experiments membranes were suspended 2 mm above the bottom of the well so that cells could be cultured on either one or both sides of the membrane. Electrospun membrane stability under tissue culture conditions was tested by placing the membrane in Cell Crowns, then incubating the membrane in culture media at 37uC for 1, 3 and 5 days. At each time point the membrane was examined using light microscopy to demonstrate that fibres remained present and attached to the mesh support. Cell culture on electrospun membranes A GEnC and/or podocyte suspension of 150,000cells/cm2 was prepared and 100 ml was pipetted onto the collagen/PCL membrane secured in Cell Crowns. A further 400 ml media was pipetted onto the membrane, and 1 ml media was pipetted outside the insert. In some cases, cells were seeded on both sides as for standard inserts above. Cells were cultured and fixed for immunofluorescence imaging as above. GEnC were incubated with a PECAM-1 antibody (R&D Systems, Minneapolis, MN), whilst podocytes were incubated with a podocin antibody (a podocyte-specific marker, Sigma). The cells were further incubat- ed for 1 hour with an appropriate secondary antibody conjugates (Alexa fluor 488 or 555, Invitrogen), washed, counterstained with DAPI, mounted and imaged as above. When cells were co- cultured, both sides of the membrane were examined and photographed. The cell types were distinguished by staining with different species primary antibodies and secondary antibodies conjugated to different fluorophores. To confirm that cells maintained viability and proliferation on the collagen/PCL a WST-1 assay (Roche Diagnostics, Mannheim, Germany) was performed 1 and 5 days after seeding. Cells were cultured at 33uC. Metabolically active cells react with tetrazolium salt in WST-1 reagent to produce a soluble formazan dye that can be observed at 450 nm. Prior to this assay, the cellular constructs were rinsed with PBS followed by incubation with media containing 10% WST-1 reagent for 2 hours at 33uC. Thereafter, aliquots were pipetted in triplicate into 96 well plates and the PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e20802