Figure 1.
IF staining for actin (green) and nuclei (blue) showing GEnC and podocyte monolayer formation in tissue culture inserts after a week at 33°C.
(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 µm.
Figure 2.
Scanning EM demonstrating morphology of GEnC (A–C) and podocytes (D–F) when co-cultured on opposite sides of a tissue culture insert porous support.
Cells were cultured for 1 week at 33°C (A and D) or a week at 33°C followed by a week at 37°C (B,C,E and F). Scale bars 100 µm (A,B,D and E) or 10 µm (C and F). Images acquired at accelerating voltage of 15 kV. GEnC and podocytes each form a monolayers under co-culture conditions.
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 33°C (A) or a week at 33°C followed by a week at 37°C (B). Bars show mean +/− 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.
Figure 4.
Appearance of GEnC cultured for 1 week at 33°C 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 µg/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 ×10.
Figure 5.
Immunofluorescence staining of (A and B) GEnC and (C and D) podocytes demonstrating monolayer formation when cells are cultured on collagen/PCL electrospun membranes for 1 week at 33°C.
GEnC are stained for PECAM-1 and podocytes for podocin. Scale bars bar 250 µM (A and C) and 50 µm (B and D).
Figure 6.
A WST-1 assay demonstrating proliferation of (A) GEnC and (B) podocytes on collagen/PCL electrospun membranes at 33°C.
Cell number is proportional to absorbance and increases over time in each a case (p<0.05, n = 3).
Figure 7.
Illustrative images of the micro-PEF nickel mesh and the mesh coated with electrospun nanofibres to form a bioartificial composite membrane.
(A) Light microscopy of micro-PEF nickel mesh (scale bar 25 µm). The black lines are the bars of the mesh, white squares are the open area. (B) Scanning EM of the micro-PEF nickel mesh coated with electrospun collagen/PCL nanofibres, scale bar 10 µm. (C) Collagen I/PCL/nickel mesh bioartificial composite membrane secured in 10 mm diameter Cell Crowns. (D,E and F) Light microscopy of the collagen/PCL/mesh composite after 1, 3 and 5 days incubation in tissue culture media at 33°C (scale bar 25 µm).
Figure 8.
Immunofluorescence demonstrating appearance of GEnC (A–C) and podocyte (D–F) cell layers after one week at 33°C on the collagen/PCL/mesh bioartificial composite membrane.
PECAM-1 on GEnC labelled red, podocin on podocytes green and nuclei blue (F only). (A and B) GEnC cultured in a single layer or (C) with podocytes co-cultured on the opposite side of the membrane. (D and E) podocytes cultured in a single layer or (C) with GEnC co-cultured on the opposite side of the membrane. Scale bars 250 µm (A and D) and 50 µm (B,C, E and F).
Figure 9.
Immunofluorescence demonstrating appearance of GEnC (A–C) and podocyte (D–F) cell layers after one week at 33°C and one week at 37°C on the collagen/PCL/mesh bioartificial composite membrane.
PECAM-1 on GEnC labelled red and podocin on podocytes green. (A and B) GEnC cultured in a single layer or (C) with podocytes co-cultured on the opposite side of the membrane. (D and E) podocytes cultured in a single layer or (C) with GEnC co-cultured on the opposite side of the membrane. Scale bars 250 µm (A and D) and 50 µm (B, C, E and F).
Figure 10.
Scanning EM demonstrating morphology of GEnC (A and B) and podocytes (C and D) co-cultured on opposite sides of the collagen/PCL/mesh bioartificial composite membrane.
Scale bars 100 µm (A and C) and 20 µm (B and D). GEnC form a uniform monolayer, comparable with those grown in monoculture or co-culture with podocytes on porous supports in tissue culture inserts (Fig. 2), whilst podocytes are less densely packed.