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Introduction The kidney glomerulus acts as the blood-filtering unit in the kidney and plays a crucial role in maintaining homeostasis. The glomerular filtration barrier is a size-selective filter composed of endothelial cells and podocytes, separated by the glomerular basement membrane. Dysfunction of the barrier can result in proteinuria, often followed by progressive renal damage and kidney failure. Aim of this study was to establish an in vitro human kidney glomerulus co-culture model using endothelial cells (ciGEnCs) and podocytes (PODO/SVTERT152) on collagen coated Melt Electrowriting (MEW) PCL membranes in a perfused bioreactor.
Methods Human urine-derived podocytes (PODO/SVTERT152) were purchased from Evercyte and conditionally immortalized human glomerular endothelial cells (ciGEnCs) provided by the University of Bristol. For the optimisation of the co-culture medium, the effect of pure HPLM (Human Plasma-Like Medium) and medium compositions, consisting of mixtures of HPLM and Endothelial Basal Medium (EBM) were tested. In addition, effects of different FCS (fetal calf serum) concentrations and addition of ATRA (all-trans retinoic acid) were evaluated on both cell types. The microporous PCL membrane was fabricated with the MEW printhead of a BioScaffolder 3.1 bioprinting machine (GeSiM, Germany) and then manually coated with collagen I. Endothelial cells were seeded first on one side of the membrane; after a growth phase of 3 days podocytes were seeded on the opposite side and then both together further cultivated under perfusion conditions in a self-designed and manufactured bioreactor. Three different bioreactors were fabricated having 3 different shear conditions (constant shear and two gradient shear bioreactors) under flow of cell culture media. Gradient shear bioreactors were included in the study to mimic the shear distribution in the native glomerulus. Cell behaviour was investigated utilising fluorescence microscopy and gene expression analyses.
Results & Discussion Both HPLM and an EBM-HPLM 1:1 mixed composition were identified as suitable candidates for the co-culture medium, using 5% FCS for growth and switching to 0.5% FCS for differentiation. The effect of collagen I crosslinking and multiple coatings showed to have a slight impact on scaffold elasticity and permeabilit. In addition, the permeability of the cell-free scaffolds and shear stress simulations of three different bioreactor designs were analysed. After 3 days of perfusion culture, successful attachment of endothelial cells and CD31 expression were observed. In addition, podocytes attached well when seeded on the opposite side of the coated membranes and synaptopodin expression proved successful differentiation. Both cell types could be successfully visualised within the same co-culture and showed typical cell morphologies. Co-culture experiments could be performed with the present set-up for up to 9 days. In conclusion, the research presented in this study establishes a starting point for developing of a novel human kidney glomerulus model. Although the flat morphology of the PCL membrane does not accurately resemble the macrostructure of glomeruli, it may provide an effective 3D microenvironment for cell-cell and cell-matrix interactions.
Acknowledgments The study was funded by the German Research Foundation (DFG, grant No. GE 1133/27-1).
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