Recombinant Spider Silk Membranes Promote Human Renal Epithelial Differentiation and Function

Porous membranes are frequently used as supports of cell monolayers in functional studies of epithelial and endothelial barriers. However, conventional polymer-based membranes such as those made of polycarbonate do not mimic the structural and biochemical properties of native basement membranes, which may limit cellular differentiation and function. Here, we use a nanofibrillar membrane made of recombinant spider silk functionalized with the integrin-binding RGD motif of fibronectin and coated with human kidney-specific laminin-521 (LN/FN-silk) as a novel substrate for renal epithelial monolayer cultures. Cellular morphology, mRNA expression, barrier properties and transporter activity were assessed using scanning and transmission electron microscopy, RNA-sequencing, lucifer yellow permeability assays, immunofluorescence staining and fluorescent probe transport assays. Immortalized renal proximal tubular epithelial cell (RPTEC/TERT1) monolayers cultured on LN/FN-silk membranes and in co-culture with human fibroblasts (fHDF/TERT166) exhibited cuboidal morphology, reduced cell death and maintained barrier integrity with tight junction expression compared to conventional polymer-based transwell membranes. In contrast to the LN/FN-silk membranes, the polycarbonate membranes released the endocrine disrupting toxicant bisphenol A, triggering estrogen-mediated signalling. Furthermore, cells on LN/FN-silk membranes showed enhanced directional anion and cation transport, compared to cells cultured on conventional membranes. In summary, the results indicate that LN/FN-silk membranes improve differentiation and functional maintenance of renal epithelial cell cultures, thereby allowing preservation of renal epithelial transport functions. Overall design: RNA-Seq of RPTEC/TERT1 monocultures on LN/FN-silk (SR), RPTEC/TERT1 / fHDF/TERT166 co-cultures on LN/FN-silk (SRF), RPTEC/TERT1 monocultures on LN/PC (PR), RPTEC/TERT1 / fHDF/TERT166 co-cultures on LN/PC (PRF) and fHDF/TERT166 monocultures (F).