Aligned Silk Sponge Fabrication and Perfusion Culture for Scalable Proximal Tubule Tissue Engineering

Chronic kidney disease and kidney failure are on the rise globally, yet there has not been a corresponding improvement in available therapies. A key challenge in a biological approach to developing kidney tissue is to identify scaffolding materials that support cell growth both <i>in vitro</i> and <i>in vivo</i> to facilitate translational goals. Scaffolds composed of silk fibroin protein possess the biocompatibility, mechanical robustness, and stability required for tissue engineering. Here, we use a silk sponge system to support kidney cells in a perfused bioreactor system. Silk fibroin protein underwent directional freezing to form parallel porous structures that mimic the native kidney structure of aligned tubules and are able to support more cells than nonaligned silk sponges. Adult immortalized renal proximal tubule epithelial cells were seeded into the sponges and cultured under static conditions for 1 week, then grown statically or with perfusion with culture media flowing through the sponge to enhance cell alignment and maturation. The sponges were imaged with confocal and scanning electron microscopies to analyze and quantify cell attachment, alignment, and expression of proteins important to proximal tubule differentiation and function. The perfused tissue constructs showed higher number of cells that are more evenly distributed through the construct and increased gene expression of several key markers of proximal tubule epithelial cell function compared to sponges grown under static conditions. These perfused tissue constructs represent a step toward a scalable approach to engineering proximal tubule structures with the potential to be used as <i>in vitro</i> models or as <i>in vivo</i> implantable tissues to supplement or replace impaired kidney function.