Recellularization of Scaffolds derived from precision-cut kidney slices.

The global rise in chronic kidney disease necessitates innovative solutions for end-stage renal dis-ease that can help to overcome the limitations of the only available treatment options, transplanta-tion and dialysis. Tissue engineering presents a promising alternative, leveraging decellularized scaffolds to retain the extracellular matrix (ECM). However, optimizing methods for decellularization and recellularization remains a challenge. Here we present novel work which builds on our previous study where we investigated several decellularization protocols. In this study we analyzed the suit-ability of decellularized scaffolds for recellularization. Precision-cut kidney slices (PCKS) were uti-lized as a model to explore the impact of different decellularization protocols on scaffold recellulari-zation. PCKS were pretreated physically followed by immersion decellularization in chemicals (CHEM-Imm). Physical pretreatments included high hydrostatic pressure (HHP-Imm) or freezing-thawing cycles (FTC-Imm). Scaffolds were recellularized, with human renal proximal tubular epithe-lial cells (RPTEC/TERT1). All scaffolds showed cell growth over the 7-day incubation period. Nota-bly, FTC-Imm demonstrated the highest expression of the tight junction protein zonula-occludens-1 (ZO-1). Moreover, as the native kidney is composed of up to 30 different cell types, we utilized arti-ficial neural networks (ANN) to investigate the distribution and attachment patterns of RPT-EC/TERT1 cells to determine if decellularized scaffolds retain cell specific attachment sites. It was revealed that, at least 97% of RPTEC/TERT1 cells were attached outside the Bowman capsules, potentially showing a clear tendency to attach to their original tubular sites. This suggests that the ECM retains instructive cues guiding the migration and attachment of the cells. Overall, our scoring system identified FTC-Imm as the most effective method.