RPTEC/TERT1 cells form highly differentiated tubules when cultured in a 3D matrix

Abstract

The proximal tubule is the primary site for renal solute reabsorption and secretion and thus a main target for drug-induced toxicity. Current nonclinical methods using 2D cell cultures are unable to fully recapitulate clinical drug responses mainly due to limited in vitro functional lifespan. Since extracellular matrices are known to be key regulators of cell development, culturing cells on classic 2D plastic surfaces inevitably results in loss of differentiation. Hence, 3D models of the human proximal tubule that recapitulate the in vivo morphology would allow for improved drug screening and disease modeling. Here, the development and characterization of a 3D proximal tubule model using RPTEC/TERT1 cells is presented. RPTEC/TERT1 cells self-assembled in matrigel to form highly differentiated and stable 3D tubular structures characterized by a branched network of monolayered cells encircling a cell-free lumen thus mimicking the proximal tubule. In vitro tubuli resembled the polarity of a proximal tubule epithelium as indicated by polar expression of Na+/K+- ATPase and ZO-3. Furthermore, 3D cultured RPTEC/TERT1 cells showed overall increased mRNA expression of xenobiotic transporters e.g. OCTs and MATEs and de novo expression of OAT3 when compared to cultures on plastics or membrane inserts. Finally, this model was used to assess delayed cisplatin-induced nephrotoxicity and demonstrated increased sensitivity when compared to 2D culture. Thus, the easy-to-use model described here may prove to be useful for mechanistic investigations, e.g. in discovery of compounds interfering with tubule formation, differentiation and polarization, as well for the detection and understanding of pharmaceutical induced nephrotoxicity.