Development and characterization of 3D models based on urine derived renal epithelial cells

Background and Aims Three-dimensional (3D) culture techniques represent a significant evolution from traditional monolayer culture methods for cell biology and regenerative medicine studies. These cell models provide a more accurate representation of human tissues, allowing a higher reliable simulation of cell-cell and cell-extracellular matrix (ECM) interactions and of the biological processes occurring in both physiological and pathological conditions. Different cell types can be combined into hybrid and multicellular 3D structures, to study how they interact and distribute. Urine derived renal epithelial cells (URECs) are a heterogeneous cells population voided in urine from the upper urinary tract, and particularly from proximal tubule; these cells are rarely detected in healthy subjects, while their number increases in presence of acute kidney damage and after kidney transplant as a response to ischemia reperfusion events and the increased cell turnover. In particular, we previously described URECs derived from kidney transplanted patients, focusing on their phenotype and their immunomodulatory properties in traditional monolayer culture [1, 2]. Here, we aimed to establish a 3D model combining URECs derived from kidney transplanted patients with Wharton's jelly mesenchymal stromal cells (WJ-MSCs), to evaluate structure, cell distribution and viability of the newly generated spheroids. For a functional characterization of the 3D model, the immunomodulatory properties were analysed by coculturing spheroids with peripheral blood mononuclear cells (PBMCs); the proliferation of CD4 and CD8 T cells, as well as the percentage of Treg cells and Granzyme B positive cells were evaluated after coculture. Method URECs and WJ-MSCs were combined to obtain spheroids by seeding cells in ultra low attachment (ULA) plates, allowing cell aggregation in 24 h Different condition and ratio were tested: URECs or WJ-MSCs only, URECs:WJ-MSCs 1:1 and 2:1 ratio. Spheroids formation and cell distribution were analysed using Incucyte, and by staining URECs and WJ-MSCs with different fluorochromes. The viability was assessed by Live/Dead assay at different time point. Spheroids were then cultured with PBMCs, and after 72 h immune cells were analysed by flow cytometry, comparing the results with activated PBMCs set as control. Results Stable spheroids were obtained combining URECs and WJ-MSCs (Fig. 1a and b), with a higher viability compared to WJ-MSCs spheres, while URECs alone could not self-aggregate (Fig. 1a, b and d). URECs were mostly located in the core of the 3D model, while WJ-MSCs were in the outer layer (Fig. 1c). During coculture with activated PBMCs (Fig. 2a), hybrid spheroids in both 1:1 and 1:2 ratio, significantly reduced CD4 and CD8 T cell proliferation (Fig. 2b), while WJ-MSCs only spheroids do not affect the proliferation rate, compared to activated PBMCs cultured without spheroids. For what concerns specific T cell subsets, hybrid spheroids reduced the percentage of Granzyme B producing cells, while increasing the CD4+CD25+FoxP3+ regulatory T cell subset (Fig. 2c). Conclusion The date obtained in our study indicate a long term stability and viability of UREC:WJ-MSCs spheroids, with a specific cell distribution into the 3D structure. Moreover, these 3D models showed promising immunomodulatory properties during coculture with PBMCs, which need to be further assessed by evaluating their effect on other immune cell subset including B cell and monocytes. These findings may provide the background for studies testing unknown effects of this new hybrid 3D cell model for cell therapy and regenerative medicine applications.