Introduction: An estimated 10% of North Americans suffer from nephrolithiasis. With rising incidence rates, there is a strong need for models to study the pathophysiology of stone disease. In vitro cell culture models are essential for mechanistic and high-throughput analysis of stone formation and drug treatment; however, there are no suitable models that adequately represent the pathophysiology of stone disease. Advancements in cell culture have given rise to three-dimensional cysts, which are hollow spheroid structures that have apical to basal polarization. These culture systems have similar physiochemical environments to in vivo models and can be cultured for the extended times required for stone formation. Here, we developed a novel 3D kidney cyst model that can generate calcium-based kidney stones. Methods: Madin-Darby canine kidney (MDCK) cells were cultured in suspension in growth factor-reduced Matrigel to generate hollow spheroid cysts. Crystal formation was induced by culturing cysts in a custom crystal-forming media for two weeks. Immunofluorescent staining was used for characterizing and visualizing the three-dimensional structure. Alendronate-fluorescein, a fluorescently labelled bisphosphonate that specifically binds to calcium-containing crystals, was used along with plain polarized light to visualize the crystal structures. Results: Stains for cellular components and structures demonstrated that the luminal spheroids were successfully formed under the culture conditions. The spheroids also have basal-to-apical polarization suggesting that crystals will form on the inside lumen of the hollow cyst. Birefringent structures resembling calcium oxalate dihydrate and calcium phosphate crystals were observed embedded along the apical face of the spheroid and protruding into the lumen. Alendronate-fluorescein staining confirmed the presence of calcium-containing crystals in the lumen of the cyst. Conclusions: We have demonstrated that hollow MDCK spheroids cultured with crystal-forming media can generate calcium-containing crystals in the spheroid lumen. This model is the first in vitro tissue culture-based model to reliably develop calcium-based crystals and can be used to investigate their pathogenesis or to perform high-throughput drug screening. Furthermore, it paves the way for personalized medicine by building the foundation for a cell system where renal tissue can be harvested and cultured to identify individual intrinsic risk factors that might contribute to stone development. SOURCE OF Funding: Canadian Institutes of Health Research (CIHR) CGS-D, The Northeastern Section of the American Urological Association (NSAUA), Lawson Health Research
.jpg "John Chmiel, MSc,BS photo")