Tubuloid Production from Primary Cultured Renal Tubular Epithelial Cells Derived from Resected Unfunctional Kidneys of Patients on Dialysis

Background: Renal tubular organoids (tubuloids) developed by our technology have the potential to serve as an alternative, more human-like model of human pathology. Tubuloids are made from patient kidney-derived primary cells, not stem cells, and mimic kidney tubules. We have previously produced tubuloids from normal and mild chronic kidney disease (CKD) kidneys. Here, cells from unfunctional kidneys of dialysis patients will be 3D engineered. Methods: 5 patients receiving hemodialysis and 5 patients with normal kidney function were joined to this study with written informed consents. Using kidneys obtained from patients by surgery for malignancy, only primary cultures of renal tubular epithelial cells were selectively grown. Growth factors and Matrigel were then added. After several days of culture, tubuloids are obtained. In primary cultured cells in 2-dimensional condition and tubuloids, Western blotting and immunostaining are used to determine renal tubular and CKD properties. Results: Primary cultured renal epithelial cells of normal kidneys show a large number of fine, swollen epithelial cells. On the other hand, those of unfunctional kidneys from dialysis patients are elongated, and some cells are giant in size. At the beginning of 3-dimentional culture, the speed of growth of normal tubuloids is faster than that of renal cells from unfunctional kidneys. Eventually, however, the size difference between normal renal tubuloids and tubuloids from dialysis patients is not very discernible. Western blotting of the completed tubuloids showed that p16, a marker of cellular senescence, was predominantly expressed in the dialysis tubuloids. KIM-1, which appears in damaged kidneys, was confirmed in two of the dialysis tubuloids strongly. Conclusion: We have established primary renal epithelial cells from patient kidneys derived from healthy kidneys and from unfunctional kidneys of hemodialysis patients, and now we have successfully produced tubuloids derived even from unfunctional kidneys. The results also revealed that tubuloids derived from unfunctional kidneys have the properties of cellular senescence. These tubuloids are expected to be used as a simple model for a new type of human pathology, more similar to humans than mice. Funding: Private Foundation Support, Government Support – Non-U.S.