Development of Human Renal Tubular Epithelial Cell Primary Cultures in Monolayers and Three-Dimensional Conditions.

Abstract

Human renal tubular epithelial cell (HRTEC) primary cultures are isolated from kidneys removed from pediatric patients undergoing nephrectomies indicated by the Pediatric Nephrology Unit of the Hospital General de Pediatría Pedro de Elizalde, Buenos Aires, Argentina. Macroscopically, the normal kidney cortex is dissected from the renal medulla and cut into fragments. Cortical fragments are then digested in Hank's solution supplemented with 0.1% collagenase type 1 and filtered through a 70 µm pore size mesh to separate the renal tubules from glomeruli. Renal tubules undergo a second digestion with collagenase to isolate epithelial cells. Finally, the enzyme digestion is stopped, and the epithelial cells are grown in flasks containing RPMI 1640 with supplements under 5% CO2at 37 °C until confluence. HRTEC cells can be grown as monolayers on 96-well plates, glass coverslips into 24-well plates, or cell-culture holders coated with collagen. Also, three-dimensional human renal tubular epithelial cell (3D-HRTEC) cultures are obtained from HRTEC cells seeded on a basement membrane (BM) matrix. Cell aggregation and formation of tubular shape structures are monitored. We have used HRTEC primary cultures for many years to study the cytotoxic action caused by Shiga toxin in the pediatric kidney, which leads to pathophysiological alterations resulting in hemolytic uremic syndrome (HUS). Both HRTEC and 3D-HRTEC cultures can be used to evaluate the effects of different toxins, synthetic drugs, biological factors, hormones, etc, specifically on the human renal proximal tubule epithelium.