Establishment of a 3-Dimensional Renal Tubular Cell Culture Model

In order to establish a cell culture system mimicking the 3-diamensional structures of renal tubule to study the pathophysiology of renal injury, distal tubular cell line, MDCK cell, was growing in the capillary fiber (CELLMAX QUAD, Cellco Incorp.) and perfused with medium at a constant flow. Basic experiments for establishment of this unique culture model were done to ensure that this system could provide as a useful system for the purpose of study. 1)Pressure measurement inside the capillary fiber: Pressure in the lumen of culture tubing perfusing with culture medium at different rate from Scale 1 through 6 showed increasing as 6, 14, 20, 28, 36, 42 mmHg. Respectively. 2) Cell metabolic condition: We sued the glucose and lactate metabolic rate to know the growing condition of MDCK cells in the fiber. The glucose concentration minimal for cell growth was kept not below 2.1mg/ml and the lactate concentration was kept not above 1.69 mg/ml.3) Cell viability study: After each experiment all the samples were studied for the viability of cell harvested from the capillary by trypan blue method. The results showed more than 90% of harvested cell were alive at each experiment. 4) Morphological study of cell growing inside the capillary: We coated the capillary lumen with gelatin by perfusing the fiber first with medium containing 0.1% gelatin. It resulted in a very well single layer cell within capillary lumen. The SCM showed the surface of MDCK cell in the flask showed less 3-D change in comparison to those cultured in capillary. It showed a single layer with cell attached to each other. The TEM showed that the cell morphology which had small villi over the apical surface and mitochondria over the within the cytoplasm. There were several vesicles transporting from the apical side toward basolateral side, and the cell and cell junction contained free space. The fiber was made of polypropylene with defined pore size. MDCK cells grew on the gelatin-Coated surface with small projection inserted into the fiber. From the above findings we confirmed that the growing of MDCK cells within a 3-D capillary fiber perfusing with medium at constant speed could successfully created a environment which was similar to the in vivo renal tubule, thus could be used for further study.