Establishment of methods to control growth of primary culture of human renal proximal tubular cells for the bio-artificial kidney device

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To prepare enough amounts of cells for producing bio-artificial kidney devices, antisense technologies were applied to the primary culture of human renal proximal tubular cells (RPTECs). RPTECs become senescent after about 15 divisions in normal culture conditions. To expand the number of cell-division beyond the limit, antisense oligo-DNAs for genes of cell-cycle regulators (p53, RB, p16, p21) were applied to the culture medium at 10 micro-mole / L. As a result, the limit of cell-divisions was increased up to about 25 cycles. It means that about 1000 times of cells cultured in normal medium could be harvested. Furthermore, application of RNAi (RNA interference) for p53 or p16 genes made cells pass through 40 cycles of cell division. About 67 million times of normal cells would be expected to be harvested at the conditions. Cells beyond the normal limit of cell-divisions continued to express gamma-GTP that is a marker of RPTECs. Because these cells are free from virus and onco-genes, they are thought to be suitable for clinical applications. So, methods to control growth of primary culture of RPTECs were established and preparation of enough amounts of functional cells for the bio-artificial kidney device could be achieved. On the other hand, we inoculated human renal cells into hollow fibers in a dialysis module, and connected the module with a goat to evaluate its functions. Transport of water, glucose, and ions between inner and outer of hollows were measured. The circulation of blood was possible to be continued for two weeks. In conclusion, basic technologies for producing the bio-artificial kidney device with the primary culture of RPTECs, and for evaluating renal functions of the device by using model animals were established so far.