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

为了制备足够数量的细胞以制造生物人工肾装置，将反义技术应用于人肾近端小管细胞的原代培养。在正常培养条件下，RPTECs在大约15次分裂后衰老。为了扩大细胞分裂数，细胞周期调控基因(p53、Rb、p16、p21)的反义寡核苷酸以10微摩尔/L的浓度加入到培养液中，使细胞分裂极限提高到约25个周期。这意味着在正常培养液中培养的细胞可获得约1000倍的细胞。此外，对p53或p16基因的RNAi(RNA干扰)的应用使细胞经历了40个细胞分裂周期。在这种条件下，预计将获得约6700万倍的正常细胞。超过正常细胞分裂极限的细胞继续表达γ-GTP，这是RPTECs的一个标志。由于这些细胞没有病毒和癌基因，它们被认为适合临床应用。因此，建立了控制RPTECs原代培养生长的方法，为生物人工肾装置制备足够数量的功能细胞奠定了基础。另一方面，我们将人肾细胞接种到透析器中的中空纤维中，并将透析器与山羊连接，以评估其功能。测量了水分、葡萄糖和离子在空穴内外的传输。血液循环有可能持续两周。综上所述，目前已经建立了利用RPTECs原代培养生产生物人工肾装置的基本技术，并利用模型动物评价了该装置的肾功能。

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.