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

为制备足够数量的细胞用于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（RPTECs）的原代培养。RPTEC在正常培养条件下约15次分裂后衰老。为了扩大细胞分裂的数量超过极限，将细胞周期调节基因（p53，RB，p16，p21）的反义寡核苷酸以10微摩尔/ L施加到培养基中。结果，细胞分裂的极限增加到约25个循环。这意味着在正常培养基中培养的细胞可收获约1000倍。此外，针对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，预计将收获约6700万倍的正常细胞。超过细胞分裂正常极限的细胞继续表达γ-GTP，其是RPTEC的标志物。由于这些细胞不含病毒和癌基因，因此被认为适合临床应用。因此，建立了RPTECs原代培养的生长控制方法，为制备足够数量的生物人工肾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模块的中空纤维中，并将模块与山羊连接以评估其功能。测量了水、葡萄糖和离子在中空结构内外之间的传输。血液循环可以持续两周。综上所述，迄今为止，建立了利用RPTECs原代培养生产生物人工肾装置以及使用模型动物评价装置的肾功能的基本技术。

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.