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

生物人工肾装置用人肾近端肾小管细胞原代培养生长控制方法的建立

• 批准号: 18590910
• 负责人: SAWADA Kaichiro
• 金额: $ 2.57万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590910/
• 关键词: bio-artificial kidney device; renal proximal tubul cells; the limit on cell division; and-sense technology; RNA interference; hollow-fiber modules; ヒト近位尿細管上皮細胞; バイオ人工尿細管; ヒト尿細管上皮細胞; アンチセンスオリゴDNA; 細胞周期調節因子; テロメラーゼ; 細胞株化

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.

为了制备足够数量的细胞用于生产生物人工肾装置，应用反义技术对人肾近端小管细胞（rptec）进行原代培养。在正常培养条件下，约15个细胞后，rptec开始衰老。为了扩大细胞分裂次数，将细胞周期调控基因（p53、RB、p16、p21）的反义寡聚dna以10微摩尔/ l的浓度加入培养基中，细胞分裂极限提高到25个周期左右。这意味着可以收获在正常培养基中培养的大约1000倍的细胞。此外，对p53或p16基因应用RNAi （RNA干扰）使细胞经历40个细胞分裂周期。在这种条件下，预计将收获大约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rptec的标记物。由于这些细胞不含病毒和癌基因，因此它们被认为适合临床应用。因此，建立了控制rptec原代培养细胞生长的方法，可以为生物人工肾装置制备足够数量的功能细胞。另一方面，我们将人类肾细胞接种到透析模块的中空纤维中，并将该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内部和外部之间的运输。血液循环可以持续两个星期。综上所述，目前已经建立了用rptec原代培养制备生物人工肾装置的基本技术，以及用模型动物评价该装置肾功能的基本技术。