Department of Pediatric Nephrology, Radboudumc, Nijmegen, The Netherlands; Department of Physiology, Radboudumc, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands; Department of Pharmacology and Toxicology, Radboudumc, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.
Department of Biomaterials Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands.
Acta Biomater. 2015 Mar;14:22-32. doi: 10.1016/j.actbio.2014.12.002. Epub 2014 Dec 17.
The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney.
对改良肾脏替代疗法的需求刺激了创新性研究，以开发基于细胞的肾脏辅助设备。此类设备的一个关键要求是形成“活膜”，由在合适的人工膜表面上具有保留的功能性有机离子转运蛋白的紧密肾细胞单层组成。在这项工作中，我们应用了一种独特的条件永生化近端小管上皮细胞（ciPTEC）系，并采用优化的涂层策略在聚醚砜（PES）膜上开发了具有功能性近端小管上皮细胞层的活膜。PES 膜用 3,4-二羟基-L-苯丙氨酸和人胶原蛋白 IV（Coll IV）的组合进行涂层。确定了最佳的涂层时间和浓度，以在保留重要血液成分的同时保留高的水传输和最佳的 ciPTEC 粘附性。通过免疫细胞化学检测紧密连接蛋白 1 来检测获得的 ciPTEC 单层。当使用 2 mg ml(-1) 3,4-二羟基-L-苯丙氨酸（4 分钟涂层，1 小时溶解）和 25 μg ml(-1) Coll IV（4 分钟涂层）的组合时，可以形成可重复的单层。开发的活膜系统中（14)C-肌酐的成功转运被用作有机阳离子转运蛋白功能的指标。加入二甲双胍或西咪替丁可显著降低肌酐的跨上皮通量，表明 ciPTEC 中存在活跃的肌酐摄取，这很可能是由有机阳离子转运蛋白 OCT2（SLC22A2）介导的。总之，这项研究表明成功开发了由 PES 膜上可重复的 ciPTEC 单层组成的活膜，这是开发生物人工肾的重要一步。
