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首页> 外文期刊>Biotechnology Progress >Development of a Renal Microchip for In Vitro Distal Tubule Models
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Development of a Renal Microchip for In Vitro Distal Tubule Models

机译：用于体外远端小管模型的肾脏芯片的开发

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Current developments in tissue engineering and microtechnology fields have allowed the proposal of pertinent tools,microchips,to investigate in vitro toxicity.In the framework of the proposed REACH European directive and the 3R recommendations,the purpose of these microtools is to mimic organs in vitro to refine in vitro culture models and to ultimately reduce animal testing.The microchip consists of functional living cell microchambers interconnected by a microfluidic network that allows continuous cell feeding and waste removal controls by fluid microflow.To validate this approach,Madin Darby Canine Kidney(MDCK)cells were cultivated inside a polydimethylsiloxane microchip.To assess the cell proliferation and feeding,the number Of inoculated cells varied from 5 to 10 x 10~5 cells/microchip(corresponding roughly to 2.5 to 5 x 10~5 cells/cm~2)and from four flow rates 0,10,25,and 50mu L/min were tested.Morphological observations have shown successful cell attachment and proliferation inside the microchips.The best flow rate appears to be 10 mu L/min with which the cell population was multiplied by about 2.2 ±0.1 after 4 days of culture,including 3 days of perfusion(in comparison to 1.7 ± 0.2 at 25 mu L/mm).At 10 mu L/min flow rate,maximal cell population reached about 2.1 ± 0.2 x 10~6(corresponding to 7 ± 0.7 x 10~7 cells/cm~3).The viability,assessed by trypan blue and lactate deshydrogenase measurements,was found to be above 90% in all experiments.At 10 mu L/min,glucose monitoring indicated a cell consumption of 16 ± 2 mu g/h/10~6 cells,whereas the glutamine metabolism was demonstrated with the production of NH3 by the cells about 0.8 ± 0.4 mu mol/day/10~6 cells.Augmentation of the flow rate appeared to increase the glucose consumption and the NH3 production by about 1.5-to 2-fold,in agreement with the tendencies reported in the literature.As a basic chronic toxicity assessment in the microchips,5 mM and 10 mM ammonium chloride loadings,supplemented in the culture media,at 0,10,and 25 mu L/ min flow rates were performed.At 10 mu L/min,a reduction of 35% of the growth ratio with 5 mM and of 50% at 10 mM was found,whereas at 25 mu L/min,a reduction of 10% with 5 mM and of 30% at 10 mM was obtained.Ammonium chloride contributed to increase the glucose consumption and to reduce the NH3 production.The microchip advantages,high surface/volume ratio,and dynamic loadings,coupled with the concordance between the present and literature results dealing with ammonia/ammonium effects on MDCK illustrate the potential of our microchip for wider in vitro chronic toxicity investigations.
机译：组织工程和微技术领域的最新发展已允许提出相关工具，微芯片来研究体外毒性。在拟议的REACH欧洲指令和3R建议的框架内，这些微工具的目的是在体外模拟器官。完善的体外培养模型并最终减少动物试验。微芯片由通过微流体网络互连的功能性活细胞微腔组成，该微腔网络允许通过流体微流进行连续的细胞喂养和废物清除控制。为验证这种方法，Madin Darby Canine Kidney（MDCK）将细胞培养在聚二甲基硅氧烷微芯片中。为了评估细胞增殖和饲养情况，接种细胞的数量从5到10 x 10〜5个细胞/微芯片不等（大致相当于2.5到5 x 10〜5个细胞/ cm〜2）。分别从四种流速0、10、25和50μL/ min进行了测试。形态学观察表明，细胞成功附着并增殖。最好的流速似乎是10μL / min，在培养4天后，其中包括3天的灌注，细胞群乘以约2.2±0.1（与之相比，在25μL时为1.7±0.2） / mm）。在10μL / min的流速下，最大细胞数达到约2.1±0.2 x 10〜6（相当于7±0.7 x 10〜7细胞/ cm〜3）。通过锥虫蓝和在所有实验中，乳酸脱氢酶的测量值均超过90％。在10μL / min的葡萄糖监测下，细胞消耗为16±2μg / h / 10〜6个细胞，而谷氨酰胺的代谢表明细胞产生的NH3约为0.8±0.4μmol / day / 10〜6个细胞。流速的增加似乎使葡萄糖消耗和NH3的产生增加了约1.5至2倍，与报道的趋势一致作为微芯片中基本的慢性毒性评估，在培养箱中补充了5 mM和10 mM氯化铵。直径在0,10和25μL / min时进行。在10μL / min时，5 mM降低了35％的生长率，而10 mM则降低了50％。 μL / min，5 mM降低10％，10 mM降低30％。氯化铵有助于增加葡萄糖的消耗并减少NH3的产生。微芯片的优势，高表面积/体积比和动态负载，再加上目前与处理氨/铵对MDCK的影响的文献结果之间的一致性，说明了我们的微芯片在更广泛的体外慢性毒性研究中的潜力。

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