Development of a renal microchip for in vitro distal tubule models
Development of a renal microchip for in vitro distal tubule models
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DOI：
10.1021/bp0603513
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发表时间：
2007-09-01
影响因子：
2.9
通讯作者：
Leclerc, Eric
中科院分区：
文献类型：
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作者：
Baudoin, Regis;Griscom, Laurent;Leclerc, Eric
目前组织工程和微技术领域的发展使得相关工具的提出成为可能，微芯片可以用来研究体外毒性。在拟议的REACH欧洲指令和3R建议的框架内，这些MicroTool的目的是模拟体外器官，完善体外培养模型，并最终减少动物试验。微芯片由功能活细胞微室组成，通过微流控网络相互连接，允许通过流体微流控持续的细胞喂养和废物清除。为了验证这种方法，在聚二甲基硅氧烷微芯片中培养了Madin Darby犬肾(MDCK)细胞。为了评估细胞的增殖和摄食，测试了接种细胞的数量从5到10×10(5)个/微芯片(大致相当于2.5到5×10(5)个/cm(2))和4个流速0，10，25和50亩L/分钟。形态观察表明，微芯片内成功地进行了细胞附着和增殖。最佳流速为10亩L/分钟，培养4天，包括灌流3天后，细胞数量增加约2.2+/-0.1倍(与25亩L/分钟的1.7+/-0.2相比)。在10亩L流量下，最大细胞数达到2.1+/-0.2×10(6)(相当于7+/-0.7×10(7)个/cm(3))。通过台盼蓝和乳酸脱氢酶检测，所有实验的活性都在90%以上。在10 mU L/min下，葡萄糖监测显示细胞消耗16+/-2 mU g/h/10(6)个细胞，而谷氨酰胺代谢显示细胞产生约0.8+/-0.4 mU/m ol/d/10(6)个细胞。流速的增加似乎使葡萄糖消耗和NH3产量增加了约1.5-2倍，这与文献中报道的趋势一致。作为对芯片的基本慢性毒性评估，在0、10和25亩L/分钟的流速下，在培养基中添加5 mM和10 mM氯化铵负载。在10亩L/min下，5 mm和10 mm的生长率分别下降35%和50%；在25亩L/min下，5 mm和10 mm的生长率分别下降10%和30%。氯化铵有助于增加葡萄糖的消耗，减少NH3的产生。微芯片的优势，高的比表面积和动态负载，再加上目前关于氨/铵对MDCK影响的文献结果的一致性，说明了我们的芯片在更广泛的体外慢性毒性研究中的潜力。
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 50 mu 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/min). 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.