Topographically-patterned porous membranes in a microfluidic device as an in vitro model of renal reabsorptive barriers.
Topographically-patterned porous membranes in a microfluidic device as an in vitro model of renal reabsorptive barriers.
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DOI：
10.1039/c3lc50199j
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发表时间：
2013-06-21
期刊：
影响因子：
6.1
通讯作者：
Charest JL
中科院分区：
文献类型：
--
作者：
Frohlich EM;Alonso JL;Borenstein JT;Zhang X;Arnaout MA;Charest JL
重吸收屏障模型需要一种手段来提供现实的生理线索，并量化跨细胞层形成的障碍。这里我们有几个用户定义的模式类型的地形图案化的多孔膜。为了证明图案化膜的实用性，我们选择了一种类型的图案，并将其应用于膜，以作为肾重吸收屏障的微流体模型中的细胞培养支持物。模型中的地形线索类似于体内发现的生理线索，而多孔结构允许量化跨细胞层的运输。通过热压印在轨道蚀刻聚碳酸酯膜上产生亚微米表面形貌，完全复制形貌特征并保留多孔结构。利用扫描电镜和图像分析仪分析了孔的大小和形状，以确定热压对孔形态的影响。将膜组装成双层微流控装置，并将人肾近端小管上皮细胞系（HK-2）和原代肾近端小管上皮细胞（RPTEC）培养至在膜上汇合。两种细胞类型的免疫荧光染色显示了指示对机械刺激有反应的重吸收屏障形成的蛋白质表达：ZO-1（紧密连接）、桩蛋白（粘着斑）和乙酰化α-微管蛋白（初级纤毛）。HK-2和RPTEC在装置中膜的脊/槽形貌方向上对齐，证明装置对细胞反应具有机械控制。这种形貌图案化的多孔膜提供了一个体外平台，在其上模拟重吸收屏障，具有理解生物转运现象、潜在疾病机制和药物毒性的有意义的应用。
Models of reabsorptive barriers require both a means to provide realistic physiologic cues to and quantify transport across a layer of cells forming the barrier. Here we have topographically-patterned porous membranes with several user-defined pattern types. To demonstrate the utility of the patterned membranes, we selected one type of pattern and applied it to a membrane to serve as a cell culture support in a microfluidic model of a renal reabsorptive barrier. The topographic cues in the model resemble physiological cues found in vivo while the porous structure allows quantification of transport across the cell layer. Sub-micron surface topography generated via hot-embossing onto a track-etched polycarbonate membrane, fully replicated topographical features and preserved porous architecture. Pore size and shape were analyzed with SEM and image analysis to determine the effect of hot embossing on pore morphology. The membrane was assembled into a bilayer microfluidic device and a human kidney proximal tubule epithelial cell line (HK-2) and primary renal proximal tubule epithelial cells (RPTEC) were cultured to confluency on the membrane. Immunofluorescent staining of both cell types revealed protein expression indicative of the formation of a reabsorptive barrier responsive to mechanical stimulation: ZO-1 (tight junction), paxillin (focal adhesions) and acetylated α-tubulin (primary cilia). HK-2 and RPTEC aligned in the direction of ridge/groove topography of the membrane in the device, evidence that the device has mechanical control over cell response. This topographically-patterned porous membrane provides an in vitro platform on which to model reabsorptive barriers with meaningful applications for understanding biological transport phenomenon, underlying disease mechanisms, and drug toxicity.