Boston University, Department of Mechanical Engineering, Boston, MA 02215, USA.
Lab Chip. 2013 Jun 21;13(12):2311-9. doi: 10.1039/c3lc50199j. Epub 2013 May 2.
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.
模型的吸收性屏障需要一种手段来提供现实的生理线索，并量化穿过形成屏障的细胞层的运输。在这里，我们有拓扑图案的多孔膜和几种用户定义的图案类型。为了演示图案膜的实用性，我们选择了一种类型的图案，并将其应用于膜中，作为肾吸收性屏障微流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穿过细胞层的运输。通过在聚碳酸酯膜上进行热压印，可以在亚微米表面上生成拓扑结构，完全复制了拓扑特征并保留了多孔结构。通过 SEM 和图像分析分析孔径和形状，以确定热压印对孔形态的影响。将膜组装成双层微流控装置，并在膜上培养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）至融合。两种细胞类型的免疫荧光染色显示出形成对机械刺激有反应的吸收性屏障的蛋白质表达：ZO-1（紧密连接），桩蛋白（焦点粘连）和乙酰化α-微管蛋白（初级纤毛）。HK-2 和 RPTEC 在装置中膜的脊/槽拓扑结构的方向上排列，这表明该装置对细胞反应具有机械控制。这种图案化多孔膜提供了一个体外平台，可以模拟吸收性屏障，对于理解生物传输现象、潜在疾病机制和药物毒性具有重要意义。
