Microfluidic cell culture and its application in high-throughput drug screening: cardiotoxicity assay for hERG channels.
Microfluidic cell culture and its application in high-throughput drug screening: cardiotoxicity assay for hERG channels.
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DOI：
10.1177/1087057110386218
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发表时间：
2011-01
影响因子：
--
通讯作者：
Beebe DJ
中科院分区：
文献类型：
--
作者：
Su X;Young EW;Underkofler HA;Kamp TJ;January CT;Beebe DJ
药物心脏毒性评价对新药的安全开发至关重要。评估一种化合物对表面心电图QT间期延长的风险，从而对危及生命的心律失常的风险进行评估，几乎在所有新药获得批准之前都是必须的。QT间期延长通常与hERG钾离子通道的电流损失有关，这是由于药物直接阻断离子通道或偶尔抑制通道蛋白的质膜表达。为了开发一种高效、可靠、具有成本效益的hERG筛选方法来检测药物介导的hERG膜运输破坏，我们展示了基于微流体的系统的使用，以提高现有方法的吞吐量和降低成本。我们通过培养稳定转染过表达hERG的HEK细胞（WT-hERG），并研究其形态、增殖率、hERG蛋白表达和对药物治疗的反应，验证了聚苯乙烯（PS）、环烯烃聚合物（COP）和聚二甲基硅氧烷（PDMS）微通道中微流体阵列平台对药物诱导的hERG运输的破坏。我们的研究结果表明，WT-hERG细胞在PS、COP和PDMS微流体通道中容易增殖。我们证明，使用从单个微通道中提取的细胞裂解液，传统的Western blot分析是可能的。Western blot分析也提供了重要证据，证明在微通道中培养的WT-hERG细胞保持了正常（孔板为基础）的hERG表达。我们进一步表明，通过使用直接通道内免疫荧光染色与使用红外扫描仪检测相结合，可以简化实验程序。最后，用五种不同的药物处理WT-hERG细胞表明，PS（和COP）微通道比PDMS微通道更适合药物筛选应用，特别是涉及疏水药物分子的测试。
Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval, and hence risk for life threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, we demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. We validate our microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP) and poly(dimethylsiloxane) (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG), and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Our results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. We demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. We further showed that experimental procedures can be streamlined by using direct in-channel immunofluorescent staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with five different drugs suggested that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.
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影响因子：
3.4
作者：
Cheng, CS;Alderman, D;Yu, WF
通讯作者：
Yu, WF
影响因子：
5
作者：
Finlayson, K;Turnbull, L;Kelly, JS
通讯作者：
Kelly, JS
影响因子：
120.1
作者：
Orloff, John;Douglas, Frank;Golub, Howard
通讯作者：
Golub, Howard
影响因子：
--
作者：
Dubin, AE;Nasser, N;Mathes, C
通讯作者：
Mathes, C
影响因子：
1.8
作者：
Paguirigan, Amy L.;Puccinelli, John P.;Beebe, David J.
通讯作者：
Beebe, David J.