心肌细胞传感器优化设计及其药物分析
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摘要
为了构建高度稳定性和一致性的心肌细胞电位传感器,从微电极阵列表面亲水性和细胞培养密度两方面对心肌细胞和微电极阵列(MEA)的耦合性进行研究.通过对MEA表面进行高分子蛋白明胶的修饰来提高MEA表面的亲水性,并重点分析不同细胞密度下构建的心肌细胞电位传感器的胞外场电位信号(EFP)信号特征.研究结果表明:心肌细胞按优化密度12万/cm2培养在经明胶修饰的MEA表面上,可促使心肌细胞和MEA形成高度紧密的耦合.在此条件下构建的心肌细胞传感器,能稳定输出一致性良好的EFP信号,电位幅值可达到约1.2mV,发放频率可达到约180次/min,信号稳定期可维持3~4d.通过选择2种典型的工具药物异丙肾上腺素和利多卡因对优化后的心肌细胞电位传感器进行分析性能的测试,实验结果表明:20μM的异丙肾上腺素和利多卡因分别大幅度增强和抑制了电位幅值和发放频率,结果与文献报导的结果相一致.该心肌细胞传感器对2种测试药物作出了快速而灵敏的响应,有望成为药物检测和分析的有效平台.
The coupling property between cardiomyocytes and microelectrode array(MEA)was studied from two aspects,including MEA surface hydrophilicity and cell culture density,in order to build a cardiomyocyte-based potential biosensor of high stability and high consistency.MEA surface hydrophilicity was improved by high molecular protein(gelatin)modification,and the characteristics of extracellular field potential(EFP)signals of cardiomyocyte-based potential biosensor under different cell densities were emphatically analyzed.Results show that the high-closely coupling between cardiomyocytes and MEA is formed after cardiomyocytes seeded on the MEA with gelatin modification at the proper cell density of1.2×105 cells/cm2.Under the optimal conditions,the cardiomyocyte-based potential biosensor presents high-stable and high-consistent EFP signals,the spike amplitude(SA)reaches about 1.2 mV,and the firing rate(FR)reaches about 180beats/min,which continues for 3-4days.Two typical tool drugs,isoprotenol(ISO)and lidocaine(LID)were applied to test the analytical performance of the optimized cardiomyocyte-based potential biosensor.Results show that SA and FR markedly increase after the treatment of 20μM ISO for 5min and significantly decrease after the treatment of 20μM LID for 5min.This biosensor performed rapid and sensitiveresponse to these two drugs and will provide a promising platform for drug detection and analysis.
The coupling property between cardiomyocytes and microelectrode array(MEA)was studied from two aspects,including MEA surface hydrophilicity and cell culture density,in order to build a cardiomyocyte-based potential biosensor of high stability and high consistency.MEA surface hydrophilicity was improved by high molecular protein(gelatin)modification,and the characteristics of extracellular field potential(EFP)signals of cardiomyocyte-based potential biosensor under different cell densities were emphatically analyzed.Results show that the high-closely coupling between cardiomyocytes and MEA is formed after cardiomyocytes seeded on the MEA with gelatin modification at the proper cell density of1.2×105 cells/cm2.Under the optimal conditions,the cardiomyocyte-based potential biosensor presents high-stable and high-consistent EFP signals,the spike amplitude(SA)reaches about 1.2 mV,and the firing rate(FR)reaches about 180beats/min,which continues for 3-4days.Two typical tool drugs,isoprotenol(ISO)and lidocaine(LID)were applied to test the analytical performance of the optimized cardiomyocyte-based potential biosensor.Results show that SA and FR markedly increase after the treatment of 20μM ISO for 5min and significantly decrease after the treatment of 20μM LID for 5min.This biosensor performed rapid and sensitiveresponse to these two drugs and will provide a promising platform for drug detection and analysis.
引文
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[2]LAVERTY H,BENSON C,CARTWRIGHT E,et al.How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines[J].British Journal of Pharmacology,2011,163(4):675-693.
[3]BOWES J,BROWN A J,HAMON J,et al.Reducing safety-related drug attrition:the use of in vitro pharmacological profiling[J].Nature Reviews Drug Discovery,2012,11(12):909-922.
[4]LEXCHIN J.Drug withdrawals from the Canadian market for safety reasons,1963-2004[J].Canadian Medical Association Journal,2005,172(6):765-767.
[5]MOSS A J,KASS R S.Long QT syndrome:from channels to cardiac arrhythmias[J].Journal of Clinical Investigation,2005,115(8):2018-2024.
[6]BRAAM S.R,TERTOOLEN L,VAN DE STOLPE A,et al.Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes[J].Stem Cell Research,2010,4(2):107-116.
[7]GIORGI M A,BOLANOS R,GONZALEZ C D,et al.QT interval prolongation:preclinical and clinical testing arrhythmogenesis in drugs and regulatory implications[J].Current Drug Safety,2010,5(1):54-57.
[8]NATARAJAN A,STANCESCU M,DHIR V,et al.Patterned cardiomyocytes on microelectrode arrays as a functional,high information content drug screening platform[J].Biomaterials,2011,32(18):4267-4274.
[9]HANCOX J C,MCPATE M J,EL HARCHI A.The hERG potassium channel and hERG screening for druginduced torsades de pointes[J].Pharmacology and Therapeutics,2008,119(2):118-132.
[10]HANSEN A,EDER A,BNSTRUP M,et al.Development of a drug screening platform based on engineered heart tissue[J].Circulation Research,2010,107(1):35-44.
[11]CHI K R.Revolution dawning in cardiotoxicity testing[J].Nature Reviews Drug Discovery,2013,12(8):565-567.
[12]ABASSI Y A,XI B,LI N,et al.Dynamic monitoring of beating periodicity of stem cell‐derived cardiomyocytes as a predictive tool for preclinical safety assessment[J].British Journal of Pharmacology,2012,165(5):1424-1441.
[13]MANDENIUS C F,STEEL D,NOOR F,et al.Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics:a review[J].Journal of Applied Toxicology,2011,31(3):191-205.
[14]FERMINI B,FOSSA A A.The impact of drug-induced QT interval prolongation on drug discovery and development[J].Nature Reviews Drug Discovery,2003,2(6):439-447.
[15]XIAO L,HU Z,ZHANG W,et al.Evaluation of doxorubicin toxicity on cardiomyocytes using a dual functional extracellular biochip[J].Biosensors and Bioelectronics,2010,26(4):1493-1499.
[16]MEYER T,KRAUSHAAR U,GUENTHER E.Microelectrode Array(MEA)high resolution electrophysiological mapping of cardiac cell,tissue and organ preparations[C]∥2009 World Congress on Medical Physics and Biomedical Engineering.Munich:IFMBE,2009:112-115.
[17]XU B,JACQUIR S,LAURENT G,et al.Phase space reconstruction of an experimental model of cardiac field potential in normal and arrhythmic conditions[C]∥2013 Annual International Conference of the IEEE.Osaka:IEEE,2013:3274-3277.
[18]KORNBLUM A,PILLEKAMP F,MATZKIES M,et al.A new model to perform electrophysiological studies in the early embryonic mouse heart[J].Cellular Physiology and Biochemistry,2013,32(1):1-10.
[19]JOHNSTONE A F,GROSS G W,WEISS D G,et al.Microelectrode arrays:aphysiologically based neurotoxicity testing platform for the 21st century[J].Neurotoxicology,2010,31(4):331-350.
[20]CASPI O,ITZHAKI I,KEHAT I,et al.In vitro electrophysiological drug testing using human embryonic stem cell derived cardiomyocytes[J].Stem Cells and Development,2009,18(1):161-172.
[21]徐莹,余辉,张威,等.基于MEMS技术的微电极阵列细胞传感器[J].自然科学进展,2007,17(9):1265-1272.XU Yin,YU Hui,ZHANG Wei,et al.Microelectrode array cell sensor based on MEMS technology[J].Process in Natural Science,2007,17(9):1265-1272.
[22]KANEKO T,NOMURA F,HATTORI A,et al.Improvement of electrical stimulation protocol for simultaneous measurement of extracellular potential with onchip multi-electrode array system[J].Japanese Journal of Applied Physics,2012,51(6S):06FK02.
[23]GUO L,ABRAMS R M,BABIARZ J E,et al.Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes[J].Toxicological Sciences,2011,123(1):281-289.
[24]ZOU Y,YAO A,ZHU W,et al.Isoproterenol activates extracellular signal-regulated protein kinases in cardiomyocytes through calcineurin[J].Circulation,2001,104(1):102-108.