RTCA技术原理及应用

郑敏xCELLigence技术的原理及其应用简介杭州高通量新药筛选中心浙江大学医学院附属一院©2012ACEABIO–ProprietaryandConfidentialACEABIO•简介–传统基于细胞水平的研究方法的局限性–ACEA公司的解决方法•技术平台:基于微电子传感器技术，通过实时监测阻抗的变化来进行实时的细胞分析–操作原理:实时细胞分析–我们监测的指标–xCELLigence系列产品–RTCA检测的特点–应用范例–独特的应用：RTCA心脏系统•总结:RTCA技术的优势xCELLigence技术介绍大纲©2012ACEABIO–ProprietaryandConfidentialACEABIO传统细胞毒性/活力检测方法工作原理及检测流程细胞培养MTTCVKT加入MTT孵育加入有机溶剂孵育加入CVTK孵育测吸光度数据处理分析单个时间点信息结果反复，不稳定新的未知蛋白/药物/基因的作用难以优化条件耗费大量时间，经费重复实验，优化条件没有直观信息做问题分析©2012ACEABIO–ProprietaryandConfidentialACEABIO00.511.522.533.5020406080100Time(hours)NormalizedCI10uM10.12CtrlDNADamaging-7.0-6.5-6.0-5.5-5.0-4.5-4.00.00.10.20.30.40.5Log[5-FU]MAbsorbance(540/620nm)DNADamagingRTCA实时动态监测细胞毒性反应检测流程细胞接种细胞增殖外界因子加入细胞效应RTCAMTT实时无标记全自动©2012ACEABIO–ProprietaryandConfidentialACEABIO细胞接种药物处理数据分析自动实时动态检测及数据分析RTCA实时无标记检测细胞接种药物处理标记&终点检测传统终点检测数据分析1-24小时BlackBox细胞毒实验-传统终点VS实时无标记检测©2012ACEABIO–ProprietaryandConfidentialACEABIO©2012ACEABIO–ProprietaryandConfidentialACEABIO微电子阻抗感应器在细胞生物学中的应用©2012ACEABIO–ProprietaryandConfidentialACEABIOZZ=Z0Z=ZcellAddCellselectrodeswithoutcellselectrodesattachedwithcells检测原理基于微电子阻抗技术的细胞检测技术Z©2012ACEABIO–ProprietaryandConfidentialACEABIO细胞指数和细胞数的关系160008000500100020004000125250CellNumberCellNumberCellIndex8.34.61.90.90.510.30.220.13CellIndex©2012ACEABIO–ProprietaryandConfidentialACEABIO00.20.40.60.810min5min20minTimeCellIndex0min5min20min细胞指数和细胞粘附力及形态的关系©2012ACEABIO–ProprietaryandConfidentialACEABIOxCELLigence系统系列产品RTCA1*96RTCA6*96RTCA3*16&CIMRTCA1*384RTCARTCAHTSystemRTCACardioInstrument©2012ACEABIO–ProprietaryandConfidentialACEABIOxCELLigence系统系列产品的匹配电子板©2012ACEABIO–ProprietaryandConfidentialACEABIO012345678901020304050Time(Hours)CellIndex10000HT108010000HT1080+Paclitaxel5000HT10805000HT1080+PaclitaxelTreatmentTreatment时间点选择在细胞试验中重要性•不同的细胞状态对于处理因素有不同的反应•RTCA系统使接种细胞后的对于细胞的检测可持续进行•RTCA为最佳处理时间的选择提供了依据©2012ACEABIO–ProprietaryandConfidentialACEABIOTraditionalCellAnalysisMethodRT-CAAssay不同时间点肿瘤细胞对抗肿瘤药物的反应性Vehicle33nM67nM100nM150nM230nM340nM500nM01234567CTR33nM67nM100nM150nM230nM340nM500nMConcentrationCellIndex0123456020406080100Time(hr)ResponseVehicle33nM67nM100nM150nM230nM340nM500nM©2012ACEABIO–ProprietaryandConfidentialACEABIOxCELLigence系统的应用•细胞增殖和细胞毒性试验•细胞贴壁和铺展•受体-配体间相互作用–RTKs–GPCRs–IgEreceptor•NK细胞介导的杀伤肿瘤细胞•肿瘤细胞的迁移和侵袭•siRNA文库筛选•心脏细胞功能和毒性监测•病毒CPE及中和实验的监测•细菌毒素介导的细胞病变的监测•……©2012ACEABIO–ProprietaryandConfidentialACEABIO利用xCELLigence系统进行细胞质控SHSY5Y012345020406080Time(hours)CellIndex10K5K2.5K1.25KGTL116012345020406080Time(hours)CellIndex10K5K2.5K1.25K012345020406080Time(hours)CellIndex10K5K2.5K0.63K012345020406080Time(hours)CellIndex10K5K2.5K0.63KA549PC3©2012ACEABIO–ProprietaryandConfidentialACEABIO00.511.522.533.544.550102030405060Time(hours)CellIndex10K5K2.5K1.25K0123450102030405060Time(hours)CellIndex10K5K2.5K1.25K0123450102030405060Time(hours)CellIndex10K5K2.5K1.25K0123450204060Time(hours)CellIndex10K5K2.5K1.25K0123456780102030405060Time(hours)CellIndex10K5K2.5K1.25K“HeLa”细胞生长曲线来源于4个不同的实验室和ATCCATCCLab1*Lab2*Lab3Lab4*Theselabswerefromthesameinstitute©2012ACEABIO–ProprietaryandConfidentialACEABIO应用xCELLigence系统研究TCRP01234567020406080Time(hours)NormalizedCI0.56uM0.19uM0.06uM0.02uMCtrl01234567020406080Time(hours)NormalizedCI50uM16.7uM5.6uM1.9uMCtrlStaurosporine5-Fluorouracil01234567010203040506070Time(Hours)NormalizedCellIndexControlDNADamagingAnti-mitoticCytostaticCytoskeletalTimedependentcellularResponseProfiling，TCRP时间依赖的细胞反应特征性图谱©2012ACEABIO–ProprietaryandConfidentialACEABIOTCRPwithKnownMechanisms00.511.522.533.5020406080Time(hours)NormalizedCICtrlColchicine012345020406080Time(hours)NormalizedCICtrlEtoposide01234567020406080Time(hours)NormalizedCICtrlHydrocortisone01234020406080Time(hours)NormalizedCICtrlEmetine012345020406080Time(hours)NormalizedCICtrlMethiothepin01234020406080Time(hours)NormalizedCICtrlAmlodipine01234567020406080100Time(hours)NormalizedCICtrlTrichostatinAShort-TermResponseLong-TermResponseAnti-mitoticsProtein-SynthesisDNADamagingNuclearHormoneHDACInhibitorsSerotoninReceptorAntagonistL-TypeVoltage-gatedCaChannelInhibitor01234020406080Time(hours)NormalizedCICtrlAzelastineAnti-Histamine©2012ACEABIO–ProprietaryandConfidentialACEABIO00.511.522.533.5020406080Time(hours)NormalizedCICtrlColchicine012345020406080Time(hours)NormalizedCICtrlEtoposide01234567020406080Time(hours)NormalizedCICtrlHydrocortisone01234020406080Time(hours)NormalizedCICtrlEmetine012345020406080Time(hours)NormalizedCICtrlMethiothepin01234020406080Time(hours)NormalizedCICtrlAmlodipine01234567020406080100Time(hours)NormalizedCICtrlTrichostatinAShort-TermResponseLong-TermResponseAnti-mitoticsProtein-SynthesisDNADamagingNuclearHormoneHDACInhibitorsSerotoninReceptorAntagonistL-TypeVoltage-gatedCaChannelInhibitor01234020406080Time(hours)NormalizedCICtrlAzelastineAnti-HistamineTCRPwithKnownMechanisms©2012ACEABIO–ProprietaryandConfidentialACEABIO利用TCRP进行药物筛选Seed4000A549CancerCellsin96wellE-PlatesTreatwithCompoundsatafinalconcentrationOf20mMMonitorthecellularresponsefor48hoursComparecytologicalprofilesOOOHOHOHOE-PlateAddCellsAddCompoundReal-TimeContinuousMon