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微米晶片在細胞生物學上的應用•細胞研究:•細胞研究的方向。•細胞生長環境。•細胞微環境的因素。•生物科技的劣勢。演講者:陳俊男07.27.2005生物技術•DNA:agarose電泳(McDonell,1977),Southernblot,clone,PCR(1971).•RNA:RT-PCR(1988),Northernblot(1994).•Protein:SDSpage(1960),westernblot(1979)•Proteomics:2DElectrophoresis(1970’s).ProteomicsProcesses生物科技的缺點:1.耗時2.費人力3.高成本4.無法在微米尺度細操控細胞,細菌5.無法及時觀徹細胞生理狀況(realtime)微米級晶片的新工具--微流道•微流道晶片(lab-On-aChip)•陣列式晶片•晶片的應用55°C.72°C95°C.cBLOOD-ON-A-CHIPC.rightpole:MitotrackerGreenFM;leftpole:MitotrackerRedCM-H2XRos.entirecell:DNA-bindingdyeHoechst33342.D.2.5hrslaterE..Blueregion:latrunculin=causethedisruptionofactinfilamentF.Phalloidin–Alexa594-labelled,and10minlatter,treatmentwithlatrunculinA.Figure1Chemotaxis.Figure2ElastomericMembranes,(Bovineadrenalcapillaryendothelialcell)Figure3.(a)Acellspreadonasquare30um30umisland;thecellwasstimulatedwithplatelet-derivedgrowthfactor(PDGF)andstainedforF-actinwithfluorophore-labeledphalloidin.(b)Cellsconfinedtovariousshapes(allwithareasof900um2)werestimulatedwithPDGFandstainedwithphalloidinand4-6-diamidino-2-phenylindoletovisualizeF-actin(green)andnuclei(blue).Imagesobtainedbyfluorescencemicroscopy.(A)18minofalivingfibroblastafterstimulationwithPDGF.(B)fibroblast,coatedwithfibronectin,stainedwithfluoresceinatedphalloidin(C)Endothelialcell,coatedwithfibronectin,stainedwithfluoresceinatedphalloidin(D)skeletalC2C12myoblast,coatedthrombospondin-1,stainedwithanti-fascinantibodies.A,B)3030misland;C,D)4040mislands.•Figure4.Elastomericsiliconemicroposts----asubstratetocellularcontractions.(a)cellsadheretothetipsofanarrayofcloselyspaced.(b)SEMshowingacellattachedtotheentiremicropostsubstrate(c)illustrationshowingtheprocessformicroprintingadhesiveproteinsonthemicroposts.(d)Differentialinterferencecontrast(top)andimmunofluorescence(bottom)micrographs:2x2arrayofpostsprintedwithfibronectin.(e),cellsonlyattachonthetips.Scalebarsindicate10m.•Figure5.Bovinecapillaryendothelialcellswereinitiallyonrectangularpatternsusing(SAMs)monolayerssurroundedbyethylene-glycol–terminated(EG-terminated)SAMs.Applicationofacathodicvoltagepulse(1.2Vfor30s)releasedthecellsfromtheconstraintsofthemicroislandsbydesorbingtheEG-terminatedSAMsandenablingproteinstoadsorbfromtheculturemediumthatallowedtheattachmentandmovementofcells.Thenumbersindicatethetimeelapsed(inminutes)afterthevoltagepulse.•Figure6.•(a)Agaroseiswickedintochannelsformedbyapoly(dimethylsiloxane)stampsealedagainstaglasscoverslipandallowedtogelbeforethestampispeeledoff.•(b)Whencellsareseededontothesesubstratescontainingbowtie-shapedwells,cellsattachandcultureaseithersinglecellsorpairs.•(c)Thesinglecell-to-cellcontactformedinthesepairscanbeblockedbyfabricatingsubstratesinwhichtheagaroseformsathinwall.PEG(chemicalfusion)Electricalfusion40xphaseHelaS3AlexaFluor®488phalloidinFibroblastThankYouForYourAttention感謝•中研院應用科學中心陳培凌老師徐昭業學長