56细胞培养

CellCultureBangmingJINBasicconceptincellcultureCellcultureCellculture:growthofcellsdissociatedfromtheparenttissuebymechanicalorenzymaticdispersal.AdvantagesUsedmolecularbiologicaltechnique,underinvitrocondition,canobservelawsofcelllifeactivitywhichcanbechangedbypeoplebutnotsubjecttoinvivoenvironment,andfurtherobservevariationofphysiologicalfunction.PrimaryCulture•PrimaryCulture：thestageofthecultureafterisolationofthecellsbutbeforethefirstsubculture.AdvantagesHistiocyteswareseparatedfromorganismandit’sbiologicalcharacterhasnotoccursweepingchange,toacertainextent,theycanreflectstateofinvivoSubcultureOnceaprimarycellissubcultured,itbecomesknownasacellline.usuallyinvolvesremovalofthemediumanddissociationofthecells,dividingaculture.Notesofsubculture•Cellinoculationdensityiswithin5×104~8×105cells/ml•Cellsarevulnerabledeathwhendensityofcelltoolow,showingthegrowthofcellsbeforereachingalongerretentionperiodCelllineProducedinthecellculturehadtheinfinitemultiplicationabilityvarietycell,thiskindofcellcanbeconductedinfinitepassageculture.常见的细胞系：NIH3T3细胞系：1963年取自小鼠胚胎细胞Hela细胞系：1952年取自人宫颈癌细胞BaseConditionandEquipmentThelivingenvironmentofcells•Temperature35~37℃•Gas5%CO2•Humidity95%air•pH7.2~7.4（酚红指示）CellCultureFlasks•25ml,squaretorticollis•25ml,triangulartorticollis•50ml,squaretorticollis•50ml,triangulartorticollisTheequipmentsincellcultureCellcultureplate6holes12holes24holes48holes96holesPetridish•35mm•60mm•100mmFreezingTube•1.5ml•1.8mlCentrifugeTube•10mL•50mLLiquidNitrogenContainerProcessCoolingBoxPhosphateBufferedSaline-Ca2+Mg2+Free(PBS)•Usedtowash/removeexcessserumthatinhibitsthefunctionofTrypsin-EDTA•Antibiotics•Penicillin（50~100Units/mL）•Streptomycin（50~100Units/mL）BalancedsaltsolutionsDigestionTrypsin•Anenzymeusedtodetachthecellsfromaculturedish.•Trypsincleavespeptidebonds(LYSorARG)infibronectinoftheextracellularmatrix.•EDTAchelatescalciumionsinthemediathatwouldnormallyinhibittrypsin.•Trypsinwillselfdigestandbecomeineffectiveifleftinwaterbathmorethan20minutesCultureMedium目前常用培养基：RPMI-1640、DMEM、TC199RPMI1640种类RPMI—1640（A）（不含谷氨酰胺、可高压灭菌）RPMI—1640（A）无酚红（不含谷氨酰胺、高压灭菌）RPMI—1640（含谷氨酰胺、除菌过滤灭菌）Dulbecco’smodifiedEagle’smediumDMEM细胞培养基•是在MEM培养基的基础上研制的•与MEM比较增加了各种成分用量，同时又分为高糖型（低于4500g/L）和低糖型(低于1000g/L)。•高糖型有利于细胞停泊于一个位置生长，适于生长较快、附着较困难肿瘤细胞等。•DMEM（A）(不含谷氨酰胺、可高压灭菌)DMEM（H）(含谷氨酰胺、高糖型、除菌过滤灭菌)DMEM（L）(含谷氨酰胺、低糖型、除菌过滤灭菌)Serum血清质量好坏是实验成败的关键•常用血清：胎牛血清、新生牛血清、小牛血清、兔血清、马血清等，胎牛血清是品质最高的。•优质血清的标准：透明，淡黄色，无沉淀物，无细菌、支原体、病毒污染。•血清的灭活（消除补体活性）56℃，30分钟。•血清的消毒：过滤除菌。•使用浓度：一般为5%—20%，最常用的10%TypesofcellcultureHeLa细胞（半贴壁细胞）N9神经胶质细胞上皮型细胞（贴壁细胞）HL-60分化细胞Giemsa染色Thegrowthprocessesofanchorage-dependentcell•Dissociationphase•Adherentphase•Latentphase•Logarthmicgrowthphase•Stagnatephase•Cellprimarycellculture•Cellsubculturing•Cellfreezing•Cellrecovering•Cellcounting•MTTBasicTechniquesforCellCulture•Chopwithcrossedscalpelsto0.5-1.0mm•Washbyresuspensionandsetting2-3XStirchoppedpiecesintrypsin/Collagenase/MechanicaldissociationMakingaPrimaryCultureRemoveallmediumWashingcellswithCa2+Mg2+-freePBSTrypsin/EDTAsolutiontodislodgecellsWashcellsandcountthecellsnumberCompletemediumtoinhibittheTrypsinactivityAddfreshmediumtoculturecellsCellsubculturing首次传代注意事项•细胞生长密度不高时，不能急于传代•原代培养的贴壁细胞需控制消化时间•吹打已消化的细胞应减少机械损伤•首次传代培养的pH应偏低些•小牛血清浓度可加大至15%～20%左右RemovevialfromliquidnitrogenfreezerImmediatelyplaceitintoa37°CwaterbathTransfercellsintoasterilecentrifugetubeCheckculturesafter24hWashingcellsAddfreshmediumtocellpelletandcultureCellsfromsuspensionCellsfrommonolayerCellrecovering冻存和复苏的原则：慢冻快融慢冻程序4℃10分钟－20℃30分钟－80℃16-18小时(或隔夜)液氮罐长期储存细胞计数步骤•将血球计数板及盖片擦拭干净，并将盖片盖在计数板上。•将细胞悬液吸出少许，滴加在盖片边缘，使悬液充满盖片和计数板之间。•静置3分钟。•镜下观察，计算计数板四大格细胞总数。细胞数/ml＝4大格细胞总数/4×104注意：压线细胞只计左侧和上方的。镜下偶见由两个以上细胞组成的细胞团，应按单个细胞计算，若细胞团占10%以上，说明分散不好，需重新制备细胞悬液。四唑盐（MTT）比色法•四唑盐（MTT）商品名为噻唑蓝；•MTT比色法的原理：活细胞中琥珀脱氢酶能将四唑盐还原成不溶干水的蓝紫色产物甲臢（formazan)，并沉淀在细胞中，而死细胞没有这种功能。二甲亚砜（DMSO）能溶解沉积在细胞中蓝紫色结晶物，溶液颜色深浅与所含的formazan量成正比。甲臢染料在A570nm处产生吸收值，用酶标仪测定OD值。细胞培养的污染和检测•细胞污染的种类细菌、酵母菌、霉菌和病毒•污染源无菌操作技术不当操作室环境不佳污染之血清ThecellscontaminatedABCA.MCF-7cellB.NIH3T3cellC.HeLacell支原体的污染和检测•Hayflick培养基直接培养法•DNA荧光染色法•PCR方法•相差显微镜观察法•测出细胞株有支原体污染时，应直接灭菌后丢弃，以避免污染其它细胞株。Thankyou构建重组表达载体：pcDNA5/FRT-IL-24pOG44质粒共转染Flp-In-CHO细胞Hygromycin筛选筛选稳定细胞株:Flp-InCHO-IL-24扩大培养收集细胞上清和细胞裂解液细胞株鉴定：半乳糖苷酶活性，westernblot，免疫荧光机制研究：抗体中和实验,STAT3磷酸化IL-24的亲和纯化及纯度和浓度鉴定IL-24重组蛋白抑制食管癌细胞增殖及作用机制分析活性分析：MTT,克隆形成,细胞凋亡技术路线