细胞发展.ppt

,刘金涛E-mail:jtliu0416华东理工大学海正研究院动物细胞与组织工程研究所,MammalianCellCulture,Contents,Antibodyproduction,Metabolismofcell,Cellculturemedium,Celllines,Historyofcellculture,DevelopmentofCellCulture,细胞培养指:从生物机体取出部分组织分散成单个细胞或直接从机体取出单个细胞在体外条件下培养，细胞能继续存活与增殖，培养过程中细胞不再形成组织。,发展与完善细胞培养技术围绕以下几个方面进行：防止污染改进培养方法设计新型培养容器设计不同的培养基等。,Typicalapplicationofmammaliancellculture,Nomenclature,原代培养：（primaryculture）首次分离组织的培养。细胞系（cellline）：原代培养物经传代成功。有限细胞系：不能继续传代或者传代次数有限（50代）连续细胞系：可以连续传代细胞株（cellstrain）：从原代培养细胞群中筛选出的具有特定性质或标志的细胞群。,TheGrowthTypeofCell,按生长方式贴壁依赖型细胞（anchorage-dependentcell）悬浮型细胞（Suspensioncell）,贴壁细胞成纤维型细胞（Fibroblast-likedcells）上皮型细胞（Epithelium-likedcells）游走型细胞,CellType,Contents,Antibodyproduction,Metabolismofcellculture,Cellculturemedium,Celllines,Historyofcellculture,Normalcell,正常细胞：正常的原代培养物进行传代形成的继代培养物,增殖能力有限，最多只可传50代左右。,贴壁依赖性细胞必须全部符合，悬浮细胞必须符合前三个,Transformedcell,培养过程中自发转化化学转变病毒转化致瘤因子转化,转化细胞：正常细胞在培养过程中获得无限增殖的能力，成为连续细胞系四种途径：,转化细胞伴随着染色体模式的改变，二倍体变成异倍体,TumorCell,来自于肿瘤组织的细胞或者经肿瘤组织与其他细胞融合产生的细胞与转化细胞相比它们是恶性的，常常是非贴壁依赖性的。,CellLine,Commonlyusedcelllines,CelllineOriginCelltypeCommentBHKBabyhamsterkidneyFibroblastAnchorage-dependent/suspension;CHOChinesehamsterovaryEpithelial.HeLaHumancervicalcarcinomaCancercellMDCKCaninekidneyEpithelialAnchorage-dependentMRC-5HumanembryoniclungFibroblastFinitelifespan,normalcells;NamalwaHumanlymphomaLymphoblast3T3MouseconnectivetissueFibroblastVigorousgrowthinsuspension;WI-38HumanembryoniclungFibroblastFinitelifespannormalcells;VeroAfricangreenmonkeykidneyFibroblastNormaldiploidcharacteristics;,ChineseHamsterOvary(CHO)Cell,Chinesehamsterovary(CHO)cells中国仓鼠卵巢细胞In1957,TheodoreT.PuckobtainedafemaleChinesehamsterandusedittoderivetheoriginalChinesehamsterovary(CHO)celllineAglycine-dependentstrain(CHO-K1)wasderivedfromtheoriginalcelllines,Sincethen,CHOcellshavebeenacelllineofchoicebecauseoftheirrapidgrowthandhighproteinproduction.,ThedevelopmentofCHOcell,1980,CHO-K1wasmutagenizedtogenerateCHO-DXB11(alsoreferredtoasCHO-DUKXorCHO-DUK-XB11),acelllinelackingDHFRactivity1983,CHO-DG44,acelllinewithdeletionsofbothDHFRallelesThesetwoDHFR-minusstrainsrequireglycine,hypoxanthine,andthymidine(GHT),DHFRisasmallmonomericenzymethatcatalyzestheconversionoffolicacid,totetrahydrofolate(THF),GeneticheterogeneityofCHOcelllines,FirstCHOCellGenome,Contents,Antibodyproduction,Metabolismofcell,Cellculturemedium,Celllines,Historyofcellculture,CellCultureMedium,1.ClassicalMedium经典培养基,2.SerumFreeMedium(SFM)无血清培养基,3.ProteinFreeMedium(PFM)无蛋白培养基,4.ChemicalDefinedMedium(CDM)CD培养基,BMEEaglesbasalmedium（基础Eagle培养基），1955年由Eagle设计，在此基础上改良的细胞培养基品种有MEM、DMEM、IMDM等MEMEaglesminimumessentialmedium（低限量Eagle培养基），1959年修改配方，是一种最基本、适用范围最广的培养基，是一种被广泛应用的培养基。DMEMDulbeccosmodificationofEaglesmedium，DMEM由Dulbecco改良的Eagle培养基，分低糖、高糖。RPMI1640RoswellParkMemorialInstitutemedium;Moore等人于1967年，针对淋巴细胞培养设计IMDMIMDM是由Iscoves改良的Eagle培养基，增加了几种氨基酸和胱氨酸量。可用于杂交瘤细胞培养，以及无血清培养的基础培养基。HamsF10,F-121963年、1969年由Ham设计，含微量元素，可在血清含量低时用，适用于克隆化培养。F10用于仓鼠、人二倍体细胞，F12适用于CHO细胞,ClassicalMedium,Serum,血清的来源有牛血清、马血清、鸡血清、羊血清及人血清。最广泛应用的为牛血清，主要是它来源充足、制备技术成熟,TheCharacteristicsofSerum,SerumFreeMedium,无血清培养基(serumfreemedium,SFM):是不需要添加血清就可以维持细胞在体外较长时间生长繁殖的合成培养基。但是它们可能包含个别蛋白或大量蛋白组分ITES:Insulin,Transferring,EthanolamineandSeleniteTraceelementGrowthfactors,1）未知组分少；2）培养基中不存在血清，对培养细胞影响小；3）杂蛋白含量少，生产的产品后期处理较容易。4）可以实现细胞的大规模悬浮培养,SFM优点：,ProteinFreeMedium,无蛋白培养基（proteinfreemedium,PFM）:即不含有动物蛋白的培养基。无血清培养基仍含有较多的动物蛋白，如胰岛素，转铁蛋白、牛血清白蛋白等,ChemicalDefinedMedium,化学成分限定的培养基(chemicaldefinedmedium,CDM):是指培养基中的所有成分都是明确的。它不含有动物蛋白和水解物或未知结构成分，而是使用了一些已知结构与功能的小分子化合物，如短肽、植物激素等。,例如：CDOptiCHOCDCHOMedium,MediumPropertiesandcomponents,Contents,Antibodyproduction,Metabolismofcell,Cellculturemedium,Celllines,Historyofcellculture,Glucose,对于连续细胞系，丙酮酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和丙酮酸羧化酶它们活性很低，导致糖酵解生成的丙酮酸很难进入TCA循环。,Glucoseisusedinmostformulationstoprovideanenergysourceaswellasaprecursorforbiosynthesis.Lacticacidisthemajorproductofglycolysis.Inmostculturesonlyasmallproportionofglucoseiscompletelyoxidizedviathetricarboxylicacid(TCA)cycle.,Glutamine,L-GlutamineisimportantasaprecursorforPeptideandproteinsynthesisaminosugarsynthesis,Purine，pyrimidine，nucleicacidandnucleotidesynthesisprovidingasourceofcarbonsforoxidation,GSGeneExpressionSystem,Expressionvectorencodingproductgene(s)plusGSgene,allowingsynthesisofglutamineanessentialnutrientOnlycellswithGSgenesurviveGSisinhibitedbyMSXwhichcanbeusedtoincreasestringencyofselectionLinkedproductgenedrivenbystrongpromoter(hCMV)togivehighexpressionHighproductivitywithoutamplification,Byproducts,Lactate,Ammonium,NH4,主要由谷氨酰胺等氨基酸脱氨产生,影响细胞生长影响细胞内氨基酸的代谢改变胞内pH影响蛋白糖基化过程,NH4的作用：,AmmoniumAltersN-GlycanStructuresofRecombinantTNFR-IgG:DegradativeVersusBiosyntheticMechanisms,lactate,LactatecanchangethemediumpHandosmolality,High-endpH-controlleddeliveryofglucoseeffectivelysuppresseslactateaccumulationinCHOfed-batchcultures,FeedingLactateforCHOCellCultureProcesses:ImpactonCultureMetabolismandPerformance,Contents,Antibodyproduction,Metabolismofcell,Cellculturemedium,Celllines,Historyofcellculture,Antibody,Antibodyproducts,1900PaulErlichproposedconceptofMagicBullet,1975GeorgeKohlerandCesarMilsteindevelophybridomatechnologyformonoclonalantibodies,Firstsuccessfulreportofmonoclonalanti-idiotypeantibodytotreathumancancer1982,1984Firstmonoclonalantibody,muromonab-CD3(DKT3)approvedbyFDA,19001905191019151920197519801985199019952000200520102015202020252030,1997FDAapprovedfirsthumanizedanti-CD20monoclonalantibodyfortreatmentofB-celllymphoma,1998FDAapprovedfirstchimericanti-HER2