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1、 刘金涛E-mail:华东理工大学海正研究院动物细胞与组织工程研究所Mammalian Cell CultureContentsAntibody productionMetabolism of cell Cell culture mediumCell lines History of cell cultureDevelopment of Cell Culture细胞培养细胞培养 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：u 防止污染u 改进培养方法u 设计新型培
2、养容器u 设计不同的培养基等。Typical application of mammalian cell culture 1、cellbiologyresearch细胞生物胞生物学研学研究究2、toxicitytesting毒理毒理实验3、productionofcomplexproteins蛋白生蛋白生产4、productionofvaccines疫苗生疫苗生产5、tissueandorganculture组织和器官培和器官培养养6、mostfrequentproduct：monoclonalantibodiesfordiagnosticsandtherapy生生产诊断断和治和治疗的的单抗抗
3、Nomenclature原代培养：（primaryculture）首次分离组织的培养。细胞系（cell line）：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（cell strain）：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。The Growth Type of Cell按生长方式按生长方式贴壁依赖型细胞（贴壁依赖型细胞（anchorage-dependent cellanchorage-dependent cell）悬浮型细胞（悬浮型细胞（Suspension cellSuspension cell）贴壁细胞贴壁细胞成
4、纤维型细胞（成纤维型细胞（Fibroblast-liked cells）上皮型细胞（上皮型细胞（Epithelium-liked cells）游走型细胞游走型细胞Cell TypeContentsAntibody productionMetabolism of cell culture Cell culture mediumCell lines History of cell cultureNormal cell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。四个特征1.无明显的基因突变2.有限的寿命3.细胞无恶性4.贴壁依赖贴壁依赖性细胞必须全部符合，悬
5、浮细胞必须符合前三个Transformed cell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Tumor Cell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。Cell LineHuman293 A549 CEM-A Hela S3 Hs68 MRC-5 WI-38 324-K H9 HEL 299 HT-1080 MarmosetB95-8Murinebalb/3T3 L929 NIH/3T3K-BALB
6、SC_1BovineMDBK BT FBLMonkeyVERO CV-1 FRhK-4 BS-K-1 RatXC MRK-49FPorcinePT-1 PK15 STHamsterBHK-21 CHOMinkMiCL Mv 1 LuEquineNBL-6FelinePG-4CanineMDCKSheepMDOCKInsectSF9Commonly used cell linesCell line Origin Cell type CommentBHK BabyhamsterkidneyFibroblastAnchorage-dependent/suspension;CHOChinesehams
7、terovaryEpithelial.HeLaHumancervicalcarcinomaCancercellMDCK CaninekidneyEpithelialAnchorage-dependentMRC-5HumanembryoniclungFibroblastFinitelifespan,normalcells;NamalwaHumanlymphomaLymphoblast3T3MouseconnectivetissueFibroblastVigorousgrowthinsuspension;WI-38 HumanembryoniclungFibroblastFinitelifespa
8、nnormalcells;Vero AfricangreenmonkeykidneyFibroblastNormaldiploidcharacteristics;Chinese Hamster Ovary(CHO)CellChinesehamsterovary(CHO)cells中国仓鼠卵巢细胞In1957,TheodoreT.Puckobtained a female Chinese hamster and used it to derive the original Chinese hamster ovary(CHO)cell lineAglycine-dependentstrain(CH
9、O-K1)was derived from the original cell linesSincethen,CHOcellshavebeenacelllineofchoicebecauseoftheirrapidgrowthandhighproteinproduction.The development of CHO cell1980,CHO-K1 was mutagenized to generateCHO-DXB11(alsoreferredtoasCHO-DUKXorCHO-DUK-XB11),a cell line lacking DHFR activity1983,CHO-DG44
10、,a cell line with deletions of both DHFR allelesThese two DHFR-minus strains require glycine,hypoxanthine,andthymidine(GHT)DHFR isasmallmonomericenzymethatcatalyzestheconversionoffolicacid,totetrahydrofolate(THF)Genetic heterogeneity of CHO cell linesFirst CHO Cell GenomeContentsAntibody productionM
11、etabolism of cell Cell culture mediumCell lines History of cell cultureCell Culture Medium1.Classical Medium 经典培养基2.Serum Free Medium(SFM)无血清培养基3.Protein Free Medium(PFM)无蛋白培养基4.Chemical Defined Medium(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MEMEaglesmi
12、nimumessentialmedium（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、
13、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Classical MediumSerumFetalbovineserum(FBS)胎牛血清1Newborncalfserum(NBCS)新生牛牛血清2Calfserum小牛血清3 3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 牛血清牛血清，主要是它来源充足、制备技术成熟Functions提供基本营养物质：氨基酸、维生素、脂类物质、核酸衍生物等。提供激素和各种生长因子：胰岛素、表皮生长因子等。提供结合蛋白：如白蛋白、转铁蛋白等提供促接触和伸展因子
14、.Drawbacks成分复杂批与批之间差异很大 含一些对细胞产生毒性的物质，如多胺氧化酶取材中可能带入支原体、病毒血清的使用使得实验和生产的标准化困难大规模生产中，血清来源越来越困难，价格昂贵The Characteristics of SerumSerum Free Medium无血清培养基无血清培养基(serum free medium,SFM):(serum free medium,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,EthanolamineandSeleni
15、teTrace elementGrowth factors1）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：Protein Free Medium无蛋白培养基（无蛋白培养基（protein free medium,PFMprotein free medium,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Chemical Defined Medium 化学成分限定的培养基化学成分限定的培养基(chemical defined medium,
16、CDM):(chemical defined medium,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：CDOptiCHOCDCHOMediumMedium Properties and componentscomponents1.Glucose 2.Amino acid 3.Vitamine4.Inorganic salts5.Trace elements6.othersproperties1.pH2.Buffer3.Osmolality4.TemperatureContentsA
17、ntibody productionMetabolism of cell Cell culture mediumCell lines History of cell cultureGlucose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.Inmostcultures
18、onlyasmallproportionofglucoseiscompletelyoxidizedviathetricarboxylicacid(TCA)cycle.GlutamineL-Glutamine is important as a precursor for nPeptide and protein synthesisnamino sugar synthesis,nPurine，pyrimidine，nucleic acid and nucleotide synthesis nproviding a source of carbons for oxidationGS Gene Ex
19、pression SystemnExpressionvectorencodingproductgene(s)plusGSgene,allowingsynthesisofglutamineanessentialnutrientnOnlycellswithGSgenesurvivenGSisinhibitedbyMSXwhichcanbeusedtoincreasestringencyofselectionnLinkedproductgenedrivenbystrongpromoter(hCMV)togivehighexpressionnHighproductivitywithoutamplifi
20、cationByproductsLactateAmmoniumNH4主要由谷氨酰胺等氨基酸脱氨产生n 影响细胞生长n 影响细胞内氨基酸的代谢n 改变胞内pHn 影响蛋白糖基化过程NH4的作用：Ammonium Alters N-Glycan Structuresof Recombinant TNFR-IgG:DegradativeVersus Biosynthetic MechanismslactateLactate can change the medium pH and osmolalityHigh-end pH-controlled delivery of glucose effecti
21、vely suppresses lactate accumulation in CHO fed-batch culturesFeeding Lactate for CHO Cell Culture Processes:Impact on Culture Metabolism and PerformanceContentsAntibody productionMetabolism of cell Cell culture mediumCell lines History of cell cultureAntibodyAntibody products 1900PaulErlichproposed
22、conceptofMagicBullet1975GeorgeKohlerandCesarMilsteindevelophybridomatechnologyformonoclonalantibodiesFirstsuccessfulreportofmonoclonalanti-idiotypeantibodytotreathumancancer19821984Firstmonoclonalantibody,muromonab-CD3(DKT3)approvedbyFDA190019051910191519201975198019851990199520002005201020152020202
23、520301997FDAapprovedfirsthumanizedanti-CD20monoclonalantibodyfortreatmentofB-celllymphoma1998FDAapprovedfirstchimericanti-HER2monoclonalantibodytherapyfortreatmentofbreastcancer1999FDAapprovedfirstconjugatedhumanizedanti-CD33immunotoxinfortreatmentofAML2001FDAapprovedfirstmurineanti-CD20radiolabeled
24、monoclonalantibodyforB-celllymphoma2003FDAapprovedfirstfullyhumananti-epidermalgrowthfactorreceptormonoclonalantibodyfortreatmentofmetastaticcolorectalcarcinomaWhy use mammalian cells？Disadvantages:Disadvantages:n Low growth ratesn High expensiven Low specific production rateAdvantages:Correct forma
25、tion of disulfide bondsCorrect glycosylationSecreted proteins Therapeutic antibodies approvedAntibody Development The Development of Stable Cell LinesCell Culture Process Monoclonal antibody downstream processCell Culture Type 2.Fed-batch Culture 流加培流加培养养 1.Batch Culture 批培批培养养 3.Perfusion Culture 灌注培灌注培养养Cell culture process operating parameters thank you！Sub titleClick to Edit TitleClick to Edit TitleSub title