细胞发展ppt课件

1、 刘金涛:jtliu0416gmail华东理工大学海正研讨院动物细胞与组织工程研讨所Mammalian Cell CultureContentsAntibody productionMetabolism of cell Cell culture mediumCell lines History of cell cultureDevelopment of Cell Culture细胞培育 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育，细胞能继续存活与增殖，培育过程中细胞不再构成组织。 开展与完善细胞培育技术围绕以下几个方面进展： 防止污染 改良培育方法 设计新

2、型培育容器 设计不同的培育基等。Typical application of mammalian cell culture 1、 cell biology research 细胞生物学研讨 2、toxicity testing 毒理实验 3、production of complex proteins 蛋白消费 4、production of vaccines 疫苗消费 5、tissue and organ culture 组织和器官培育 6、most frequent product： monoclonal antibodies for diagnostics and therapy 消费诊

3、断和治疗的单抗Nomenclature原代培育： primaryculture初次分别组织的培育。细胞系cell line：原代培育物经传代胜利。有限细胞系：不能继续传代或者传代次数有限50代 延续细胞系：可以延续传代细胞株cell strain：从原代培育细胞群中挑选出的具有特定性质或标志的细胞群。The Growth Type of Cell按生长方式贴壁依赖型细胞anchorage-dependent cell悬浮型细胞Suspension cell贴壁细胞成纤维型细胞Fibroblast-liked cells上皮型细胞Epithelium-liked cells游走型细胞Cell T

4、ypeContentsAntibody productionMetabolism of cell culture Cell culture mediumCell lines History of cell cultureNormal cell正常细胞：正常的原代培育物进展传代构成的继代培育物增殖才干有限，最多只可传50代左右。四个特征无明显的基因突变有限的寿命细胞无恶性贴壁依赖贴壁依赖性细胞必需全部符合，悬浮细胞必需符合前三个Transformed cell培育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：正常细胞在培育过程中获得无限增殖的才干，成为延续细胞系四种途径：转化细胞伴随着染色

5、体方式的改动，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交融产生的细胞与转化细胞相比它们是恶性的，经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 SC_1BovineMDBK BT FBLMonkeyVERO CV-1 FRhK-4 BS-K-1 RatXC MRK-49FPorcinePT-1 PK15 STHamsterBHK-2

6、1 CHOMinkMiCL Mv 1 LuEquineNBL-6FelinePG-4CanineMDCKSheepMDOCKInsectSF9Commonly used cell linesCell line Origin Cell type CommentBHK Baby hamster kidney Fibroblast Anchorage-dependent / suspension; CHO Chinese hamster ovary Epithelial .HeLa Human cervical carcinoma Cancer cellMDCK Canine kidney Epit

7、helial Anchorage-dependentMRC-5 Human embryonic lung Fibroblast Finite lifespan, normal cells; Namalwa Human lymphoma Lymphoblast3T3 Mouse connective tissue Fibroblast Vigorous growth in suspension; WI-38 Human embryonic lung Fibroblast Finite lifespan normal cells; Vero African green monkey kidney

8、Fibroblast Normal diploid characteristics; Chinese Hamster Ovary(CHO)CellChinese hamster ovary(CHO)cells 中国仓鼠卵巢细胞In 1957, Theodore T. Puck obtained a female Chinese hamster and used it to derive the original Chinese hamster ovary (CHO) cell lineA glycine-dependent strain (CHO-K1) was derived from th

9、e original cell linesSince then, CHO cells have been a cell line of choice because of their rapid growth and high protein production. The development of CHO cell1980, CHO-K1 was mutagenized to generate CHO-DXB11 (also referred to as CHO-DUKX or CHO-DUK-XB11), a cell line lacking DHFR activity1983, C

10、HO-DG44, a cell line with deletions of both DHFR allelesThese two DHFR-minus strains require glycine, hypoxanthine, and thymidine (GHT)DHFR is a small monomeric enzyme that catalyzes the conversion of folic acid, to tetrahydrofolate (THF) Genetic heterogeneity of CHO cell linesFirst CHO Cell GenomeC

11、ontentsAntibody productionMetabolism of cell Cell culture mediumCell lines History of cell cultureCell Culture Medium 1. Classical Medium 经典培育基 2. Serum Free Medium(SFM) 无血清培育基 3. Protein Free Medium(PFM) 无蛋白培育基 4. Chemical Defined Medium(CDM) CD 培育基BME Eagles basal medium根底Eagle培育基，1955年由Eagle设计，在此

12、根底上改良的细胞培育基种类有MEM、DMEM、IMDM等MEM Eagles minimum essential medium低限量 Eagle 培育基，1959 年修正配方，是一种最根本、适用范围最广的培育基，是一种被广泛运用的培育基。DMEM Dulbeccos modification of Eagles medium ，DMEM 由 Dulbecco 改良的 Eagle 培育基，分低糖、高糖。RPMI 1640 Roswell Park Memorial Institute medium; Moore等人于1967年，针对淋巴细胞培育设计IMDM IMDM是由Iscoves改良的Eag

13、le培育基，添加了几种氨基酸和胱氨酸量。可用于杂交瘤细胞培育，以及无血清培育的根底培育基。Hams F10,F-12 1963年、1969年由Ham设计，含微量元素，可在血清含量低时用，适用于克隆化培育。F10用于仓鼠、人二倍体细胞，F12适用于CHO细胞Classical MediumSerumFetal bovine serum (FBS) 胎牛血清1Newborn calf serum (NBCS) 新生牛牛血清2Calf serum 小牛血清33血清的来源有牛血清、马血清、鸡血清、羊血清及人血清。最广泛运用的为 牛血清，主要是它来源充足、制备技术成熟Functions提供根本营养物质：

14、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.Drawbacks成分复杂批与批之间差别很大 含一些对细胞产生毒性的物质，如多胺氧化酶取材中能够带入支原体、病毒血清的运用使得实验和消费的规范化困难大规模消费中，血清来源越来越困难，价钱昂贵The Characteristics of SerumSerum Free Medium无血清培育基(serum free medium,SFM):是不需求添加血清就可以维持细胞在体外较长时间生长繁衍的合成培育基。但是它们能够包含个别蛋白或大量蛋白组分ITES

15、: Insulin, Transferring, Ethanolamine and SeleniteTrace elementGrowth factors1 未知组分少； 2 培育基中不存在血清，对培育细胞影响小； 3 杂蛋白含量少，消费的产品后期处置较容易。4 可以实现细胞的大规模悬浮培育SFM 优点：Protein Free Medium无蛋白培育基protein free medium, PFM:即不含有动物蛋白的培育基。无血清培育基仍含有较多的动物蛋白，如胰岛素，转铁蛋白、牛血洁白蛋白等Chemical Defined Medium 化学成分限定的培育基(chemical define

16、d medium, CDM):是指培育基中的一切成分都是明确的。 它不含有动物蛋白和水解物或未知构呵斥分，而是运用了一些知构造与功能的小分子 化合物，如短肽、植物激素等。例如：CD OptiCHOCD CHO MediumMedium Properties and componentscomponentsGlucose Amino acid VitamineInorganic saltsTrace elementsotherspropertiespHBufferOsmolalityTemperatureContentsAntibody productionMetabolism of cell

17、Cell culture mediumCell lines History of cell cultureGlucose对于延续细胞系，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。Glucose is used in most formulations to provide an energy source as well as a precursor for biosynthesis.Lactic acid is the major product of glycolysis. In most cultures only a sm

18、all proportion of glucose is completely oxidized via the tricarboxylic acid (TCA) cycle.GlutamineL-Glutamine is important as a precursor for Peptide and protein synthesisamino sugar synthesis, Purine，pyrimidine ，nucleic acid and nucleotide synthesis providing a source of carbons for oxidationGS Gene

19、 Expression System Expression vector encoding product gene(s) plus GS gene, allowing synthesis of glutamine an essential nutrient Only cells with GS gene survive GS is inhibited by MSX which can be used to increase stringency of selection Linked product gene driven by strong promoter (hCMV) to give

20、high expression High productivity without amplificationByproductsLactateAmmoniumNH4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 改动胞内pH 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

21、-end pH-controlled delivery of glucose effectively 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 cultureAn

22、tibodyAntibody products 1900 Paul Erlich proposed concept of Magic Bullet1975 George Kohler and Cesar Milstein develop hybridoma technology for monoclonal antibodiesFirst successful report of monoclonal anti-idiotype antibody to treat human cancer 1982 1984 First monoclonal antibody, muromonab-CD3(D

23、KT3) approved by FDA 1900 1905 1910 1915 1920 1975 1980 1985 1990 1995 2000 2005 2021 2021 2020 2025 20301997 FDA approved first humanized anti-CD20 monoclonal antibody for treatment of B-cell lymphoma1998 FDA approved first chimeric anti-HER2 monoclonal antibody therapy for treatment of breast cancer1999 FDA approved first conjugated humanized anti-CD33 immunotoxin for treatme