細(xì)胞發(fā)展ppt課件

1、 劉金濤:jtliu0416gmail華東理工大學(xué)海正研討院動(dòng)物細(xì)胞與組織工程研討所Mammalian Cell CultureContentsAntibody productionMetabolism of cell Cell culture mediumCell lines History of cell cultureDevelopment of Cell Culture細(xì)胞培育 指:從生物機(jī)體取出部分組織分散成單個(gè)細(xì)胞或直接從機(jī)體取出單個(gè)細(xì)胞在體外條件下培育，細(xì)胞能繼續(xù)存活與增殖，培育過程中細(xì)胞不再構(gòu)成組織。 開展與完善細(xì)胞培育技術(shù)圍繞以下幾個(gè)方面進(jìn)展： 防止污染 改良培育方法 設(shè)計(jì)新

2、型培育容器 設(shè)計(jì)不同的培育基等。Typical application of mammalian cell culture 1、 cell biology research 細(xì)胞生物學(xué)研討 2、toxicity testing 毒理實(shí)驗(yàn) 3、production of complex proteins 蛋白消費(fèi) 4、production of vaccines 疫苗消費(fèi) 5、tissue and organ culture 組織和器官培育 6、most frequent product： monoclonal antibodies for diagnostics and therapy 消費(fèi)診

3、斷和治療的單抗Nomenclature原代培育： primaryculture初次分別組織的培育。細(xì)胞系cell line：原代培育物經(jīng)傳代勝利。有限細(xì)胞系：不能繼續(xù)傳代或者傳代次數(shù)有限50代 延續(xù)細(xì)胞系：可以延續(xù)傳代細(xì)胞株cell strain：從原代培育細(xì)胞群中挑選出的具有特定性質(zhì)或標(biāo)志的細(xì)胞群。The Growth Type of Cell按生長方式貼壁依賴型細(xì)胞anchorage-dependent cell懸浮型細(xì)胞Suspension cell貼壁細(xì)胞成纖維型細(xì)胞Fibroblast-liked cells上皮型細(xì)胞Epithelium-liked cells游走型細(xì)胞Cell T

4、ypeContentsAntibody productionMetabolism of cell culture Cell culture mediumCell lines History of cell cultureNormal cell正常細(xì)胞：正常的原代培育物進(jìn)展傳代構(gòu)成的繼代培育物增殖才干有限，最多只可傳50代左右。四個(gè)特征無明顯的基因突變有限的壽命細(xì)胞無惡性貼壁依賴貼壁依賴性細(xì)胞必需全部符合，懸浮細(xì)胞必需符合前三個(gè)Transformed cell培育過程中自發(fā)轉(zhuǎn)化化學(xué)轉(zhuǎn)變病毒轉(zhuǎn)化致瘤因子轉(zhuǎn)化轉(zhuǎn)化細(xì)胞：正常細(xì)胞在培育過程中獲得無限增殖的才干，成為延續(xù)細(xì)胞系四種途徑：轉(zhuǎn)化細(xì)胞伴隨著染色

5、體方式的改動(dòng)，二倍體變成異倍體Tumor Cell來自于腫瘤組織的細(xì)胞或者經(jīng)腫瘤組織與其他細(xì)胞交融產(chǎn)生的細(xì)胞與轉(zhuǎn)化細(xì)胞相比它們是惡性的，經(jīng)常是非貼壁依賴性的。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 中國倉鼠卵巢細(xì)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 經(jīng)典培育基 2. Serum Free Medium(SFM) 無血清培育基 3. Protein Free Medium(PFM) 無蛋白培育基 4. Chemical Defined Medium(CDM) CD 培育基BME Eagles basal medium根底Eagle培育基，1955年由Eagle設(shè)計(jì)，在此

12、根底上改良的細(xì)胞培育基種類有MEM、DMEM、IMDM等MEM Eagles minimum essential medium低限量 Eagle 培育基，1959 年修正配方，是一種最根本、適用范圍最廣的培育基，是一種被廣泛運(yùn)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，針對(duì)淋巴細(xì)胞培育設(shè)計(jì)IMDM IMDM是由Iscoves改良的Eag

13、le培育基，添加了幾種氨基酸和胱氨酸量?？捎糜陔s交瘤細(xì)胞培育，以及無血清培育的根底培育基。Hams F10,F-12 1963年、1969年由Ham設(shè)計(jì)，含微量元素，可在血清含量低時(shí)用，適用于克隆化培育。F10用于倉鼠、人二倍體細(xì)胞，F(xiàn)12適用于CHO細(xì)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血清的來源有牛血清、馬血清、雞血清、羊血清及人血清。最廣泛運(yùn)用的為 牛血清，主要是它來源充足、制備技術(shù)成熟Functions提供根本營養(yǎng)物質(zhì)：

14、氨基酸、維生素、脂類物質(zhì)、核酸衍生物等。提供激素和各種生長因子：胰島素、表皮生長因子等。 提供結(jié)合蛋白：如白蛋白、轉(zhuǎn)鐵蛋白等提供促接觸和伸展因子.Drawbacks成分復(fù)雜批與批之間差別很大 含一些對(duì)細(xì)胞產(chǎn)生毒性的物質(zhì)，如多胺氧化酶取材中能夠帶入支原體、病毒血清的運(yùn)用使得實(shí)驗(yàn)和消費(fèi)的規(guī)范化困難大規(guī)模消費(fèi)中，血清來源越來越困難，價(jià)錢昂貴The Characteristics of SerumSerum Free Medium無血清培育基(serum free medium,SFM):是不需求添加血清就可以維持細(xì)胞在體外較長時(shí)間生長繁衍的合成培育基。但是它們能夠包含個(gè)別蛋白或大量蛋白組分ITES

15、: Insulin, Transferring, Ethanolamine and SeleniteTrace elementGrowth factors1 未知組分少； 2 培育基中不存在血清，對(duì)培育細(xì)胞影響??； 3 雜蛋白含量少，消費(fèi)的產(chǎn)品后期處置較容易。4 可以實(shí)現(xiàn)細(xì)胞的大規(guī)模懸浮培育SFM 優(yōu)點(diǎn)：Protein Free Medium無蛋白培育基protein free medium, PFM:即不含有動(dòng)物蛋白的培育基。無血清培育基仍含有較多的動(dòng)物蛋白，如胰島素，轉(zhuǎn)鐵蛋白、牛血潔白蛋白等Chemical Defined Medium 化學(xué)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確的。 它不含有動(dòng)物蛋白和水解物或未知構(gòu)呵斥分，而是運(yùn)用了一些知構(gòu)造與功能的小分子 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對(duì)于延續(xù)細(xì)胞系，丙酮酸脫氫酶、磷酸烯醇式丙酮酸羧化酶和丙酮酸羧化酶它們活性很低，導(dǎo)致糖酵解生成的丙酮酸很難進(jìn)入TCA循環(huán)。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脫氨產(chǎn)生 影響細(xì)胞生長 影響細(xì)胞內(nèi)氨基酸的代謝 改動(dòng)胞內(nèi)pH 影響蛋白糖基化過程N(yùn)H4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