动物细胞培养及单克隆抗体

920944生产规模培养昆虫细胞〔英万Agathos,5.N.…了B ioteehnol.Ady一1901,9(i)一51一58〔译自DBA,1991,10(15),91一08839〕 文章讨论了以下内容:昆虫细胞培养在生物技术中的应用;昆虫细胞培养的生物加工开发状况,培养基开发;用于昆虫细胞培养的培养器构建及发展方向。昆虫培养是药物和农药有效生产的良好寄主,适合于大规模技术,以满足(A)昆虫病毒生防因子和(B)对人及动物有医疗价值的重组蛋白的有计划生产的需要。其间最重要的系统是用杆状病毒载体转染的鳞翅目昆虫细胞。双翅目品系也具有发展前途(如黄猩猩果蝇、Aedes azbo尹￡e乙。…     

昆虫细胞(Sf21)悬浮培养过程中生长限制性基质间歇补加技术的应用

基于Sf21昆虫细胞在悬浮培养过程中所表现出的生长代谢特征,提出以培养液中残糖浓度作为控制参数,并利用限制性基质(葡萄糖和蛋白水解物)的间歇补加技术调控细胞生长的方案。实际控制表明：与批培养相比,Sf21细胞在两种具代表性的昆虫细胞培养基(IPL-41和TC-100)中的生长期和稳定期都得到了有效的延长。TC-100培养液中最高细胞培养密度由3.0×10⁶ cells/mL提高到6.5×10⁶ cells/mL;IPL41培养液中最高细胞培养密度则由7.05×10⁶ cells/mL提高到9.0×10⁶cells/mL。由于限制性基质的间歇补加技术是利用较确定的营养成分来代替复杂昂贵的补料培养基,因此更适合于昆虫细胞的大规模高密度培养。     

无载体固定化培养模式下HEK293细胞的生长和代谢特征

利用HEK293细胞在悬浮培养体系中下具有聚集成团的体外培养特性,在250ml的spinner flask搅拌式细胞培养瓶中以悬浮细胞团的形式实施HEK293细胞的无载体固定化培养,以细胞密度、细胞活力、细胞团粒径分布和葡萄糖比消耗率 (qglc)、乳酸比产率 (qlac)、乳酸转化率 (Ylac/glc)、氨基酸消耗为观察指标,同时设置静止培养体系作为参照,考察无载体固定化培养模式下的HEK293细胞生长和代谢特征。观察结果表明,HEK293细胞在搅拌式细胞培养瓶中无载体固定化培养和在组织培养瓶中静止贴壁培养表现为基本相同的细胞生长和代谢特征,平均粒径小于300μm的细胞团中的物质传递能够满足HEK293细胞维持正常生长和代谢的基本需要。HEK293细胞的无载体固定化培养便于实施灌注操作、提高生物反应器单位体积的生产效率。     

昆虫细胞（Sf21）悬浮培养过程中生长限制性基质间歇补加技术的 …

基于ｒ２１昆虫细胞在浮过程中所表现出的生长代谢特征,提出以培养液中残糖浓度作为控制参数,并利用限制性基质（葡萄糖和蛋白水解物）的间歇补加技术调控细胞生长的方案。实际控制表明：与批培养相比,Ｓ１ｆ２１细胞在两种具代表性的昆虫水解物）的间歇补加调控细胞生长的方案。实际控制表明：与批培养相比,Ｓｆ２１细胞在两种具代表性的昆虫细胞培养基（ＩＰＬ－４１和ＴＣ－１００）中的生长期和稳定期部都到有效的延长。ＴＣ     

细胞工程

880936微载体培养动物细胞的流体动力学效应〔英」/Cr。ughan,M.S.…了Bioteehn-01.Bioeng一1987,29(1)一130一141〔译自CBA,1987,(5),2041〕 对于微载体培养中的流体动力学现象作了研究,以便更好地设计大规模生产用的反应器。文中用新的概念和理论模型对新旧资料作了分析。(梁博文)88093了通过对营养物的控制降低废物的分泌:在血清培养哺乳动物细胞时使产物和细胞产率达到最大值的可能采取的方案〔英〕/G laeken,M.W.…了Bioteehnol.Bioeng一1986,28(9)一1376~1389〔译自CBA,1987,(5),2118〕 在培养基中生长的哺乳动物细胞会分泌一…     

体外培养和遗传育种

863229通过植物细胞培养生产结晶利血平[日〕了Bioteehnol.Japan Newssurviee一1985,3(9)一8〔译自CBA,1985,(9),3112〕 日本Sekisui化学合成工业公司研究从印度萝芙木(Ra。功olfia serpe。tina)的细胞培养物生产利血平,并分离出了结品。 (杜允)863230植物原生质体:植物生物技术的一个新工具〔英〕/Rao,P.S.了C。rr.Sei一1985,54一335一336〔译自DBA,1985,嫂 (15),85一07553〕 成功地培养了檀香和印度娃几藤(重要药用植物)2个植物系统的原生质体,并获得了再生植株。用酶液处理叶肉细胞,分离花生(Arachish夕力ogaea)的原生质体,产生了…     

生物防治

924473从摇瓶到发酵镶:大规模昆虫细胞培养的挑战〔会,英〕/Belisle,B.W.…f In Vitro一1992,25(s),Pt.2。一43A〔译自DBA,1092,11 (11),92一06235〕 棒状病毒作为生防因子,要满足市场要求,还需克服成本高的问题。采用经调整的市售无血清培养基,研究了在5个鳞翅目细胞系中小规模、大规模生产胞外及包含体LdMNPV(舞毒蛾核多角体病毒)和AcMNPv(首稿银纹夜蛾核多角体病毒)。采用AeMNPV和草地夜蛾(S,o己。尹tera fr。夕玄-尹”己a) Sfg细胞作为模型系统,培养细胞,规模为100ml~401。细胞密度和存活率达到相当于所报道的含血清培养基的…     

梅花鹿体细胞航天诱变后的生物学特性变化分析

本研究以自主研发的聚偏二氟乙烯膜(PVDF)细胞培养技术进行了梅花鹿(Cervus nippon)体细胞的航天培养及生物诱变,并分析了其细胞在航天诱变后的生长曲线和核型特性。采用耳缘皮肤组织块贴壁培养的方法进行体细胞的原代培养;胰蛋白酶消化的方法完成细胞的传代培养;依照程序降温的方法完成细胞的冷冻保存;每天计数24孔板每孔的活细胞数并利用Excel 97-2003绘制出细胞的生长曲线,采用IBM SPSS Statistics 23统计软件回归分析程序中的非线性回归分析子程序Logistic曲线模型分析细胞生长曲线的拟合度;以常规染色体标本制备技术,利用细胞遗传工作站(AI)软件对梅花鹿体细胞的染色体核型进行分析。实验结果表明,新开发的PVDF膜细胞培养技术可有效保持梅花鹿体细胞在太空飞行过程中的存活并成功回收、传代建系。通过细胞传代培养观察发现,该体细胞经航天诱变后保持正常的成纤维细胞特征,生长曲线呈"S"型,航天诱变组体细胞在培养4 d进入对数生长期,与对照组体细胞在培养2 d进入对数生长期相比其细胞增殖速度减慢。拟合生长曲线分析表明,航天诱变组细胞生长拟合度值与对照组相似,但细胞增殖拐点时间、拐点细胞量分析结果证明,航天诱变后的体细胞增殖速度减慢。核型分析显示,航天诱变梅花鹿体细胞染色体数保持2n=66,染色体形态正常,核型为66(XX),其中32对常染色体,1对性染色体。本研究建立了哺乳动物在航天运行条件下的细胞培养PVDF新技术,并分析了航天诱变对于梅花鹿细胞生物学特性变化的影响,为以后进行相关研究提供了基础信息和技术支持。     

悬浮培养CHO 细胞生产重组人促红细胞生成素条件的优化*

目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO)。方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量。结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础。结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本。     

组成型过量表达hTERT 对Vero 细胞体外培养生物学特性的影响

目的:考察组成型过量表达人端粒酶催化亚单位(hTERT)对Vero细胞在无血清培养体系中的细胞形态、生长和代谢的影响。方法:以组成型过量表达hTERT的Vero细胞系T1为研究对象,以活细胞密度和细胞活力为主要观察指标,结合细胞形态和贴附伸展动态,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养和悬浮培养体系中的细胞生长;以葡萄糖比消耗速率(qglc)、乳酸比生成速率(qlac)、乳酸转化率(Ylac/glc)和谷氨酰胺比消耗率(qgln)为反映细胞代谢的主要观察指标,考察T1细胞和野生型Vero细胞在静止贴附培养、微载体固定化培养的细胞代谢。结果:hTERT组成型过量表达在降低Vero细胞的贴附伸展能力和对血清的依赖程度的同时,提高了细胞无血清批次培养后期的细胞活力和活细胞密度,并赋予了T1非贴附依赖性生长的能力。hTERT组成型过量表达未对Vero细胞的代谢产生明显的影响。结论:hTERT组成型过量表达可降低Vero细胞的贴附生长依赖性和对血清的依赖程度,是有应用潜力的改良哺乳动物细胞体外培养性状的技术途径。     

Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.

Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.     

Comparative characterization of growth and recombinant protein production among three insect cell lines with four kinds of serum free media

Three insect cell lines, Sf9, Sf21 and Tn5B1-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was appropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5B1-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3. times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammonium ion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line, respectively. The maximum specific β-galactosidase production rate was 4.5. fold that of the Sf9 cell line, a 3 times higher protease activity per cell.     

Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins

Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1-4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1-4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1-4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1-4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.     

Maximizing production of estrogen receptor beta with the baculovirus expression system

Steroid hormone/nuclear receptor expression in cultured insect cell lines is routinely driven by a baculovirus vector. An advantage of the baculovirus production of these receptors is that large amounts of functional receptors are obtained for subsequent in vitro studies. Most laboratories produce nuclear receptors in Spodoptera frugiperda (Sf)9 cells. However, no one has determined whether this cell line is optimal for the production of any nuclear receptor. We compared the time course and level of estrogen receptor beta (ER beta) production from a baculovirus in two S. frugiperda cell lines, IPLB-SF21AE (Sf21) and Sf9, and two Trichloplusia ni cell lines, Tn368 and BTI-TN5b1-4 (High Five). Cells were harvested at various times (0.5-5 days) after infection. ER beta expression and activity was determined by specific [3H]estradiol (E2) binding, Western blot analysis, and estrogen response element (ERE) binding in vitro. The highest functional, bioactive ER beta expression both at the earliest time after infection and in the amount of ER beta produced/cell was with the Sf21 cell line. Baculovirus expressed ER beta-bound EREs with high affinity in a DNA sequence-dependent manner. We conclude that Sf21 cells are the best-suited cells for ER beta production.     

Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion

Summary The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9^P35AcV5-1 and Sf9^P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9^P35AcV5-1 and Sf9^P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.     

Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: influence of ammonia, time of harvest, temperature, and dissolved oxygen

Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this possibility in the insect cell/baculovirus system, structures of N-linked oligosaccharides attached to SEAP (human secreted alkaline phosphatase), expressed under various culture conditions in BTI Tn5B1-4 cells, were characterized using FACE (fluorescence-assisted carbohydrate electrophoresis). Parameters varied were time of harvest, ammonia added during infection, dissolved oxygen, and temperature. It was found that glycosylation in the insect cell/baculovirus expression system is a robust, stable system that is less perturbed by variations in culture conditions than the level of protein expression. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increased the amount of alpha-mannosidase resistant structures from 4.1% at 34 hours postinfection (h pi), to 5.0% at 100 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to N-acetylhexosaminidase digest, although a small amount was insensitive to both mannosidase and N-acetyl-hexosaminidase digests. Lowering the temperature from 28 degrees C to 24 degrees C or even 20 degrees C, resulted in a twofold increase in oligosaccharides containing terminal alpha(1,3)-mannose residues. This condition did not affect the amount of mannosidase-resistant structures. However, this could result in more complete glycosylation of recombinant proteins in the BTI Tn5B1-4 cell line, because more structures with the potential for further processing would be produced.     

Improved production of recombinant tumstatin in stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

We describe the expression and in vitro activity of recombinant tumstatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells. Recombinant tumstatin was secreted into a culture medium with a molecular weight of 29 kDa. Recombinant tumstatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant tumstatin expressed in stably transformed Tn 5B1-4 cells was approximately 0.76 microg/ml. A maximum production level of 4.0 mg/l recombinant tumstatin was obtained in a T-flask culture of Tn 5B1-4 cells, 6 days after cultivation. We also investigated the individual effects of both dimethyl sulfoxide (DMSO) and sodium butyrate on recombinant tumstatin production in stably transformed Tn 5B1-4 cells. Supplementing cultures with DMSO and sodium butyrate separately increased recombinant tumstatin production in stably transformed Tn 5B1-4 cells by 117 and 32%, respectively.     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1–4 CELLS

Abstract  The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B1–4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins, however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B1–4 cells. The growth characteristics, productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B1–4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing ß -galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B1–4 cells.     

Expression and characterization of recombinant beta-secretase from Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Gal beta1,4-GlcNAc alpha 2,6-sialytransferase

Beta-Secretase (betaSEC) was expressed in Trichoplusia ni BTI Tn5B1-4 (Tn5B1-4) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Gal beta1,4-GlcNAc alpha 2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 57 to 59 k Da. A lectin blot analysis indicated that recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells (Tn5B1-4 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two-dimensional electrophoresis revealed that recombinant beta-secretase from Tn5B1-4 beta SEC/GalT-ST cells had a lower isoelectric point than beta-secretase from control Tn5B1-4 betaSEC cells (Tn5B1-4 cells transformed only with beta-secretase cDNA). The enzyme activity of recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells was enhanced up to 77% compared to control Tn5B1-4 betaSEC cells. The concentrations at half-maximum inhibition (IC(50)) values estimated from inhibition analyses using purified beta-secretases from Tn5B1-4/betaSEC and Tn5B1-4/betaSEC/GalT-ST cells were 32 and 290 nM, respectively.