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1.
When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO. 相似文献
2.
本文以我国重要水产养殖动物中国明对虾(Fenneropenaeus chinensis)贴壁培养和悬浮培养的血细胞、植块培养的类淋巴器官(Oka器官)细胞和卵巢细胞为材料,通过磷酸钙共沉淀法、脂质体介导的转染(脂染)和电穿孔法等多种方法进行了导入EGFP基因的实验。结果表明,通过脂染可以成功地将质粒DNA导入悬浮培养的血细胞、植块培养的Oka器官细胞和卵巢细胞,并使报告基因EGFP得到表达。 相似文献
3.
Serum-free large-scale transient transfection of CHO cells 总被引:11,自引:0,他引:11
Derouazi M Girard P Van Tilborgh F Iglesias K Muller N Bertschinger M Wurm FM 《Biotechnology and bioengineering》2004,87(4):537-545
To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 x 10(6) cells/ml were transfected with 2.5 microg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10 相似文献
4.
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。 相似文献
5.
Pham PL Perret S Doan HC Cass B St-Laurent G Kamen A Durocher Y 《Biotechnology and bioengineering》2003,84(3):332-342
Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection. 相似文献
6.
适于无血清贴壁培养的抗凋亡宿主细胞系CHO-IVB2的构建 总被引:3,自引:0,他引:3
应用无血清培养基培养CHO细胞时,由于没有血清提供各种贴壁因子,细胞以悬浮的方式生长。在实际的大规模细胞培养中,CHO细胞往往以贴壁方式培养,要么贴壁于悬浮的微载体中,要么贴壁于固定的聚酯盘状介质或中空纤维中,而很少直接悬浮于培养基中。在无血清培养基中,Vitronectin单一组分可以促使CHO细胞的贴壁和扩增。通过双表达lgf-1和Bcl-2基因,已经构建了可以在无蛋白培养基IMEM中抗凋亡生长的细胞株CHO-IB3。在此基础上,构建了可以同时表达Igf-1、Vitronectin和Bcl-2三个蛋白的三顺反子表达载体pCI—NII—IVB。将该载体转染于CHO—dhfr^-细胞中,构建了一个细胞株CHO—IVB2。该细胞株可以在无蛋白培养基中抗凋亡生长,适于以贴壁的方式大规模培养,用于大量生产外源目的蛋白。 相似文献
7.
Tsoncheva VL Todorova KA Ivanov IG Maximova VA 《Zeitschrift für Naturforschung. C, Journal of biosciences》2005,60(7-8):644-648
A simple method for spontaneous transfection into mammalian cells (both adherent and suspension in culture) with plasmid DNA is described. This method does not require any specific DNA carrier or technical device and can be applied for obtaining both transient and stably transfected cells. The efficiency of spontaneous transfection is slightly lower in comparison with that of the conventional calcium phosphate and lipofectin transfection methods and does not depend on the type of cell culture used. 相似文献
8.
The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells. 相似文献
9.
In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI)
to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes
were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI
transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method).
The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression
levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the
ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several
different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained
with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA
concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared
to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared
to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1
(CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression
in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production
of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments,
however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
Yasuhiro Tomooka Walter Imagawa Satyabrata Nandi Howard A. Bern 《Journal of cellular physiology》1983,117(3):290-296
The effect of lithium on the growth of mammary epithelial cells from adult virgin and midpregnant BALB/c or BALB/cfC3H mice was tested in a serum-free collagen gel culture system. The serum-free medium consisted of a 1:1 mixture of Ham's F12 and Dulbecco's Modified Eagle's medium supplemented with insulin, transferrin, cholera toxin, epidermal growth factor (EGF), and bovine serum albumin fraction V (BSA V). A multifold increase in cell number occurred during 10–12 days of culture in this medium. In dose-response studies in which the concentration of each component of this serum-free medium was varied in turn, the addition of LiCL (10 mM) enhanced growth at most concentrations of each factor. However, LiCL could not enhance growth in the absence of insulin or BSA V, but could replace EGF. The optimal concentration of LiCl was 5–10 mM; higher concentrations (20–80 mM) were toxic. KCl (1–10 mM) when added to the serum-free medium slightly stimulated growth; the addition of NaCl to the medium had little effect on growth. LiCl did not enhance the growth of cells from spontaneous mammary tumors of BALB/cfC3H mice. 相似文献
11.
Dame MK Spahlinger DM DaSilva M Perone P Dunstan R Varani J 《In vitro cellular & developmental biology. Animal》2008,44(7):245-252
Skin from Gottingen minipigs was used as a source of tissue for organ and cell culture and compared to human skin for growth conditions and sensitivity to irritants. Optimal organ culture conditions were determined, based on the preservation of the histological structure. These included serum-free, growth factor-free conditions with a calcium concentration of 1.5mM. Formulations in which the calcium concentration were low (0.075-0.15mM) failed to support tissue viability (even in the presence of dialyzed serum). Epidermal keratinocytes were grown from tissue explants and as single cells from enzyme-disrupted tissue. Optimal keratinocyte growth was achieved using a serum-free, growth factor-supplemented culture medium with a calcium concentration of 0.15mM. Fibroblasts were optimally grown from explant cultures using a medium with 1.5mM calcium and 10% fetal bovine serum. The conditions that were optimal for maintenance of intact pig skin, as well as for the isolated cells, are the same conditions that have been shown previously to be optimal for intact human skin and skin cells. In additional studies, pig skin keratinocytes and fibroblasts were exposed to a panel of contact irritants and contact sensitizers. Using growth inhibition as the response, the median effective dose values with each agent were very similar to the values previously determined for human epidermal keratinocytes and human dermal fibroblasts. Taken together, these data suggest that the skin from the Gottingen minipig can be used as a surrogate for human skin in ex vivo skin safety studies. 相似文献
12.
瞬时基因表达可溶性的VEGFR2: I-IV 总被引:1,自引:0,他引:1
通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。 相似文献
13.
通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。 相似文献
14.
Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening. 相似文献
15.
犬肾细胞MDCK无血清贴壁及单细胞悬浮培养 总被引:1,自引:0,他引:1
旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程,以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象,对2 656个可编码区进行分析,获得特异性靶点序列,并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性,筛选获得9个DNA序列,并设计了4对引物。经验证2对引物的特异性较好,其中引物SA3的基因组DNA检测限为13.7 fg/μL,菌体检测限为9.25×102 CFU/mL。结果验证 相似文献
16.
The effect of the glucocorticoids, insulin, and glucose concentration on glycogen deposition in adult rat liver parenchymal cells maintained in a chemically defined, serum-free medium has been studied. Increasing the medium concentration of glucose from 5.6 mM to 30.6mM in the absence of hormones increased cellular glycogen content from 6.5 to 51 μg of glycogen per mg of cell protein. Treatment of the cells with insulin increased the glycogen content by 15 to 30% at medium glucose concentrations above 10.6 mM. The addition of the synthetic glucocorticoid, dexamethasone, to the culture medium resulted in 40 to 105% increases in glycogen content at glucose concentrations greater than 5.6 mM. The addition of dexamethasone and insulin together in the culture medium resulted in an increase in glycogen content that was greater than the additive effect of each hormone alone. This established that glucose concentrations above 10.6 mM stimulate glycogen deposition in the absence of any hormonal stimulus. In addition, glucocorticoids directly stimulate glycogen deposition at glucose concentrations which are greater than physiological (5.6 mM). 相似文献
17.
近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h? 相似文献
18.
Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium 总被引:6,自引:0,他引:6
C A Reznikoff L J Loretz D M Pesciotta T D Oberley M M Ignjatovic 《Journal of cellular physiology》1987,131(3):285-301
Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
19.
Calfection: a novel gene transfer method for suspension cells 总被引:2,自引:0,他引:2
We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates. 相似文献
20.
Ding Huang Wen-Juan Peng Qian Ye Xu-Ping Liu Liang Zhao Li Fan Kang Xia-Hou Han-Jing Jia Jian Luo Lin-Ting Zhou Bei-Bei Li Shi-Lei Wang Wen-Ting Xu Ze Chen Wen-Song Tan 《PloS one》2015,10(11)
Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted. 相似文献