共查询到20条相似文献,搜索用时 0 毫秒
1.
CHO工程细胞 (11G-S) 悬浮培养的无血清培养基的设计 总被引:1,自引:1,他引:1
以悬浮适应的表达重组尿激酶原 (Pro-urokinase,pro-UK) CHO工程细胞系11G-S为对象,采用Plackett-Burman实验设计及响应面分析法,设计支持CHO工程细胞 (11G-S) 悬浮生长的无血清培养基。以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计对影响细胞生长的培养基添加成分进行考察,确定了3种对细胞生长明显促进作用的培养基添加成分:胰岛素、转铁蛋白及腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种适用于CHO工程细胞 (11G-S) 悬浮培养的无血清培养基SFM-CHO-S。11G-S细胞在SFM-CHO-S批次悬浮培养的细胞最大生长密度达到4.12×106 cells/mL,pro-UK的最大累积活性达到5 614 IU/mL,培养效果优于商品化的同类无血清培养基。 相似文献
2.
Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA
Analysis of variance
- BSA
bovine serum albumin
- CHO
Chinese hamster ovary
- FBS
fetal bovine serum
- MTT
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
- PBS
phosphate buffered saline
- SFM
serum-free medium 相似文献
3.
Eun Jung Kim No Soo Kim Gyun Min Lee 《In vitro cellular & developmental biology. Animal》1999,35(4):178-182
Summary To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical
optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10% serum
medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium
was prepared by supplementing Dulbecco’s modified Eagle’s medium and Ham’s nutrient mixture F12 with hypoxanthine (10 mg/l)
and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting
abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine
were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated
by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 × 105 cells/ml, a maximum viable cell concentration of 6.4×105 cells/ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1×105 cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium.
In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique
and weaning of cells. 相似文献
4.
Development of a serum-free medium for the production of humanized antibody from chinese hamster ovary cells using a statistical design 总被引:1,自引:0,他引:1
Eun Jung Kim No Soo Kim Gyun Min Lee 《In vitro cellular & developmental biology. Animal》1998,34(10):757-761
Summary To develop serum-free (SF) media for the production of humanized antibody from recombinant Chinese hamster ovary (rCHO) cells,
a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing
α-minimal essential medium (α-MEM) with Fe(NO3)3·9H2O, CuCl2, ZnSO4·7H2O, and Na2SeO3 which are generally contained in SF medium formulations. Insulin, transferrin, and ethanolamine were also supplemented to
the basal medium to determine their optimal concentrations. From this statistical analysis, serine, phenylalanine, and tyrosine
were identified as important determinants for cell growth. Also, putrescine, linoleic acid, and hydrocortisone were shown
to be important for both cell growth and antibody production. The SF medium was formulated by supplementing the basal medium
with components showing positive effects on cell growth and/or antibody production. Cell growth and antibody production in
this SF medium were comparable to those in α-MEM supplemented with 5% dialyzed fetal bovine serum. Taken together, the results
obtained here show that a Plackett-Burman design facilitates the development of SF media for rCHO cells aimed at producing
a humanized antibody. 相似文献
5.
研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。 相似文献
6.
Jong Youn Baik Tae Kwang Ha Young Hwan Kim Gyun Min Lee 《Biotechnology progress》2011,27(6):1680-1688
To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 相似文献
7.
Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture,
and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free
medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous
growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine
growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced
it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal
cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free
medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and
large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
8.
Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary
to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems
used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have
been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The
Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale
production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been
evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target
protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information
needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3,
an SFM developed in our laboratory, as a CHO culture medium. When CHO cells were cultured in MED-3 instead of an SSM, several
genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like
growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several
key supplements-including insulin, transferrin, ethanolamine, and selenium-were removed from MED-3, theIGF expression was consistently down-regulated and cell growth decreased proportionately. Based on these results, we concluded
that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain
a high level ofIGF expression. The data presented in this study, therefore, might provide useful information for the design and development
of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally
for protein production in pharmaceutical and biomedical research. 相似文献
9.
Dyring C 《Cytotechnology》1997,24(3):183-191
A recombinant Chinese hamster ovary (CHO) cell clone, S1, stably expressing human insulin-like growth factor binding protein-1
(hIGFBP-1), was treated with polyethylene glycol (PEG), resulting in cell fusion, in order to further enhance the protein
expression by increasing the gene copy number and/or the amount of organelles important to the protein expression/-secretion.
Both the fused cell line, Peg1, and its mother cell line, S1, were adapted to serum-free growth in suspension and were characterised
with respect to growth and productivity. Peg1 was easier to adapt to the serum-free suspension conditions and had a higher
viability during the adaptation period than S1. Furthermore, Peg1 showed a stable productivity of hIGFBP-1 that was twice
as high as that for S1 under both adherent and suspension conditions. A considerable difference in the specific productivity
(up to 3–4 times) was noticed during the growth phase. PEG fusion experiments have earlier been studied in our laboratory
with CHO cells producing recombinant factor VIII and our results correlates very well with the results obtained with the factor
VIII producing cells. Surprisingly, it was possible to obtain high producing recombinant cell lines, which were stable for
more than 4 months.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
Mitsuo Satoh Shinji Hosoi Seiji Sato 《In vitro cellular & developmental biology. Plant》1990,26(11):1101-1104
Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or
aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells
increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular
proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide,
was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium.
In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although
amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases.
This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology
for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization). 相似文献
11.
Erkki Hltt Pirkko Pohjanpelto 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(4):321-327
We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed. 相似文献
12.
In this study, we found that adding sodium propionate to batch culture medium resulted in cell growth suppression in a dosedependent
manner, while it enhanced follicle-stimulating hormone (FSH) production in Chinese hamster ovary (CHO) cells. In a pseudo-perfusion
culture, with the exchange of fresh medium containing 2.5 mM sodium propionate, a final FSH of 532 μg was obtained, which
was approximately 1.5-fold higher than the control. Overall, the results demonstrate that the application of sodium proplonate
for the commercial production of recombinant proteins in rCHO cells is feasible. 相似文献
13.
Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis. 总被引:1,自引:0,他引:1 下载免费PDF全文
Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins. 相似文献
14.
Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media 下载免费PDF全文
Adaptation of dihydrofolate reductase (DHFR)‐deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time‐consuming and labor‐intensive process because nonadapted DHFR‐deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension‐adapted DHFR‐deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2–359 μM enhanced cell growth in in‐house CDM. Addition of 3.6 μM THF to in‐house CDM resulted in a more than 2.5‐fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9‐fold enhancement of maximum viable cell density. An anchorage‐ and serum‐dependent DHFR‐deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in‐house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension‐adapted DHFR‐deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR‐deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539–1546, 2016 相似文献
15.
16.
主要考察流加培养基中不同营养成分、流加起始时间及初始接种密度对11G-S细胞无血清流加培养的影响。在研究中以悬浮适应的表达尿激酶原 (Pro-urokinase,Pro-UK) CHO工程细胞系11G-S为研究对象,在100 mL的摇瓶中无血清悬浮流加培养11G-S细胞,同时以活细胞密度、细胞活力及Pro-UK活性为评价依据。结果表明在培养基中氨基酸、无血清添加成分及无机盐对促进细胞生长、细胞活力维持及蛋白表达起着较为重要的作用;且流加起始时间为72 h及初始接种密度为3×105~4×105 cells/ 相似文献
17.
Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium 总被引:2,自引:0,他引:2
Francois Gasser Philippe Mulsant Michel Gillois 《In vitro cellular & developmental biology. Plant》1985,21(10):588-592
Summary A new synthetic medium (referred to as GC3) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's
F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and
selenium (1×10−7
M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium)
also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading.
Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high
density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium. 相似文献
18.
19.
Kahina Mellahi Florian Cambay Denis Brochu Michel Gilbert Michel Perrier Sven Ansorge Yves Durocher Olivier Henry 《Biotechnology progress》2019,35(1):e2742
Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 6 cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 6 cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019 相似文献
20.
In order to develop a serum-free (SF) medium for the production of erythropoietin (EPO) by suspension culture of recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with Fe(NO3)3.9H2O, CuCl2 and ZnSO4.7H2O which are generally contained in SF medium formulations. Insulin, transferrin and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, glutamate, serine, methionine, phosphatidylcholine, hydrocortisone and pluronic F68 were identified as positive determinants for cell growth. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth in suspension culture. An EPO titer in this optimized SF medium was 79% of that in IMDM supplemented with 5% dialyzed fetal bovine serum (dFBS). Furthermore, the in vitro and in vivo biological activities of EPO produced in the SF medium were comparable to those produced in the serum-supplemented medium. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for the production of EPO by suspension culture of rCHO cells. 相似文献