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1.
Chinese hamster ovary (CHO) cells were cultivated in a compact loop bioreactor using MEM-alpha medium supplemented with 10% fetal calf serum. Effects of physical and chemical environments, i.e., pH in the medium, stirring speed of impellers, temperature and partial pressure of oxygen (pO2) upon growth of suspended cells in the bioreactor were determined in batch cultures. Growth behavior was characterized by specific rates of growth (mu), glucose consumption (qG) and lactate production (qL), and the yield coefficients (cell yield from glucose, YX/G, and lactate yield from glucose, YL/G). An effect of medium osmolality was also evaluated with T-flask monolayer cultivation. The best growth was observed at pH 7.6, 37 degrees C, 400 rpm, 50-100% saturation with oxygen and 320 mOsmol kg-1. Corresponding to the previous work with a human melanoma cell line, the sophisticated cultivation and process control systems have been improved for CHO cells. 相似文献
2.
N Kurano C Leist F Messi C Gandor S Kurano A Fiechter 《Journal of biotechnology》1990,16(3-4):245-258
Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium. 相似文献
3.
Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10% fetal bovine serum (FBS) were adapted to 0.5% FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10(-7) M and 10(-8) M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5% serum proliferate, though at a slower rate than cells in 10% serum. Treatment with MMC revealed that at 10(-7) M, MMC is cytotoxic to cells to both the media; at 10(-8) M, MMC is non-cytotoxic to cells in both media. 相似文献
4.
Jae Choon Kim Jin Hyun Seong Brian Lee Yas Hashimura Daniel Groux Duk Jae Oh 《Biotechnology and Bioprocess Engineering》2013,18(4):801-807
Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The kLa value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a kLa value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 106 cells/mL) andwas similar to the STR (9.7 × 106 cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system. 相似文献
5.
The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar Km values for this lipid. 相似文献
6.
Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion
culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in
the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below
32°C resulted in growth suppression. However, specific productivity of FSH, q
FSH, increased as culture temperature decreased, and the maximum q
FSH of 43.4 ng/106 cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures,
we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 107 cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer
of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably
due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical
for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising. 相似文献
7.
Nyberg GB Balcarcel RR Follstad BD Stephanopoulos G Wang DI 《Biotechnology and bioengineering》1999,62(3):324-335
Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum-free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical "consistency index." The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate-supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account. 相似文献
8.
Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells. 相似文献
9.
High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium 总被引:2,自引:0,他引:2
A recombinant CHO cell line, CHO2DS, was immobilized on porous microcarrier Cytopore 1 and cultivated in 1 l modified Super-spinner and 2 l stirred tank bioreactor with the perfusion of a low-cost chemically defined protein-free medium DF6S. CHO2DS cells could enter into the inner space and grew both in the inner space and on the surface of Cytopore 1 in DF6S and produced prothrombin at 22 mg l–1 after 10 days. From a seeding density of 5.7 × 105 cells ml–1, the highest viable cell density of CHO2DS was 1.12 × 107 cells ml–1. 相似文献
10.
Vlajkovic SM Wang CJ Soeller C Zimmermann H Thorne PR Housley GD 《The international journal of biochemistry & cell biology》2007,39(4):810-817
Membrane-bound NTPDase2 is a member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family involved in the regulation of P2 receptor signaling. NTPDase2 has broad substrate specificity for extracellular nucleotides, but hydrolyses nucleoside 5'-triphosphates with high preference over nucleoside 5'-diphosphates. In this study, we have sought to determine how enzyme substrates acting on P2 receptors affect intracellular NTPDase2 trafficking. To achieve this, Chinese hamster ovary (CHO) cells were transiently transfected with rat-specific NTPDase2 cDNA tagged with green fluorescent protein (GFP), to allow direct visualisation of subcellular localisation and trafficking of NTPDase2. Cells were superfused with NTPDase2 substrates (ATP and UTP) and synthetic nucleotide analogues (ATPgammaS and ADPbetaS), and confocal image stacks were acquired at regular time intervals. NTPDase2 incorporation into the plasma membrane was determined by comparative analysis of fluorescence intensity in the cytosolic and membrane compartments. GFP-tagged NTPDase2 was fully functional and ATP and ATPgammaS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPbetaS were ineffective. The increased ATP hydrolysis rate correlated with increased NTPDase2 trafficking to the plasma membrane. ATP-induced NTPDase2 trafficking was mediated by activation of endogenous P2X receptors involving Ca2+ entry rather than by P2Y receptor-induced release of Ca2+ from intracellular stores. Our results suggest that P2X receptor activation stimulates insertion of latent NTPDase2 into the plasma membrane. The increase in surface-located NTPDase2 may reflect a regulatory mechanism counteracting excessive stimulation and desensitisation of P2 receptors. 相似文献
11.
We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20-30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+-triggered exocytosis that is both massive and rapid. Ca2+-triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells. 相似文献
12.
Objective
to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors.Results
Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 106 cells ml?1, while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 107 cells ml?1. Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l?1. Considering the LDH content of viable cells measured to be 110 U per 109 cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R2 ≥ 0.95) for the three types of cell populations.Conclusion
The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.13.
We have selected Chinese hamster ovary (CHO) cells resistant to infection by encephalomycarditis (EMC) virus. Thus far, we have obtained five lines resistant to EMC, all of which manifest different phenotypes. Three of the five are not persistently infected with virus, while two lines produce infectious virus and grow in its presence. The nonpersistently infected lines exhibit different resistance profiles to the other viruses we have tested, and they are stable in nonselective growth conditions. Their resistance appears to be due to a genetic alteration in the cell. 相似文献
14.
The glycosylation patterns of recombinant therapeutic glycoproteins can be engineered by overexpression of glycosyltransferases in the host cells used for glycoprotein production. Most prior glycosylation engineering experiments have involved constitutive expression of cloned glycosyltransferases. Here we use tetracycline-regulated expression of two glycosyltransferases, N-acetylglucosaminlytransferases III and V (GnTIII and GnTV) to manipulate glycoform biosynthesis in Chinese hamster ovary (CHO) cells and to study the effect of glycosyltransferase overexpression on this host. The amount of GnTIII and GnTV in these cells, and the glycosylation patterns of several cellular glycoproteins, could be controlled simply by manipulating the concentration of tetracycline in the culture medium. Using this system, it was found that overexpression of either GnTIII or GnTV to high levels led to growth inhibition and was toxic to the cells, indicating that this may be a general feature of glycosyltransferase overexpression. This phenomenon has not been reported previously, probably due to the widespread use of constitutive promoters, and should be taken into account when designing vectors for glycosylation engineering. The growth inhibition effect sets an upper limit to the level of glycosyltransferase overexpression, and may thereby also limit the maximum extent of in vivo modification of poorly accessible glycosylation sites. Also, such inhibition implies a bound on constitutive glycosyltransferase expression which can be cloned. 相似文献
15.
Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation. 相似文献
16.
When Chinese hamster ovary (CHO) cells are shifted from medium which contains serum into serum-free medium, they complete one cell doubling and arrest in the G1 phase of the cell cycle. During the first 72 hr of arrest, there is little change in intracellular adenosine 3':5'-phosphate (cAMP) level, and the cells retain their usual epithelial-like morphology. After 96 hr, the cAMP level doubles, the magnitude of the prostaglandin E1-induced changes in cAMP increases threefold, and the cells convert from a rounded, epithelial-like shape to an elongated, fibroblast-like form. The fact that these biochemical and cellular transitions are subsequent to the growth arrest shows that the cAMP increase is not the cause of the growth arrest but is consistent with a role for cAMP in the control of cell morphology. In addition, these changes point to the importance of the G1 phase for initiating cAMP-related events. 相似文献
17.
Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells. 总被引:13,自引:7,他引:6
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Six X-ray-sensitive (xrs) strains of the CHO-K1 cell line were shown to revert at a very high frequency after treatment with 5-azacytidine. This suggested that there was a methylated xrs+ gene in these strains which was structurally intact, but not expressed. The xrs strains did not complement one another, and the locus was autosomally located. In view of the frequency of their isolation and their somewhat different phenotypes, we propose that the xrs strains are mutants derived from an active wild-type gene. However, there is in addition a methylated silent gene present in the genome. Azacytidine treatment reactivated this gene. We present a model for the functional hemizygosity of mammalian cell lines, which is based on the inactivation of genes by de novo hypermethylation. In contrast to results with xrs strains, other repair-defective lines were found not to be reverted by azacytidine. 相似文献
18.
Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
《Process Biochemistry》2010,45(12):1845-1851
Chinese hamster ovary (CHO) cells are widely used in producing therapeutic proteins. Gene amplification techniques are frequently used in improving protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used for the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and found one BAC clone (Cg0031N14) containing a CHO genomic DNA sequence adjacent to Dhfr. The BAC clone contained a large palindrome structure with a small inverted repeat in the junction region. To investigate the effect of the palindrome structure derived from the BAC clone Cg0031N14 on Dhfr amplification in CHO cells, we constructed plasmids that contain part or the whole junction region of the palindrome structure. The transfected CHO DG44 cells containing part or the whole junction region of the palindrome structure could adapt quickly to high methotrexate (MTX) concentrations. Moreover, the cells containing the whole junction region of the palindrome structure showed a high ratio of GFP-positive cells during gene amplification. On the basis of these results, we estimated that the junction region plays an important role in gene amplification in CHO cells. 相似文献