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1.
The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×106 to 6–12×106 cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.  相似文献   

2.
The polyamine spermine was assessed for enhancement of IgM production by human-human hybridoma, HB4C5 cells, under serum-free conditions. IgM production of HB4C5 cells was stimulated approximately 6-fold by the addition of 7.3 mM of spermine. The facilitating effect was observed soon after inoculation. In spite of suppression of cell growth, the IgM production rate was accelerated for at least 5 days without medium change. Moreover, laser confocal microscopic analysis revealed that the IgM content inside HB4C5 cells was increased by spermine treatment. These findings suggest that spermine enhances specific IgM productivity of the hybridoma line. Spermine also facilitated IgM production by human peripheral blood lymphocytes under serum-free conditions. This result implies that spermine enhances immunoglobulin production of not only specific hybridoma lines, but also non-specific immunoglobulin producers. Immunoglobulin production stimulating activity of spermine was accelerated 2-fold by the addition of DNA whith a chain length of about 400–7000 base pairs (bp). However, degraded short-chain DNA fragments (less than 200 bp) did not facilitate the immunoglobulin production stimulating activity of spermine. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

3.
Kim HG  Sung KD  Ham MS  Chung KH  Chung KH  Lee HY 《Cytotechnology》1995,17(3):165-172
The addition of 2.8 g/ml algal extracts enhanced both scu-PA production and cell growth in a serum-free medium, compared to a conventional serum-free medium for the cultivation of recombinant CHO cells. The growth rate and scu-PA production were relatively lower in the serum-free medium than 5% serum containing medium: however, specific scu-PA production rate was higher in the serum-free medium due to the long-term period of cultivation (3.66×10–4 vs. 2.48×10–4 IU/cell/day). Overall scu-PA production rate was also greater in an enforced serum-free medium as 25,000 IU/day over 50 d of perfusion cultivation. The conversion ratio of scu-PA to tcu-PA was greatly reduced in the serum-free medium during perfusion cultivation (10% compared to 20% conversion in a serum containing medium).  相似文献   

4.
Protein production capabilities ofTrichpolusia ni (TN 368) cells andSpodoptera frugiperda (Sf9) cells were compared in GTC100 medium in suspension culture using as a vector a genetically engineeredAutographa californica nuclear polyhedrosis virus. TN 368 produces more -galactosidase than Sf9, on a per cell basis (2.2×105 and 1.7×105 units/ 106 cells1 respectively). In growth experiments serum-free medium supported a higher maximum Sf9 cell density (4±1×106 cells/ml) than the serum- based media (1.5±5×106 cells/ml in GTC100 and 2±1×106 cells/ml in TNM-FH). However, using a cell density of 5×05 cells/ml, the productivity per cell varied, from a low of 4.5×104 units in EX-CELL-400 medium to a high of 7.6×104 units in TNM-FH. The TN 368 cells were twice a large as Sf9 cells and appeared to be more shear sensitive than Sf9 cells.  相似文献   

5.
Highly purified recombiaant basic fibroblast growth factor (rbFGF) and acidic FGF (aFGF) stimulated the proliferation of human-human (h-h) hybridomas to the extent of over four-fold from a low cell density such as 1×103 cells per ml in a serum-free medium in 24-well plates. The stimulatory effect of rbFGF was also observed in various lymphoid cell lines. Expecting that FGF could be an autocrine growth factor, we introduced bFGF gene into a h-h hybridoma using an expression plasmid induced by dexamethasone. The transformed cells thus obtained, HPO-75.11bbFGF-7, were able to grow well from a low inoculum density in a serum-free medium and antibody production was also increased when bFGF gene expression was induced. The transformed cells could grow at clonal density in a serum-free medium in 96-well plates, though the original cells could not. We also obtained a more practical transfectant, HPO-75.29-H74, using a high-shear stress adapted clone as the recipient and an expression plasmid having bFGF gene under the control of metallothioneine-I promoter. The HOP-75.29-H74 cells were capable of growing and producing human monoclonal antibody against hepatitis B virus surface antigen from an inoculum density of 1×103 cells per ml in an agitation vessel without addition of an inducer.  相似文献   

6.
The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 g/ml lactoferrin, 10M ethanolamine, 35 /ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.Abbreviations MoAb monoclonal antibody  相似文献   

7.
Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×107 cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×107 cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.  相似文献   

8.
Keen MJ  Rapson NT 《Cytotechnology》1995,17(3):153-163
A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11* cells which had been engineered to express a humanised antibody, CAMPATH*-1H, were routinely grown using serum-containing medium. From a seeding density of 105 cells ml–1, cells grown in static culture with serum reached a maximal cell density of 6.5×105 cells ml–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l–1 after 11 days in culture. CHO 3D11* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×105 cells ml–1 reached a maximal viable cell density of 2.16×106 cells ml–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids  相似文献   

9.
Immunoglobulin production stimulating activity of polyamines was investigated. Spermidine, thermine and triethylenetetraamine (TETA) stimulated IgM production of human–human hybridoma, HB4C5 cells under serum-free condition. IgM production of HB4C5 cells was accelerated 5.9-, 5.3-, and 3.7-fold by spermidine at 4.5 mM, thermine at 2 mM and TETA at 2.5 mM, respectively. However, putrescine did not enhance IgM production. Spermidine enhanced IgM productivity of the hybridoma cells in spite of its growth suppression activity. TETA also inhibited cell proliferation and the effect on the acceleration of IgM productivity disappeared during 5 days because of its cytotoxicity. On the other hand, thermine facilitated IgM productivity of the hybridoma cells without growth suppression. The laser confocal microscopic analysis revealed that IgM content inside HB4C5 cells was increased by thermine. This result suggests that thermine facilitates IgM synthesis in hybridoma cells.  相似文献   

10.
Summary Five mouse-human hybridomas, H2, H3, V1, V2 and V6 cells secreting anti-virus human monoclonal immunoglobulin G (IgG) were cultured in serum-free media at high density in a settling perfusion culture vessel with an inner cell sedimentation zone. The H2, H3 and V6 cells reached a density of 107 cells/ml in 0.5% (w/v) BSA-ITES-eRDF (see Materials and methods). The H2 cells reached only 6.8 × 106 cells/ml in the absence of bovine serum albumin (BSA), but the addition of 0.2% (w/v) Pluronic F68 increased the maximum cell density to 1.1 × 107 cells/ml, which was the same level as in BSA including medium. On the other hand, Pluronic F68 showed no stimulative effect on the growth of H2 cells in static culture. Pluronic F68 also increased the maximum cell density of V2 cells from 4.6 × 106 cells/ml to 6.9 × 106 cells/ml even in the presence of 0.5% (w/v) BSA.  相似文献   

11.
Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×107 cells/ml, when the specific perfusion rate was set to 2.3 vol day-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.  相似文献   

12.
We screened for immunoglobulin production stimulating factors (IPSFs) in polysaccharides using human-human hybridoma cells, HB4C5, cultured in serum-free medium. Among polysaccharides, citrus pectin, locust bean gum, and chitosan stimulated IgM production of HB4C5 cells. Especially chitosan showed the strongest IPSF activity; 100 ng/ml of chitosan stimulated IgM production approximately 5-fold. Chitosan had several characteristics as IPSF, as follows. 1) For the IPSF activity, 70-90% deacetylation was essential. 2) Chitosan oligomers (n = 5, 6, 7) and chitin oligomers (n = 5, 6, 7) showed no IPSF activities. 3) The IPSF activity of chitosan was inhibited by glucosamine, one of the constitutive sugars of chitosan. 4) Chitosan stimulated IgM production of human lymphocytes in serum-free culture, but not IgG or IgA, nor in serum-supplemented culture.  相似文献   

13.
A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 104 cells/ml to 1.6 × 106 cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 107 cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 106 cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 109 cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.  相似文献   

14.
A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator — an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day–1 had been reached, viable cell density stabilized at around 10×106 cells ml–1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 g 1×106 cells–1 h–1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production –100 mg l–1 d–1.  相似文献   

15.
Hu S  Deng L  Wang H  Zhuang Y  Chu J  Zhang S  Li Z  Guo M 《Cytotechnology》2011,63(3):247-258
The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 105 cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.  相似文献   

16.
Keen MJ 《Cytotechnology》1995,17(3):193-202
Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×106 cells ml–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×105 cells ml–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×106 cells ml–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum  相似文献   

17.
The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×107 cells ml–1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.  相似文献   

18.
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   

19.
Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U ml–1 (2 ng ml–1) of the interleukin.Abbreviations IL-1,2, and 6 interleukin-1, 2 and 6 - rhIL-6 recombinant human interleukin-6 - MCAb monoclonal antibody - TNP trinitrophenyl - unit (U) of interleukin-6 A unit (U) is equivalent to the amounts of IL-6 which gives one-half maximal IgM secretion by SKW6-CL4 cells (1U ml–1=200 pg ml–1)  相似文献   

20.
For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 107 cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.  相似文献   

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