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  • Articles  (28)
  • hybridoma  (28)
  • Springer  (28)
  • 2000-2004  (6)
  • 1995-1999  (22)
  • 1945-1949
  • Process Engineering, Biotechnology, Nutrition Technology  (28)
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  • Articles  (28)
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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 23 (1997), S. 231-239 
    ISSN: 1573-0778
    Keywords: apoptosis ; hybridoma ; amino acids ; starvation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two mouse hybridoma cell lines cultured in different basal media withthe iron-rich protein-free supplement were subjected to deliberatestarvation by inoculation into media diluted with saline to 50% or less.In the diluted media the growth was markedly suppressed and a largefraction of cells died by apoptosis. The cells could be rescued fromapoptotic death by individual additions of amino acids, such as glycine,L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine,L-histidine, D-serine, β-alanine or taurine. Amino acids withhydrophobic or charged side chains were without effect. The apoptosispreventing activity manifested itself even in extremely diluted media,down to 10% of the standard medium. The activity of L-alanine in theprotection of cells starving in 20% medium was shown also in semicontinuousculture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, andthe apoptotic index dropped from 37% in the control to 16%. It wasconcluded that the apoptosis-preventing amino acids acted as signalmolecules, rather than nutrients, and that the signal had a character ofa survival factor. The specificity of present results, obtained with twodifferent hybridomas, supports our view (Franěk and Chládková-Šrámková, 1995) that the membranetransport macromolecules themselves may play the role of therecognition elements in a signal transduction pathway controlling thesurvival of hybridoma cells.
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  • 2
    ISSN: 1573-0778
    Keywords: apoptosis ; Bcl-2 ; fixed-bed ; hollow fibre ; hybridoma ; perfusion ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 34 (2000), S. 131-139 
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-xL ; cell growth ; cell viability ; hybridoma ; myeloma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.
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  • 4
    ISSN: 1573-0778
    Keywords: batch culture ; conditioned medium ; growth ; hybridoma ; inoculum ; protein productivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Apart from gas concentrations, temperature, and pH, generally only the initial conditions can be manipulated in batch culture. Inoculum size and initial conditioned medium concentration represent two important considerations for optimal batch production. Two hybridoma cell lines were used to assess the impact of these initial conditions on population growth and monoclonal antibody productivity in suspension batch culture. Varying initial cell concentration over the range of 1.0 × 105 cells mL-1 to 3.0 × 105 cells mL-1 did not affect maximum product titre or maximum volumetric cell-hours attained. Initial percent of conditioned medium up to 40 percent strongly impacted on population growth and productivity, with initial levels of 30 to 40% conditioned medium reducing or eliminating lag phase and increasing average viable cell density. However, specific productivity and product titre declined with increasing initial percent conditioned medium, even on a per volume of fresh medium basis. Glutamine and glucose depletion or ammonia toxicity could cause depressed product titres when conditioned medium is used. Glutamine and glucose levels can easily be replenished in conditioned medium at minimal cost, and ammonia can be removed. Specific productivity was higher during cyclic batch operating mode than during batch operating mode. This may be because cyclic batch operating mode results in an incidental volume of conditioned medium at the beginning of each cycle. A two stage, cyclic-batch/batch operating mode can be employed to fully utilize medium and maximize product titre.
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  • 5
    ISSN: 1573-0778
    Keywords: glucose ; glutamine ; hybridoma ; nucleotides oxygen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of media concentrations of glucose andglutamine on the intracellular nucleotide pools andoxygen uptake rates of a murine antibody-secretinghybridoma cell line were investigated. Cells takenfrom mid-exponential phase of growth were incubated inmedium containing varying concentrations of glucose(0–25 mM) and glutamine (0–9 mM). The intracellularconcentrations of ATP, GTP, UTP and CTP, and theadenylate energy charge increased concomitantly withthe medium glucose concentration. The total adenylatenucleotide concentration did not change over a glucose concentration range of 1–25 mM but therelative levels of AMP, ADP and ATP changed as theenergy charge increased from 0.36 to 0.96. Themaximum oxygen uptake rate (OUR) was obtained in thepresence of 0.1–1 mM glucose. However at glucoseconcentrations 〉1 mM the OUR decreased suggestinga lower level of aerobic metabolism as a result of theCrabtree effect.A low concentration of glutamine (0.5 mM) caused asignificant increase (45–128%) in the ATP, GTP,CTP, UTP, UDP-GNac, and NAD pools and a doubling ofthe OUR compared to glutamine-free cultures. Theminimal concentration of glutamine also caused anincrease in the total adenylate pool indicating thatthe amino acid may stimulate thede novosynthesis of nucleotides. However, all nucleotidepools and the OUR remained unchanged within the rangeof 0.5–9 mM glutamine.Glucose was shown to be the major substrate forenergy metabolism. It was estimated that in thepresence of high concentrations of glucose (10–25 mM),glutamine provided the energy for the maintenance ofup to 28% of the intracellular ATP pool, whereas theremainder was provided by glucose metabolism.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 21 (1996), S. 111-120 
    ISSN: 1573-0778
    Keywords: hybridoma ; extracellular and intracellular amino acids ; glucose ; lactate ; batch culture ; enriched media
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This paper presents batch culture data of the murine hybridoma, AFP-27, cultured in conventional basal media and in a nutrient-rich modified version. Expression of antibody was fivefold higher in the enriched formulation, with significant product secretion in the decline phase. Cultures were initiated at conventional inculation densities (1 ∼ 2 × 105 viable cells ml−1) and high inoculation densities (1.5 ∼ 1.7 × 106 viable cells ml−1). Amino acid levels have been reported for all cultures, with apparent differences described. Relative levels of intracellular amino acids are also reported, with significant accumulation of proline, glycine and alanine. The results have significance in the design of enriched media which are clearly beneficial for commercial production of antibodies from hybridomas.
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  • 7
    ISSN: 1573-0778
    Keywords: cell counting ; CHO ; crystal violet ; hybridoma ; trypan blue ; Vero-cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Nuclear counts determined by crystal violet staining from samples of stationary or microcarrier cultures of hybridomas, CHO or Vero cells were consistently and significantly higher than cell concentrations determined by the trypan blue or Coulter counter methods. This difference was attributed to the presence of a significant proportion of binucleated cells, which are assumed to be 35% of the cell population in the stationary phase of Vero cultures. The proportion of such cells during exponential growth was variable. However, continuous sub-culture of these cells induced a degree of synchrony during growth which resulted in a cyclic variation of the difference between the cell and nuclei counting techniques. This data indicates that care should be taken in interpreting cell culture profiles based solely on crystal violet nuclei staining counts.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 26 (1998), S. 139-152 
    ISSN: 1573-0778
    Keywords: batch kinetics ; cell cycle ; cell-hours ; hybridoma ; population parameters ; productivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Several methods exist for assessing population growth and protein productivity in mammalian cell culture. These methods were critically examined here, based on experiments with two hybridoma cell lines. It is shown that mammalian cell culture parameters must be evaluated on the same basis. In batch culture mode most data is obtained on a cumulative basis (protein product titre, substrate concentration, metabolic byproduct concentration). A simple numerical integration technique can be employed to convert cell concentration data to a cumulative basis (cell-hours). The hybridoma lines used in this study included a nutritionally non-fastidious line producing low levels of MAb and a nutritionally fastidious hybridoma with high productivity. In both cases the cell-hour approach was the most appropriate means of expressing the relationship between protein productivity and cell population dynamics. The cell-hour approach could be used as the basis for all metabolic population parameter evaluations. This method has the potential to be used successfully for both prediction and optimization purposes.
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  • 9
    ISSN: 1573-0778
    Keywords: acyclic nucleoside phosphonate ; cell cycle ; hybridoma ; specific MAb production rate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20–27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration.
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  • 10
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; cell death ; hybridoma ; osmolarity ; pH ; shear ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses.
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  • 11
    ISSN: 1573-0778
    Keywords: antibody production ; human monoclonal antibody ; hybridoma ; retinoic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10-7 M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas.
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 17 (1995), S. 193-202 
    ISSN: 1573-0778
    Keywords: Y0 ; hybridoma ; myeloma ; protein-free ; culture medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract 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.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 21 (1996), S. 81-89 
    ISSN: 1573-0778
    Keywords: apoptosis ; hybridoma ; amino acids ; starvation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two mouse hybridoma cell lines cultured in different basal media with the iron-rich protein-free supplement were subjected to deliberate starvation by inoculation into media diluted with saline to 50% or less. In the diluted media the growth was markedly suppressed and a large fraction of cells died by apoptosis. The cells could be rescued from apoptotic death by individual additions of amino acids, such as glycine, L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine, L-histidine, D-serine, β-alanine or taurine. Amino acids with hydrophobic or charged side chains were without effect. The apoptosis preventing activity manifested itself even in extremely diluted media, down to 10% of the standard medium. The activity of L-alanine in the protection of cells starving in 20% medium was shown also in semicontinuous culture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, and the apoptotic index dropped from 37% in the control to 16%. It was concluded that the apoptosis-preventing amino acids acted as signal molecules, rather than nutrients, and that the signal had a character of a survival factor. The specificity of present results, obtained with two different hybridomas, supports our view (Franěk and Chládková-Šrámková, 1995) that the membrane transport macromolecules themselves may play the role of the recognition elements in a signal transduction pathway controlling the survival of hybridoma cells.
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  • 14
    ISSN: 1573-0778
    Keywords: apoptosis resistant ; bag–1 ; bcl–2 ; COS–1 ; hybridoma ; protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA-λ containing SV40 ori and immunoglobulin λ gene for transiently expressing λ protein. The bcl–2 expressing COS–1 cells produced more λ protein than the mock transfected COS–1 cells after 4 days posttransfection. Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants. Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone. Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.
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  • 15
    ISSN: 1573-0778
    Keywords: antibody consistency ; hollow fibre bioreactor ; hybridoma ; monoclonal antibody
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This paper analyses the performance of MAbMaxTM/TricentricTM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation.
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  • 16
    ISSN: 1573-0778
    Keywords: antibody productivity ; apoptosis ; BAG-1 ; Bcl-2 ; cell survival ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures.
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  • 17
    ISSN: 1573-0778
    Keywords: hybridoma ; glutamate ; cystine ; transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Glutamic acid was found to be growth inhibitory to a murinelymphocyte hybridoma in a concentration-dependent manner from 3to 12 mM glutamate. At 12 mM glutamate there was a 70% decreasein the specific growth rate of the cells. Attempts to alleviateinhibition or adapt cells to growth in glutamate-based mediawere unsuccessful. It is proposed that elevated glutamate levelsimpair adequate uptake of cystine, a critical amino acid for thesynthesis of glutathione. Glutathione is required by cells toprevent intracellular oxidative stress. The measured rate ofuptake of U-14C L-cystine into the cells was found to havethe following parameters: Km = 0.87 mM, Vmax = 0.9nmole/mg cell protein per min. The uptake was sodiumindependent and resembled the previously described x- ctransport system, with elevated glutamate levels causingextensive inhibition. Glutamate at a concentration of 1.4 mMcaused a 50% decrease in cystine uptake from the serum-freegrowth medium. Glutamate was taken up from the external medium(Km = 20 mM and Vmax = 12.5 nmole/mg cell protein permin) by the same transport system in a stereo specific, sodiumindependent manner. Of the amino acids examined, it was foundthat cystine and homocysteic acid were the most extensiveinhibitors of glutamate uptake and that inhibition was competitive. Metabolic profiles of the cells grown in culturescontaining enhanced glutamate levels revealed an overallincrease in net production of alanine, serine, asparagine andaspartate. A substantially increased specific consumption ofglutamate was accompanied by a decreased consumption of cystine,valine and phenylalanine.The combined kinetic and metabolic results indicate thatglutamate and cystine are taken up by the anionic transportsystem x- c. The increasing levels of glutamate in themedium result in a decreased transport of cystine by this systemdue to competitive inhibition by glutamate.
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  • 18
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    Cytotechnology 24 (1997), S. 213-218 
    ISSN: 1573-0778
    Keywords: gene deletion ; hybrid antibody ; hybridoma ; immunoglobulin light chain ; monoclonal antibody
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Monoclonal antibodies (mAbs) of the IgG class produced by mouse hybridomas raised with NS-1 myelomas have been shown to contain two types of immunoglobulin light (κ) chains derived from the myelomas and antigen-stimulated spleen lymphocytes, and the hybridomas produce three mAb species with light chain heterogeneity (Abe and Inouye, 1993). In the present study, 9 hybridoma lines secreting homogeneous mAbs have been isolated from 63 lines cloned from an established hybridoma line producing three mAbs. They secrete homogeneous mAbs containing light chains derived from either myeloma or spleen cells. They contain either κ gene derived from the respective cells, and the other gene was deleted during the cultivation. The deletion frequency of the κ gene of myelomas is 3 times higher than that of spleen cells, although 80–85% of hybridomas reach the stable state containing both κ genes.
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  • 19
    ISSN: 1573-0778
    Keywords: adaptation ; hybridoma ; monoclonal antibody ; protein free medium ; suspension culture ; weaning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A simple protein free medium was formulated and tested in suspension culture using three hybridoma cell lines. The medium, referred to as CDSS (Chemically Defined Serum Substitutes), consisted of the basal medium DMEM:Ham F12, 1:1, with HEPES (D12H), plus pluronic F68, trace elements, ferric citrate, ascorbic acid, and ethanolamine. No protein or lipid components were added. All three cell lines were weaned off serum using CDSS and a commercially available protein free medium PFHM-II. Data shown here indicated that normally cells took 1–7 weeks to wean off serum and an additional 2–7 weeks to adapt to suspension culture. After adaptation the cells were able to grow well in suspension culture using both protein free media and in the main performed better than serum containing controls. The stability of the three hybridoma cells for antibody production following freeze/thaw procedures and long term subculturing was also tested. All three lines were frozen using our protein free CDSS medium (containing 0.75% bovine serum albumin and 10% dimethyl sulfoxide) in liquid nitrogen for up to one year. Cells thawed from these stocks recovered well and were able to maintain good growth and antibody production characteristics. One line was shown to grow using our protein free CDSS medium in suspension culture for 12 weeks without loss of antibody productivity.
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  • 20
    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal antibody ; stirred tank perfusion culture ; potassium acetate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To increase the yield of monoclonal antibody in a hybridoma culture, it is important to optimize the combination of several factors including cell density, antibody productivity per cell, and the duration of the culture. Potassium acetate enhances the production of antibodies by cells but sometimes depresses cell density. The production of anti-(human B-type red blood cell surface antigen) antibody by Cp9B hybridoma was studied. In batch cultures, potassium acetate inhibited Cp9B cells growth and decreased the maximal cell density but the productivity of antibody per cell was increased. The balance of the two effects resulted in a slight decline of antibody production. In a stirred tank bioreactor, the inhibitory effect of potassium acetate on cell density was overcome by applying the perfusion technique with the attachment of a cell-recycling apparatus to the bioreactor. In such a reactor, potassium acetate at 1 g l-1 did not cause a decrease in the cell density, and the antibody concentration in the culture supernatant was increased from 28 μg ml-1 to 38 μg ml-1. Potassium acetate also suppressed the consumption of glucose and the accumulation of lactate in batch cultures, but the glucose and lactate levels were kept stable by applying the perfusion technique in the stirred tank bioreactor.
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  • 21
    ISSN: 1573-0778
    Keywords: antibody productivity ; apoptosis ; bcl-2 ; fed batchculture ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mouse hybridoma 2E3 transfected with human bcl-2 gene survived longer with increasing expression level of bcl-2 when cultured in DME medium supplemented with 9% serum. One of the transfectants, 2E3BCMGbcl-2, overexpressed bcl-2 and could maintain viable cell density higher than the initial density for more than four days at a low 0.5% serum concentration. In comparison a mock transfectant 2E3BCMG remained viable for only one day. However, both hybridomas died out within a day in serum-free medium. These results suggested that bcl-2 needed a small amount of some serum components to suppress apoptosis of the hybridoma. Overexpression of bcl-2 also suppressed apoptosis of the hybridoma induced by glutamine deprivation. When hybridoma 2E3BCMGbcl-2 was inoculated in DME medium supplemented with 9% serum and cultured for 10 d with additional 2% serum feed at day 4 of the culture, viable cell density increased 2-fold and antibody produced 3-fold, in comparison with mock transfected 2E3 cultured in the same manner. The mock transfectant with additional feed of serum at day 4 of the culture showed no difference in viable cell density and antibody production. These results suggested that the mock transfectant committed to apoptosis before day 4 of the culture and the additional serum at day 4 could not reverse the commitment.
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  • 22
    ISSN: 1573-0778
    Keywords: apoptosis ; Bcl-2 ; diluted medium ; hybridoma ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps.
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  • 23
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 30 (1999), S. 27-36 
    ISSN: 1573-0778
    Keywords: agitation ; fatty acids ; hybridoma ; linoleic acid ; lipid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The murine hybridoma (CC9C10) was subjected to high shear rates in a spinner flask to determine the effect of various culture additives on cell survival. At 500 rpm, the half-life of the viable cell concentration in a low protein serum-free medium was 50 min. Both bovine serum albumin and Pluronic F-68 had a significant effect in protecting cells under these conditions. The effects of the two supplements were additive, so that in the presence of both supplements there was minimal cell damage at 500 rpm. The survival rate of cells grown in media supplemented with linoleic acid improved significantly under high stirring rates. Cells grown for one passage in 50 μM linoleic acid and stirred at 500 rpm had a significantly higher survival rate than control cells. For cells grown over 5 passages in 25 μM linoleic acid, the survival rate at 470 rpm was ×3 greater than that determined for control cells. This difference gradually decreased at higher stirring rates up to 610 rpm when the half-life of the viable cell population was reduced to ∼10 min. Supplementation of cultures with linoleic acid has previously been shown to result in incorporation into all three cellular lipid fractions - polar, non-polar and free fatty acid (Butler et al., 1997). Our explanation for the increased survivability of the cells at high agitation rates in the presence of linoleic acid is that the structural lipid components of the cell including the outer membrane attained a higher unsaturated/saturated ratio which was more robust than that of control cells.
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  • 24
    ISSN: 1573-0778
    Keywords: antibody production ; human monoclonalantibody ; hybridoma ; lung cancer ; vitamin A acetate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The antibody productivity of the human–human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 μg/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 106 cells/ml. However, when the cell density was over 107 cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human–human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human–human hybridomas is discussed.
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  • 25
    ISSN: 1573-0778
    Keywords: fed batch ; hybridoma ; macromolecular composition ; monoclonal antibody ; substrate limitation ; target specific growth rate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.
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  • 26
    ISSN: 1573-0778
    Keywords: cell culture ; hollow fiber bioreactor ; hybridoma ; micro bioreactor ; optimization ; T-flask
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.
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  • 27
    ISSN: 1573-0778
    Keywords: CHO ; dissolved oxygen (DO) ; essential amino acids ; hybridoma ; intracellular amino acids ; Monod constants (KS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the KS-values for the essential amino acids were calculated using Monod equations via computer simulation. The KS-values were in the range of 10 mmol L−1 and the pool of most intracellular amino acids remained constant at about 10–100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected.
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  • 28
    ISSN: 1573-0778
    Keywords: adaptation ; antibody production rate ; hybridoma ; intracellular amino acids ; osmotic pressure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The time length required for the adaptation of AFP-27 hybridoma cells to high osmotic pressure and the effect of a gradual increase of osmotic pressure on monoclonal antibody production were investigated. When the cells were subjected to an increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg- 1, the intracellular content of osmoprotective free amino acids reached a maximum level 6 h after the osmotic pressure was increased to 366 mOsmol kg-1. The same time period of 6 h incubation at 366 mOsmol kg-1 was required to obtain a high growth rate of AFP-27 cells at 440 mOsmol kg-1 when the cells were subjected to a two-step increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg-1 and then to 440 mOsmol kg-1. The time length for the physiological adaptation of the cells to 366 mOsmol kg-1 was consequently estimated to be 6 h. Osmotic pressure during batch cultivation was gradually increased from 300 mOsmol kg-1 to 400 mOsmol kg-1 with an adaptation time of at least 6 h. The specific growth rates following a gradual increase of osmotic pressure were higher than those at a constant osmotic pressure of 400 mOsmol kg-1, while the specific monoclonal antibody production rate increased with the increase in the mean osmotic pressure. As a result, the cells grown under a gradual increase of osmotic pressure produced higher amounts of monoclonal antibodies than did those grown under constant osmotic pressure.
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