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1
Damico, Nicole. "Preparing and Cloning a Natural Killer Cell Hybridoma". Youngstown State University / OhioLINK, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1004458025.
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Wilson, James Samuel. "Process intensification of hybridoma cell fermentation". Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/12155.
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Monoclonal antibodies can be produced in culture fluid by the fermentation of specificially selected hybridoma cells. Hybridoma cells exhibit suspension type fermentation characteristics and therefore the simplest method for large scale fermentation is that of the stirred tank fermenter. However, such is the growing demand for monoclonal antibodies, methods for increasing the production capacity of a commercial process are being developed. This study examines some of the current process intensification methods in relation to an established production facility. As well as examining the actual productivity increases possible with any method, the applicability of that method to a commercial environment is taken into account. Hollow-fibre systems are investigated, with a potential increase in productivity which was outweighed by the significant retooling and retraining costs. Gel Bead entrapment systems are shown to have great promise, as they can be readily placed into existing equipment and production methods. However, all methods examined, including alginate bead entrapment, were found unsuitable for hybridoma cell culture. A novel method for cell entrapment was developed, using an agarose/alginate gel mixture which allowed greatly improved growth and consistent antibody production. The entrapment method was examined in a continuous chemostatic system. This system was then scaled-up and applied to the existing facility, to give a 25L airlift operating in a chemostatic mode at a rate of 1.2-1.5 day-1.
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Amribt, Zakaria. "Macroscopic modelling of hybridoma cell fed-batch cultures with overflow metabolism: model-based optimization and state estimation". Doctoral thesis, Universite Libre de Bruxelles, 2014. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209279.
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Monoclonal antibodies (MAbs) have an expanding market for use in diagnostic and therapeutic applications. Industrial production of these biopharmaceuticals is usually achieved based on fed-batch cultures of mammalian cells in bioreactors (Chinese hamster ovary (CHO) and Hybridoma cells), which can express different kinds of recombinant proteins. In order to reach high cell densities in these bioreactors, it is necessary to carry out an optimization of their production processes. Hence, macroscopic model equations must be developed to describe cell growth, nutrient consumption and product generation. These models will be very useful for designing the bioprocess, for developing robust controllers and for optimizing its productivity.This thesis presents a new kinetic model of hybridoma cell metabolism in fed batch culture and typical illustration of a systematic methodology for mathematical modelling, parameter estimation and model-based optimization and state estimation of bioprocesses.
In the first part, a macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a nonlinear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed.
In the next step, the effort is directed to the maximization of biomass productivity in fed-batch cultures of hybridoma cells based on the overflow metabolism model. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder-Mead simplex optimization algorithm. Two different objective functions (performance criteria) are considered for optimization; the first criterion to be maximized is the biomass productivity obtained at the end of the fed-batch culture, the second criterion to be minimized is the difference between global substrate consumption and the maximum respiratory capacity.
The optimal multi exponential feed rate trajectory improves the biomass productivity by 10% as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture so as to control the hybridoma cells at the critical metabolism state, which allows maximizing the biomass productivity. The robustness analysis of optimal feeding profiles obtained with different optimization strategies is considered, first, with respect to parameter uncertainties and, finally, with respect to model structure errors.
Finally, the overflow metabolism model is used to develop an extended Kalman filter for online estimation of glucose and glutamine in hybridoma cell fed-batch cultures based on the considered available measurements (biomasses (on-line), lactate and ammonia (on-line or off-line)). The observability conditions are examined, and the performances are analysed with simulations of hybridoma cell fed-batch cultures. Glutamine estimation sensitivity is enforced by minimizing a cost function combining a usual least-squares criterion with a state estimation sensitivity criterion.
Doctorat en Sciences de l'ingénieur
info:eu-repo/semantics/nonPublished
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Faraday, David Brian Foster. "The mathematical modelling of the cell cycle of a hybridoma cell line". Thesis, University of Surrey, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341620.
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Costello, Mark Eugene. "Growth and productivity of hybridoma cell lines in vitro". Thesis, Manchester Metropolitan University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280629.
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El, Debs Bachir. "Functional single-cell hybridoma screening using droplet-based microfluidics". Strasbourg, 2011. http://www.theses.fr/2011STRA6182.
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µm de diamètre) pour la culture de cellules mammifères. On a utilisé ce système pour sélectionner spécifiquement des cellules hybridomes sécrétant des anticorps inhibant l’Enzyme de Conversion de l’Angiotensine. -1 (ECA-1). L’émulsion contenant les cellules encapsulées dans les gouttes a été incubé pendant 6 heurres pour obtenir une quantité considérable d’anticorps avec l’ECA-1. Ensuite, cette émulsion a été réinjectée dans une puce microfluidique, fusionnée avec des gouttes contenant un mélange réactionnel permettant l’obtention d’un signal fluorescent d° à l’activité de l’ECA-1. Les gouttes ayant une faible intensité de fluorescence ont été triées. Une variance considérable dans le taux d’expression d’anticorps a été constatée au niveau mono-cellulaire au sein d’une même lignée de cellules hybridomes o_ les cellules exprimant un taux élevé d’anticorps ont été isolées et cultivées. Ce système permet le criblage de 5_104 cellules par heure et pourra être utiliser pour le criblage de lymphocytes B non immortaliséesThis thesis describes a microfluidic platform allowing the functional screening of hybridoma cells on the single-cell level. In this system, individual cells from a heterogeneous population are encapsulated into aqueous microdroplets of a water-in-oil emulsion and assayed directly for the release of antibodies inhibiting drug targets. The microfluidic setup comprises a novel fully integrated chip which allows reinjection, fusion and sorting of droplets sufficiently large (~100 µm in diameter) for the cultivation of mammalian cells. We successfully used this device for the specific selection of hybridoma cells releasing antibodies inhibiting angiotensin converting enzyme-1 (ACE-1). After cell encapsulation, the resulting emulsion was incubated off-chip for 6h to obtain significant antibody concentrations. Subsequently, the droplets were reinjected into another chip, fused with a second droplet species containing all components of a fluorescence assay for ACE-1 activity, and droplets with low fluorescence intensity (indicating ACE-1 inhibition) were sorted. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be sorted and recultivated. The approach enabled screening more than 5_104 cells per hour and should even be applicable to non-immortalized primary B-cells, as no cell proliferation is required
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Okerlund, Linda Susan. "Energy consumption among static and proliferating hybridoma cell populations". Diss., The University of Arizona, 1991. http://hdl.handle.net/10150/185447.
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To investigate the effects of proliferation on metabolism and cell product yields, proliferating and growth-limited EPOBF7 hybridoma cells have been compared as to their growth rates, energy demand, relative energy distributions, and monoclonal antibody (MAb) yield. Medium deprivation of leucine or serum was used to prevent growth. Energy consumption rates were determined in cell suspensions from rates of glucose consumption, lactate production, and oxygen utilization. In addition, the energy consumption of pathways critical to cell growth and survival were estimated from the relative decreases occurring in oxygen and glucose consumption upon pathway inhibition. The overall rate of energy consumption was significantly lower among growth-limited cultures. In addition, the distributions of oxidative and glycolytic energy among cellular synthetic pathways differed significantly between the culture conditions. Non-growing cultures also produced significantly lower antibody yields. Cell growth was also investigated using ³¹p nuclear magnetic resonance (NMR) spectroscopy of cells grown and maintained in bioreactor culture. Saturation transfer methods detected measurable transfer between inorganic phosphate (P(i)) and the gamma resonance of ATP. This transfer rate could be correlated with cellular growth rates within the reactor. Transfer of magnetization from the gamma resonance of ATP to P(i) was also detected, although the rate of this transfer did not appear to be related to the growth rate. The ratio of these transfer rates was consistently near 4. This information is believed to suggest the importance of other reactions through which ATP may donate its terminal phosphate. Cells in bioreactor culture were found to grow more slowly and produce lower levels of monoclonal antibody when compared to cells proliferating in suspension. such phenomena may be a function of diffusion limitations within the reactor such that the cells cannot obtain the nutrient supply required for optimal cell growth or product formation.
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Futch, William S. Jr. "Dissection of Microphage Activation Using T Cell Hybridoma Derived Lymphokines". VCU Scholars Compass, 1985. http://scholarscompass.vcu.edu/etd/4566.
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Macrophage phenotype/function can be modulated by various T-cell lymphokines (LK). The alteration of macrophage phenotype is a result of LK concentration, duration of exposure, and the level of macrophage activation when obtained from in vivo sources through elicitation by either sterile irritants or immune cellular mechanisms. In order to dissect macrophage activation into discrete signals T cell hybridomas were constructed by fusing HAT sensitive BW5147 cells with nylon-wool purified, con A stimulated T cells. The resulting T cell hybrids were screened for their ability to: (a) protect macrophages from the cytopathic effect of Naegleria; (b) induce class II MHC gene product (Ia antigen) expression; (c) increase cytostasis and tumoricidal activity; and (d) alter ectoenzyme profiles on either resident or thioglycollate (TG) elicited macrophages. Two hybridomas (T-3 and T-9) were selected for further evaluation because of their activity patterns. Supernatants from T-3 and T-9 were compared with cloned Y-interferon (γ-IFN) for alteration of biological activities. Both T-3 and T-9 were able to protect resident macrophage cells from Naegleria but had no protective effect on TG-macrophages. T-9 supernatant had patterns of activity similar to γ-IFN while T-3 patterns were different. The addition of anti-Y-IFN removed T-9 cytostatic activity while not affecting T-3 induced activity. The LK inducing protection from the cytopathic effect of Naegleria lysate is not γ-IFN but another molecular moiety. It was also shown that γ-IFN does not protect TG-macrophages from the destructive effects of adenylate cyclase produced by Bordetella pertussis. We conclude that activation of macrophages for the destruction of tumor cells and activation for protection against amoeba and bacteria occur via different biological pathways. Furthermore, we have proposed an association between the cell cycle and the responsiveness of resident and TG-elicited macrophages to specific LK.
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Balcarcel, R. Robert. "Effects of rapamycin and insulin on the cell cycle and apoptosis of hybridoma cell cultures". Thesis, Massachusetts Institute of Technology, 1999. http://hdl.handle.net/1721.1/85361.
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Rousseau, Fanny. "Systèmes microfluidiques pour la génération d'hybridomes et d'anticorps monoclonaux". Electronic Thesis or Diss., université Paris-Saclay, 2025. http://www.theses.fr/2025UPASQ013.
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Les anticorps sont des molécules produites par le système immunitaire, caractérisées par une grande spécificité de liaison pour un antigène donné, ce qui en fait des outils biologiques puissants pour des applications thérapeutiques et de diagnostic. La production en quantité d'anticorps monoclonaux in vitro est rendue possible en 1975 grâce à la technique des hybridomes. Bien que cette technique soit simple, facile à implémenter et peu coûteuse, ses faibles rendements limitent aujourd'hui son utilisation et ont donné lieu à des méthodes plus modernes de production d'anticorps, qui présentent toutefois de nouvelles limitations.Dans ce contexte, l'objectif de cette thèse est d'améliorer la technique des hybridomes actuelle afin de la repositionner comme une technologie innovante et attractive. En particulier, il s'agit d'implémenter trois dispositifs microfluidiques, intervenant aux différentes étapes du processus de production d'anticorps monoclonaux, pour en optimiser les rendements et en faciliter la procédure.La première partie de ce projet est consacrée à l'identification et à l'isolement des cellules sécrétrices d'anticorps issues de rates de souris immunisées. Après fusion avec les cellules de myélome, ces cellules favorisent la génération d'hybridomes sécrétant des anticorps spécifiques de l'antigène cible. Le but est ainsi de pouvoir réaliser des fusions cellulaires ciblées pour limiter la génération d'hybridomes non fonctionnels. Pour cela, après identification par cytométrie en flux à l'aide d'un panel de marqueurs membranaires ad hoc, l'isolement des cellules d'intérêt est réalisé à l'aide d'un tri magnétique intégré en puce microfluidique.La deuxième partie de la thèse porte sur le développement d'une puce microfluidique dédiée à la fusion cellulaire par voie chimique, en utilisant le polyéthylène glycol (PEG). Ce dispositif vise à optimiser les conditions de fusion entre des cellules de rate et de myélome, et à améliorer les rendements de la méthode conventionnelle. Une version alternative de cette puce, adaptée à la fusion cellulaire par électroporation est également démontrée.Enfin, la dernière partie de cette thèse est consacrée à la sélection des hybridomes sécréteurs d'anticorps spécifiques de la cible d'intérêt par criblage en microfluidique de gouttes. Cette partie, réalisée en collaboration avec l'entreprise strasbourgeoise MicroOmix, permet de simplifier et d'accélérer les étapes post fusion de sélection des clones d'intérêt employées dans la technique des hybridomesAntibodies are molecules produced by the immune system and are characterized by their high binding affinity and specificity for a given antigen, thus making them powerful biological tools for therapeutic and diagnostic applications. The in vitro production of antibodies was made possible in 1975 by the development of the hybridoma technology. This technique is simple, easy to implement and inexpensive, but its use has been limited by low yields, which has led to the emergence of more modern methods that present their own set of challenges.The central aim of this thesis is to unlock the existing hybridoma technique, repositioning it as an efficient and appealing technology. In particular, the objective is to implement three microfluidic devices at each step of the monoclonal antibody production process in order to optimise yields and facilitate the procedure.The first part of this project is focused on the identification and isolation of antibody-secreting cells from the spleen of immunized mice. Following fusion with myeloma cells, this cell subset may facilitate the generation of hybridomas with the ability to secret antibodies that are specific to the target antigen. Our aim is thus to perform targeted fusions between myeloma and antibody-secreting cells, thereby preventing the generation of non-functional hybridomas. To achieve this objective, the cells of interest are identified by flow cytometry using a dedicated surface markers panel. These cells subset are then isolated using an integrated microfluidic magnetic cell-sorting chip.The second part of the thesis concerns the development of a microfluidic chip dedicated to chemical cell fusion, using polyethylene glycol (PEG). The objective of this device is to optimize the conditions for efficient fusion between spleen and myeloma cells, and to improve the yields of the conventional method in tube. An alternative version of this chip, adapted for cell fusion by electroporation, is also demonstrated.Finally, the last part of this project illustrates the potential of droplet-based microfluidics for the single-cell selection and high-throughput screening of hybridomas that secret antigen-specific antibodies. This demonstration, carried out in collaboration with the Strasbourg-based company MicroOmix, aims to simplify and accelerate the post-fusion steps of the hybridoma technology
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Minter, Ralph. "Development of antibody technology to identify natural killer cell surface antigens in Xenopus laevis". Thesis, Durham University, 1999. http://etheses.dur.ac.uk/4598/.
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Natural killer (NK)-like lymphocytes have recently been identified in thymectomised (Tx) Xenopus which are capable of spontaneous cytotoxicity towards the MHC- deficient, allogeneic thymus tumour cell line B(_3)B(_7). This Thesis describes attempts to raise antibodies to Xenopus NK cell surface antigens by phage display and hybridoma technology. The phage display technique was optimised for raising antibodies to novel, cellular antigens in a trial run using the Xenopus thymus tumour cell line B(_3)B(_7). Having isolated a phage antibody which was shown by flow cytometry to bind B(_3)B(_7) cells, the technique was then used to try and raise antibodies to Xenopus NK cells. Isolation of an NIC-specific phage antibody was not achieved but phage antibody XL-6 was raised, which bound an antigen on Xenopus lymphocytes. Phage antibody XL-6, and soluble scFv derived from this, were able to identify a putative mature T cell population in the thymus and may be specific for an amphibian homologue of the mammalian leukocyte common antigen CD45. Hybridoma technology was used to isolate three monoclonal antibodies, 1F8, 4D4 and 1G5, which were shown by flow cytometric analysis to identify a putative NK cell population in control and Tx Xenopus. Following immunomagnetic purification, 1F8- positive spleen cells from control and Tx animals were shown to kill the MHC- deficient tumour target B(_3)B(_7), confirming that this antibody was specific for Xenopus NK cells. Western blotting experiments showed that 1F8, 4D4 and 1G5 identified a doublet of protein bands at 72 and 74 kilodaltons in Xenopus gut lymphoid lysates. Initial attempts to isolate cDNA encoding a Xenopus NK cell surface antigen through immunoscreening a xenopus gut cDNA expression library with antibody 1G5 were unsuccessful as was an attempt to clone a Xenopus homologue of the mammalian NK receptor NKR-Pl by PGR.
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Barnabé, Norman C. J. "The relationship between intracellular nucleotides and hybridoma cell culture productivity and viability". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ31964.pdf.
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Dempsey, Jonathan H. "Studies on the growth and antibody production of a hybridoma cell line". Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/13612.
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In this thesis a number of features of hybridoma cells have been investigated experimentally to obtain a better understanding of their growth and antibody secretion in vitro, with a view to improving the efficiency of antibody production. Initially the effect of immune derived mediators on the growth and secretion of hybridomas was explored as some of these mediators have been shown to play an important role in the growth and antibody secretion of B-cells in vivo. In particular interleukin-2 and interleukin-6 were studied and were shown, in some cases, to improve the rate of cell growth, but they had little or no effect on the level of antibody secretion. Using flow cytometry, the expression of antibody on the surface of a hybridoma cell was investigated. The presence of antibody on the cell surface was correlated with overall antibody secretion and to the stage of the cell cycle. It was shown that antibody that is in the process of being secreted from the cell can be detected and that it is related not only to the overall secretion of antibody but also to the stage of the cell cycle. Attempts to synchronize hybridoma cells in one phase of the cell cycle by well documented chemical means were unsuccessful, and a technique was developed to isolate a synchronous population using flow cytometry. A synchronous cell population was cultured and samples were analysed for cell surface antibody, stage of the cell cycle and antibody secretion at various points in the cell cycle.
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Thorpe, Jane Stewart. "An examination of the growth inhibitory metabolites of a murine hybridoma cell line". Thesis, University of Surrey, 1989. http://epubs.surrey.ac.uk/848121/.
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This thesis reports the results of studies into the effects of various growth inhibitory metabolites upon a murine hybridoma cell line. The cells were examined in vitro and subjected to many different such metabolic stresses as the effects of waste products of normal metabolism such as lactic acid and ammonia. Data presented suggests that these factors alone are not soley responsible for the inhibitory effects observed upon cell growth at high cell concentrations. A cytotoxic effect was also produced by the hybridomas on addition of a dilution of medium that had been exposed to a high concentration of the test cell line for a short period of time. The effect was determined to be not due to the concentrations of lactic acid and ammonium ions present, and further analyses of this medium were carried out. Separation was effected by means of F.P.L.C. and cytotoxic materials were identified in several peaks derived from the medium. The material was identified as being proteinaceous, and proved cytotoxic at high dilutions. The presence of such proteins may be of importance in high cell concentration bioreactors, as they may be retained and so accumulate and prevent optimisation of culture conditions and hence product formation. Further studies may prevent such products being formed or alleviate their effect upon cellular growth.
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Okeson, Carl D. "Glutamine replenishment and ammonia removal in hybridoma cell cultures via immobilized glutamine synthetase". Thesis, The University of Arizona, 1999. http://hdl.handle.net/10150/278706.
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Hybridoma cells utilize glutamine as their primary nitrogen source and excrete ammonia as a metabolic waste product. This ammonia can quickly accumulate to toxic levels in hybridoma culture media, and can severely reduce monoclonal antibody production (Ozturk et al., 1991). The enzyme glutamine synthetase (E.C. 6.3.1.2), which catalyzes the reaction UNFORMATTED EQUATION FOLLOWS: NH₄⁺ + L-glutamate + ATP Mg²⁺ → L-glutamine + ADP +Pᵢ +H⁺, UNFORMATTED EQUATION ENDS was evaluated as a means of reducing ammonia and replenishing glutamine in hybridoma culture medium. The effect of each enzyme reactant on soluble glutamine synthetase activity was quantified, and enzymatic reaction equilibrium evaluated. Enzyme reaction rates in two culture media, both with and without serum, were compared. Glutamine synthetase was immobilized via three different methods, and their effects compared. Cell sensitivity to each enzymatic reactant was studied. Finally, immobilized glutamine synthetase was incorporated in a hybridoma cultivation, and its effect on culture characteristics evaluated.
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Abeydeera, W. P. Piyadasa. "The use of carbon-13 NMR spectroscopy in quantitative studies of hybridoma cell metabolism". Thesis, Queensland University of Technology, 1992. https://eprints.qut.edu.au/36967/1/36967_Abeydeera_1992.pdf.
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An attempt has been made for the first time to identify and quantify the products of glucose metabolism of hybridoma cells which have considerable industrial relevance in the production of monoclonal antibodies. Two hybridoma cell lines (SP01 and G26) have been grown in two different serum- and protein-free media in batch suspension culture, which again is representative of an industrial production process.
In this study, specifically labelled 1-13C enriched glucose was used to quantitatively monitor the fate of carbon (C1) from glucose under aerobic conditions. To achieve reliable quantitative results 13C NMR has been developed for studying the concentrated supernatants from such cultures. Experimental parameters for the quantitative analysis by PFT 13C NMR have been appropriately selected after having gathered relevant information from the natural abundance 13C
spectra. The C labelled experiments revealed that under aerobic conditions, the predominant incorporation of C1 of glucose was into lactate and alanine. For SP01 cells, ~ 6()0/4 of the total lactate originated from 45% of the total glucose utilized while 8% of the glucose consumed was converted to alanine leaving a large percentage ( ~47%) of glucose unaccounted for, which could well enter into alternative metabolic pathways. Also, the methyl carbon of alanine was found to have originated largely from glucose catabolism.
The data from G26 cells showed a sharp decline in the amount of lactate originating from glucose but this misleading observation can be attributed to the underestimation of 13C intensities of metabolites in the presence of paramagnetic species. A similar interference, but to a much lesser degree, was observed for SP01 cells. This interference has been explained in terms of a Ferric-citrate-ligand complex which could variably influence the T1s and NOEs of metabolite carbons resulting in inaccurate intensity measurements. This observation indicates a major limitation to the use of the NMR for the type of studies performed in this project but advice is provided on how to minimise this problem.
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Gaillard, Isabelle. "Mise en œuvre de la chromatographie liquide haute performance dans le cadre du suivi hors ligne et en ligne des protéines de cultures d'hybridomes : caracterisation de la méthode chromatographique et comparaison avec le test ELISA". Vandoeuvre-les-Nancy, INPL, 1993. http://www.theses.fr/1993INPL022N.
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Depuis la mise au point des techniques de formation des lignées d'hybridomes, des efforts importants sont fournis pour améliorer la productivité des anticorps monoclonaux et pour mieux comprendre le métabolisme cellulaire. Les méthodes analytiques jouent un rôle important dans le contrôle des procédés en permettant la détermination quantitative et qualitative de plusieurs paramètres et variables. L'objectif général de cette étude consiste à contribuer à l'amélioration de la maitrise des bioprocédés en proposant une méthode analytique capable de quantifier les protéines présentes dans les cultures d'hybridomes. Afin d'atteindre cet objectif, les potentialités de l'HPLC ont été testées. Les protéines considérées dans cette étude sont d'une part, les composes protéiques propres au milieu de culture et d'autre part, les anticorps monoclonaux secrétés par les cellules. La première partie de cette étude présente l'optimisation des conditions opératoires de la méthode chromatographique. Les deux modes chromatographiques mis à profit sont l'HPLC d'affinité et la RP-HPLC. Une étude comparative des performances de la méthode par HPLC et du test ELISA est exposée dans une seconde partie. La troisième partie concerne l'application de la méthode chromatographique aux suivis des protéines de cultures cellulaires d'hybridomes. Enfin, la réalisation couplage automatise réacteur-échantillonneur-HPLC et son application à la détermination en ligne de la concentration des IgG d'une culture discontinue, constituent la dernière partie de ce travail
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Reissig, Alexander. "Evaluation of on-line cell viability and L-lactate measurements in soft sensor for mammalian cell cultures". Thesis, Linköpings universitet, Teknisk biologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-112918.
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Increasing demand on more effective cell culture reactors has driven optimization works to increase output of products. This has led to development of soft sensors that uses mathematical formulas to increase the available information for the parameters during runs. In the project two parameters was evaluated for use in such a soft sensor, viability by measuring on-line capacitance with Aber probe and L-lactate production using BioSenz apparatus. To determine how well these could be used both were used on batch reactors measuring on a mouse-mouse B cell hybridoma culture which produced IgG1. On-line measurements were performed by probes which measured directly on the cell suspension or withdrew sterile sample from the reactor. Measuring viability gave results with low error, which can be concluded to the variation in reference cell count, but it could not be determined if measuring L-lactate production with BioSenz works in reactors of this size. More work needs to be done on other types of reactors, like fed-batch or perfusion, or lower working volumes.
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Modha, Kishor. "An investigation into the effects of inhibitors of DNA biosynthesis on monoclonal antibody production in hybridoma cell cultures". Thesis, University of Surrey, 1990. http://epubs.surrey.ac.uk/843235/.
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The results of this investigation demonstrated that antibody production in MAK 33 cells was not growth dependent suggesting that antibody synthesis could be maintained in the absence of cell division. The inhibition of cell growth with specific inhibitors of DNA synthesis enhanced the cell specific antibody production rate (CSAPR). Studies on the metabolism of MAK 33 cells under growth inhibited conditions showed that the rise in CSAPR was correlated with the rise in intracellular ATP concentration and the increase in the uptake rates of glucose and glutamine. These results suggested that one of the key factors initiating the rise in CSAPR was the increase in the availability of ATP for antibody synthesis. The rise in intracellular ATP concentration was proposed to be a consequence of the following changes in cell metabolism; 1. Increase in anaerobic glycolysis demonstrated by a concurrent rise in glucose consumption and lactate production. 2. The salvage of energy normally utilised in the processes pertaining to cell division (DNA synthesis and mitosis). Also as part of this investigation a screening assay was developed which could identify compounds with the potential to arrest cell division via the inhibition of DNA synthesis without impairing antibody synthesis. Upon the application of some of the compounds identified by the screening assay to 0.5 litre stirrer cultures, it was discovered that both CSAPR and antibody yield could be enhanced if growth was arrested in mid-exponential phase. These results demonstrated that antibody yield of a batch culture was a function of both viable cell number and CSAPR. Both parameters should be considered with equal importance when designing production strategies intended to improve the antibody yield.
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Pelletier, François. "Mise au point d'observateurs d'état pour le suivi de cultures de cellules animales". Vandoeuvre-les-Nancy, INPL, 1995. http://www.theses.fr/1995INPL135N.
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L’objectif de ce travail est l'élaboration d'outils d'estimation d'état simples d'emploi pour l'évaluation de la composition des milieux de culture cellulaires. La première partie concerne le développement d'un nouvel estimateur appelé observateur à base de bilans de matière construit sur le principe de l'observateur asymptotique. Il peut être appliqué à n'importe quel type de culture. Il ne nécessite aucun réglage. La possibilité de prendre en compte certaines lois cinétiques connues permet de le faire fonctionner avec un nombre restreint de mesures. La seconde partie traite de la mise au point d'une nouvelle variante du filtre de Kalman, le filtre de Kalman auto-ajustable. Le réglage du filtre de Kalman est délicat et peut conduire à des problèmes de stabilité de l'erreur d'estimation et de convergence des valeurs estimées vers les valeurs réelles. Le filtre de Kalman auto-ajustable assura la stabilité des erreurs d'estimations avec un nombre réduit de réglages. Les deux nouveaux observateurs ont été appliqués à trois cultures d'hybridomes productrices d'anticorps monoclonaux. À partir de deux mesures expérimentales (glucose et lactate déshydrogénase ou ammoniaque et lactate déshydrogénase), la composition du milieu de culture est évaluée par les deux techniques. L’observateur à base de bilans de matière a donné de bons résultats et ceux obtenus avec le filtre de Kalman auto-ajustable sont acceptables tant que l'on ne sort pas du domaine de validité du modèle
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Pullen, A. M. "Studies on Peyer's patch T cell hybridomas". Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233317.
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The initial objective of this thesis was to generate Peyer's patch T cell hybridomas producing lymphokines that regulate IgA-secreting B lymphocytes. Unprimed Peyer's patch cells were fused with BW5147. Karyotype analysis and fluorescent staining of the thy-1.2 marker confirmed the generation of hybridomas. It was envisaged that these hybridomas would be tested for their effects on IgA production by LPS-stimulated B cells. However, when the panel of hybridomas was available for testing there were technical difficulties with this assay. Sendai virus-primed Peyer's patch T cells were used in a subsequent fusion, which was screened using an antigen-specific in vitro helper assay. A number of hybridomas stimulated the production of anti-Sendai antibodies by primed B cells. The same hybridomas secreted IL-2 on stimulation by syngeneic spleen cells in the absence of added virus. Recombinant IL-2 replaced the hybridomas in stimulating primed B cells in the helper assay. These studies showed that several of the hybridomas had apparent auto-reactivity. This was not due to viral contamination of the animal stocks since spleen cells from isolator-reared syngeneic mice gave similar results. The genes responsible for stimulating the hybridomas were mapped to the I-region of the MHC. It was important to elucidate whether these T cells were truly auto-reactive or whether they were in fact in vitro artefacts. Hybridomas adapted to grow in serum free medium and subsequently tested for their response to syngeneic cells in the abscence of serum, did not produce IL-2. The addition of foetal calf serum restored the response. The component of foetal calf serum which is necessary for the stimulation of the hybridomas has been partially purified. It can be separated from the main serum protein components by HPLC on a DEAE ion exchange column. It is eluted by high salt which suggests that it is highly acidic or is bound strongly by hydrophobic interactions. The material is trypsin sensitive. It is labile at 4o C and is unstable to freezing and thawing and this has hampered its further purification. The mode of action of the component has been studied using pulsing experiments and it has been shown to act on the stimulator cells and not on the hybridomas. The data suggests that the hybridomas do not recognise a self-antigen alone, but rather that they recognise a component of the xenogeneic serum as antigen with self-MHC restriction. However it has not been formally excluded that the foetal calf serum component stimulates the expression or processing of an auto-antigen.
22
Oancea, Adriana Ecaterina. "Immunoglobulin heavy chain gene expression in hybridoma cells". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0010/NQ28030.pdf.
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Godwin, Aella Natasha. "Enhanced production considering temperature effects on new hybridomas for bovine natural killer cell monoclonal antibodies". Pullman, Wash. : Washington State University, 2008. http://www.dissertations.wsu.edu/Thesis/Fall2008/A_Godwin_112008.pdf.
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Thesis (M.S. in chemical engineering)--Washington State University, December 2008.Title from PDF title page (viewed on Mar. 4, 2009). "School of Chemical Engineering and Bioengineering." Includes bibliographical references.
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Keen, Michael John. "Low-protein media for specialised mammalian cells". Thesis, London South Bank University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336367.
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Cherlet, Marc. "De l'intérêt de la cytométrie en flux pour l'étude de cultures d'hybridomes en bioréacteurs : cinétiques de croissance et décès cellulaires, de production d'anticorps et d'évolution du pH intracellulaire". Vandoeuvre-les-Nancy, INPL, 1995. http://www.theses.fr/1995INPL065N.
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Ces travaux visent à démontrer l'intérêt d'une technique performante d'analyse des cellules, la cytométrie en flux, pour la maitrise optimale de procèdes de cultures de cellules d'hybridomes en réacteurs, grâce à la quantification de divers paramètres cellulaires tels que le pH, l'ADN, les anticorps cytoplasmiques et membranaires, le volume. Ces analyses permettent des études cinétiques détaillées des processus de croissance et décès cellulaires, et de production d'anticorps, en fonction de diverses conditions opératoires. La première partie traite du cas particulier du pH intracellulaire. Grâce au protocole mis au point, les évolutions du pHi sont suivies au cours de cultures discontinues et continues en conditions standard ou sub-optimales de croissance, sous l'effet du pH, de l'osmolarité et de la teneur en ions ammonium du milieu. La deuxième partie précise l'évolution des divers paramètres cellulaires accessibles par la cytométrie en flux dans le cas d'une culture discontinue standard. L'apoptose est mise en évidence comme phénomène principal du décès cellulaire. La dernière partie s'intéresse à la production des anticorps sous l'effet de facteurs stimulants, comme l'acide butyrique, l'osmolarité et les ions ammonium. La relation entre les vitesses spécifiques de production d'anticorps et de croissance cellulaire est discutée en fonction des conditions opératoires et de l'état physiologique des cellules, dont le cycle cellulaire. Le développement d'une procédure de double marquage des IgG membranaires et de l'ADN est présenté, ainsi que son application lors d'une culture discontinue standard afin de déterminer le profil des vitesses de sécrétion d'anticorps en fonction de la position des cellules dans le cycle cellulaire
26
Hayter, P. M. "An Investigation Into Factors That Affect Monoclonal Antibody Production by Hybridomas in Culture". Thesis, University of Surrey, 1989. http://epubs.surrey.ac.uk/1056/.
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Charbonneau, Joel R. "Control of apoptosis in murine B cell hybridomas during stationary batch culture". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ61251.pdf.
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Samaraweera, Preminda. "Oligosaccharides of mouse immunoglobulin-M: Structural variations in hybridoma and myeloma cells". Diss., The University of Arizona, 1988. http://hdl.handle.net/10150/184496.
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Many protein-linked oligosaccharides are believed to impart biological specificities to the molecules. The knowledge of detailed structural characteristics of oligosaccharides is essential for understanding their functions. In order to develop methodology for characterization of oligosaccharides of glycoproteins, and to compare glycosylation patterns of different immunoglobulins, oligosaccharides of IgM from two cell lines, MOPC 104E and PC 700, were analyzed. Homogeneous preparations of glycopeptides carrying individual glycosylation sites of the heavy chain were obtained from the two IgM's. The oligosaccharides of these glycopeptides were prepared by hydrazinolysis, and fractionated by HPLC under conditions that resolve oligosaccharides by charge and size, and by affinity chromatography on Concavalin A-Sepharose. Structures of some of these oligosaccharides were determined by 400 MHz NMR spectroscopy. HPLC fractionation by charge resolved oligosaccharides with zero, one, two, and three sialic acids. As indicated by HPLC analyses, oligosaccharides at all the glycosylation sites of both the IgM's were highly heterogeneous. A comparative study on oligosaccharides prepared by peptide-N-glycosidase F digestion of glycopeptides showed a similar degree of heterogeneity. Therefore, it was concluded that the observed heterogeneity of oligosaccharides was not an artefact caused by hydrazinolysis. Major differences between the glycosylation patterns of the two IgM's were evident from analyses of the oligosaccharides by both chromatographic techniques and NMR spectroscopy. MOPC IgM contained a high proportion of sialylated oligosaccharides when compared to PC IgM. Furthermore, the major oligosaccharide structures of MOPC IgM were of triantennary type whereas PC IgM contained biantennary oligosaccharides as its major species. In both the IgM's, a decreased trend of oligosaccharide processing was observed from the N-terminus to the C-terminus.
29
Stoian, Alina. "Modélisation et simulation de l'atmosphère d'une enceinte membranaire pour des tests de toxicité". Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20026.
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Un problème fondamental pendant l'évaluation in vitro de la toxicité de composés organiques volatils (COVs) est le manque de connaissance de l'évolution de la concentration des COVs à laquelle les systèmes vivants sont exposés au cours des études expérimentales. Ce travail présente un nouveau dispositif expérimental conçu pour étudier l'exposition des systèmes vivants aux COVs. Le dispositif est formé de deux compartiments séparés par une membrane hydrophobe poreuse et permet des durées relativement longues de manipulations sans restreindre la respiration cellulaire. Une modélisation théorique qui couple la conservation de masse et du moment entre les différentes phases et la respiration des cellules hybridomes (ATCC CRL-1606) au sein du dispositif a été développée. Le modèle permet de prédire l'évolution de la concentration des COVs, de l'oxygène et du dioxyde de carbone dans le dispositif. Les résultats simulés pour le transfert des COVs ont revélé une bonne concordance avec les résultats expérimentaux et ont montré que le type de membrane et son diamètre, le coefficient de partage des COVs et la hauteur de la phase liquide ont une influence significative sur l'évolution de la concentration de ceux-ci dans la phase liquide. Néanmoins la disponibilité de l'oxygène au niveau des cellules dépend principalement de la densité cellulaire initiale, de la vitesse spécifique de consommation de ce gaz et de la hauteur du liquide alors que les paramètres liés à la membrane ont une influence sur le contrôle du pHA major problem during in vitro evaluation of the toxicity of volatile organic compounds (VOC) is the lack of knowledge of the evolution of the concentration of such compounds during the course of experimental studies with living systems. This work presents the design of a novel experimental device for the study of cell culture exposure to VOCs. The device is made of two compartments separated by a porous hydrophobic membrane and allows relatively long durations of handling without restricting cellular breathing. A theoretical modeling which couples mass and moment conservation between the different phases inside the device with the breathing kinetics of hybridoma cells (ATCC CRL-1606) was developed. The model allows predicting the evolution of the concentration of the VOCs, the oxygen and the carbon dioxide inside the device. The simulations of the mass transfer of the VOCs simulated presented a good agreement with experiments and showed that the type of membrane and its diameter, the VOCs partition coefficient and the height of the liquid phase have a significant influence on the evolution of their concentration in the liquid phase. Nevertheless, the availability of oxygen for the cells depends mainly on the initial cellular density, the specific kinetics of consumption of this gas and on the height of the liquid phase, whereas the parameters related to membrane have an influence on the control of the pH
30
Sekhon, Harbuksh S. "The creation of T cell hybridomas to map human GAD65 epitopes in NOD mice". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0009/MQ34413.pdf.
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Burns, John W. "Design, construction, modelling and control of a dual-hollow fibre bioreactor for hybridoma cells". Thesis, University of Edinburgh, 1991. http://hdl.handle.net/1842/13277.
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This thesis describes research carried out by the author between 1988 and 1991 at the Department of Chemical Engineering, University of Edinburgh, under the supervision of Dr Donald Glass and Dr Bruce Ward. The aim of the research was to design, build, operate and control a dual-hollow fibre bioreactor. The principle behind the design is that of the blood supply system in animals. The nutrients are supplied in one set of fibres to the growth region, similar to the arteries in the blood system, and another separate set of fibres takes waste products away from the growth region, in a manner analogous to the venous system. The design, construction and operation of the bioreactor is described. The development of novel building techniques are explained, covering new ground in fibre bioreactor construction. The monitoring equipment required is described with a number of successful experimental runs demonstrating the data collection capabilities of the apparatus. During the research, areas of work not initially envisaged were explored, with the aim to provide a basis for future control strategies. This included the development of a fibre testing rig, so that different fibres and various medium preparations could be tested outside a reactor system. This was done due to the lack of basic information available on fibre performance. This leads into work on the modelling the bioreactor by means of a numerical solution run on a computer. The model provides new areas of simulation, the fouling of fibres and the changing nutrient concentrations supplied to the bioreactor. The work is now at a stage where experimental work and modelling work should be brought closer together to help understand problems experienced in both areas.
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Woodland, David L. "An investigation into the loss of antigen-specific lytic activity in cytolytic T cell hybridomas". Thesis, University of Bath, 1986. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370154.
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Liu, Xiaochuan. "The cell biology and physiology of cytoplasmic male sterility in Petunia hybrida". Thesis, University of Reading, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328541.
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VAZQUEZ, MORENO LUZ. "BIOCHEMICAL AND GENETIC STUDIES OF ANTIBODY (IMMUNOGLOBULIN-M) PRODUCING CELLS (GLYCOPROTEINS, U-CHAIN, HYBRIDOMAS)". Diss., The University of Arizona, 1985. http://hdl.handle.net/10150/187920.
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We have chosen the murine immunoglobulin M (IgM) as system to study glycoprotein biosynthesis and carbohydrate processing. Secreted IgM heavy chain (m) has five glycosylation sites which location and structures have been determined. m chain variable region (VH) is involved in antigen binding, while the constant region (CH) is responsible for the effector functions in which the carbohydrate plays an important role. We have determined the carbohydrate structures present at each glycosylation site of IgM produced by a hybridoma cell line (PC 700) and its derived mutants and compared them to IgM from myeloma cell MOPC 104E. PC 700 mutants secrete altered IgM. The alterations include: deletion of one or more constant domains (mutants: 128, 313, and 562) and m chain hyperglycosylation (mutants 21 and 38). Gene analysis indicated that deletions can arise from two different mechanisms. One of these involve a major gene change (mutant 128), while others come from base point mutations (mutants 313 and 562). Cells 21 and 38 did not appear to have m gene insertions. Determination of purified single glycosylation site structures show that PC 700 m chain is processed only to biantennary. Heavy chain protein fragmentation and carbohydrate studies indicate that mutants 21 and 38 alterations are due to an increase in oligosaccharide processing and reduction of unprocessed structures. There is a trend of processing going from PC 700 < 21 < 38. In addition, our results show how growth cell conditions can affect the carbohydrate processing without altering the determinants of m chain oligosaccharide structures. Studies on the IgM molecule illustrate the need for precisely define structure-function relationships. This would allow the selection of the best antibodies for studies such as those involved in immunotherapy.
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Hossain, Ishrat. "Preparation of monoclonal antibodies against immunoglobulin kappa of AL-amyloidosis and characterization of antibody producing hybridoma cells". Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-334412.
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Lewis-Smith, Aidan C. "Molecular and genetic variation in cell cultures and regenerated plants of Petunia hybrida". Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/15209.
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Ma, Jin Graduate School of Biomedical Engineering Faculty of Engineering UNSW. "The generation of monoclonal antibodies to investigate perlecan turnover in cells and tissues". Publisher:University of New South Wales. Graduate School of Biomedical Engineering, 2008. http://handle.unsw.edu.au/1959.4/39171.
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Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.
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Heilmann, Katja. "Wechselwirkungen von Immunzellen mit synthetischen und biomimetischen Oberflächen". Phd thesis, Universität Potsdam, 2006. http://opus.kobv.de/ubp/volltexte/2006/884/.
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Die vorliegende Arbeit wurde im Zeitraum von Oktober 2002 bis November 2005
an dem Institut für Biochemie und Biologie der Universität Potsdam in Kooperation
mit dem Institut für Chemie des GKSS Forschungszentrums in Teltow
unter der Leitung von Herrn Prof. Dr. B. Micheel und Herrn Prof. Dr. Th.
Groth angefertigt.
Im Rahmen dieser Arbeit wurden die Wechselwirkungen von Immunzellen mit
verschiedenen Kultursubstraten untersucht. Dafür wurden drei verschiedene Hybridomzelllinien
eingesetzt. Eine Hybridomzelllinie (K2) ist im Laufe dieser Arbeit
hergestellt und etabliert worden.
Der Einsatz von synthetischen und proteinbeschichteten Kulturoberflächen führte bei Hybridomzellen zu einer deutlich gesteigerten Antikörpersynthese im Vergleich zu herkömmlichen Zellkulturmaterialien. Obwohl diese Zellen in der Regel als Suspensionszellen kultiviert werden, führten die eingesetzten Polymermembranen (PAN, NVP) zu einer verbesserten Antikörpersynthese (um 30%) gegenüber Polystyrol als Referenz. Es konnte gezeigt werden, dass es einen Zusammenhang zwischen der Produktivität und dem Adh asionsverhalten der Hybridomzellen gibt.
Um den Einfluss von Proteinen der extrazellulären Matrix auf Zellwachstum und Antikörpersynthese von Hybridomzellen zu untersuchen, wurden proteinbeschichtete Polystyrol-Oberflächen eingesetzt. Für die Modifikationen wurden Fibronektin, Kollagen I, Laminin und BSA ausgewählt. Die Modifikation der Polystyrol-Oberfläche mit geringen Mengen Fibronektin (0,2-0,4 µg/ml) führte zu einer beträchtlichen Steigerung der Antikörpersynthese um 70-120%. Für Kollagen I- und BSA-Beschichtungen konnten Steigerungen von 40% beobachtet werden. Modifikationen der Polystyrol-Oberfläche mit Laminin zeigten nur marginale Effekte. Durch weitere Versuche wurde bestätigt, dass die Adhäsion der Zellen an Kollagen I- und Laminin-beschichteten Oberflächen verringert ist. Die alpha2-Kette des alpha2beta1-Integrins konnte auf der Zelloberfläche nicht nachgewiesen werden. Durch ihr Fehlen wird wahrscheinlich die Bindungsfähigkeit der Zellen an Kollagen I und Laminin beeinflusst. Durch die Ergebnisse konnte gezeigt werden, dass Hybridomzellen nicht nur Suspensionszellen sind und das Kultursubstrate das Zellwachstum und die Produktivität dieser Zellen stark beeinflussen können. Der Einsatz von synthetischen und proteinbeschichteten Kultursubstraten zur Steigerung der Antikörpersynthese kann damit für die industrielle Anwendung von großer Relevanz sein. Für die Modellierung einer Lymphknotenmatrix wurden Fibronektin, Kollagen I, Heparansulfat und N-Acetylglucosamin-mannose in verschiedenen Kombinationen an Glasoberflächen adsorbiert und für Versuche zur In-vitro-Immunisierung eingesetzt. Es konnte gezeigt werden, dass die Modifikation der Oberflächen die Aktivierung und Interaktion von dendritischen Zellen, T- und B-Lymphozyten begünstigt, was durch den Nachweis spezifischer Interleukine (IL12, IL6) und durch die Synthese spezifischer Antikörper bestätigt wurde. Eine spezifische Immunreaktion gegen das Antigen Ovalbumin konnte mit den eingesetzten Zellpopulationen aus Ovalbumin-T-Zell-Rezeptor-transgenen Mäusen nachgewiesen werden. Die In-vitro-Immunantwort wurde dabei am stärksten durch eine Kombination von Kollagen I, Heparansulfat und N-Acetylglucosamin-mannose auf einer Glasoberfläche gefördert. Die Etablierung einer künstlichen Immunreaktion kann eine gesteuerte Aktivierung bzw. Inaktivierung von körpereigenen dendritischen Zellen gegen bestehende Krankheitsmerkmale in vitro ermöglichen. Durch die Versuche wurden Grundlagen für spezifische Immunantworten erarbeitet, die u.a. für die Herstellung von humanen Antikörpern eingesetzt werden können.
In this scientific work the interactions of immune cells with different culture substrata were investigated. Therefore, three hybridoma cell lines were tested, one cell line (K2) was established during this work. The application of synthetic and protein-coated culture surfaces lead to a significantly increased synthesis of monoclonal antibodies in comparison to usual tissue polystyrene. Although hybridoma cells were normally cultured in suspension applied polymer membranes like PAN and NVP induced an increase by 30%. Furthermore, an influence of cell adhesion and antibody synthesis could be shown. To investigate the influence of extracellular matrix proteins on growth and antibody synthesis of hybridoma cells tissue culture polystyrene was coated with fibronectin, collagen I, laminin and bovine serum albumine in different concentrations. Modifications with fibronectin (concentrations between 0.2 and 0.4 µg/ml) improved the yield of monoclonal antibodies considerably by 70-120%. Coating cell culture plates with collagen I and bovine serum albumine induced an increase by 40%. The coating with laminin showed only marginal effects. Further experiments approved a decreased adhesion of hybridoma cells on collagen I and laminin coated surfaces. FACS analysis showed a reduced presence of the alpha2-chain of the alpha2/beta1-integrin responsible for mediating the binding to collagen I and laminin. Probably, the binding affinity to collagen I and laminin coated surfaces was influenced by this. The results showed a high impact of modified culture substrata on antibody synthesis even if hybridoma cells were cultured in suspension normally and this could be an approach for industrial application. The second part of this work comprised the creation of a lymph node paracortex related surface. Different matrix proteins like fibronectin, collagen I, heparane sulfate and a sugar named N-acetylglucosamine-mannose were coated in different combinations on glass surfaces to create a matrix. Dendritic cells were cultivated on these surfaces and get activated with ovalbumin. After that naïve T- and B-cell populations were added and it could be shown nicely that the modifications of the culture surface were essential for activation and interaction of dendritic cells, T- and B-cells which resulted in the secretion of specific interleukins (IL12, IL6) and specific antibodies (anti-ovalbumin-antibodies). In these experiments a specific immune respone to ovalbumin in vitro could be detected if the cells were isolated from ovalbumin-receptor-transgenic-mice (TgNDO11.10). This In-vitro-immunization was triggered at most if cells were cultured on a surface coated with a combination of collagen I, heparane sulfate and N-acetylglucosamine-mannose. These experiments could be basics for controlled specific immune reactions in vitro which could be used for the production of human antibodies or for the controlled activation or inactivation of immune cells.
39
Meng, Qiang-hua. "Characterization of the reactivities of SLE and normal-derived human hybridoma lupus anticoagulant, anti-phospholipid and anti-dDNA autoantibodies with platelets and endothelial cells". Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74217.
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The mechanisms by which lupus anticoagulant and antiphospholipid autoantibodies cause hemostatic abnormalities in patients with systemic lupus erythematosus (SLE) are poorly understood. We have approached this problem by investigating the binding and functional effects of human hybridoma lupus anticoagulant, anti-phospholipid and anti-DNA autoantibodies derived from SLE patients on platelets and endothelial cells. Most lupus anticoagulant antibodies did not bind to intact platelets and endothelial cells in vitro, while many antiphospholipid and anti-DNA antibodies were reactive. A comparison of SLE and normal-derived autoantibodies demonstrated that platelet-binding autoantibodies derived from SLE patients exhibited greater antigen specificity and platelet cytotoxicity than similar antibodies derived from normal individuals. By Western blotting analysis, many SLE-derived polyspecific antibodies reacted specifically with individual platelet proteins, whereas normal-derived polyspecific antibodies did not. One SLE-derived antibody, 9604 was found to react with ADP-activated platelets but not resting platelets. The reactive components in platelets were reducible polypeptides of approximately 200,000 and 32,000 molecular weight. These data suggest that some SLE autoantibodies may be able to interact with platelets and result in cell lysis or dysfunction in vivo.
40
Perani, Angelo. "Cultures d'hybridomes : production d'anticorps monoclonaux : étude de l'apoptose et application à la toxicité de surfactifs". Vandoeuvre-les-Nancy, INPL, 1995. http://docnum.univ-lorraine.fr/public/INPL_T_1995_PERANI_A.pdf.
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La production d'anticorps monoclonaux par des cultures d'hybridomes peut être optimisée en utilisant des milieux sans-sérum ou en effectuant des cultures en masse. Par ailleurs, la production d'anticorps peut être améliorée en maintenant les cultures en vie pendant des temps de plus en plus longs. Ceci nous a donc amené à étudier la mort des cellules par apoptose et nécrose. Trois hybridomes différents (A49, A64 et B146) ont été adaptés à plusieurs types de milieux sans sérum suivant une technique dite d'adaptations lentes. Nous décrivons ensuite un autre type d'adaptations qui ont été réalisées en un temps plus court: adaptations rapides. Nous avons, par ailleurs, comparé trois hybridomes (AFRC MAC 65, HF2 x 653 et OX 19) dans 3 systèmes de culture différents (boites, flacons à agitation et systèmes comportant des tubes à dialyse), en observant leur croissance et l'état du matériel génétique (empreinte digitale et fragmentation de l'ADN). Un certain nombre d'expériences complémentaires ont été réalisées afin de tester les propriétés du système de culture à base de tubes à dialyse: utilisation de différents tubes à dialyse, utilisation de différentes souches cellulaires ; culture dans un milieu sans sérum ; utilisation d'antiprotéases, « scaling up ». Nous avons étudié la mort cellulaire programmée ou apoptose chez les hybridomes à l'aide de techniques diverses: électrophorèse sur gel, observations morphologiques et suivi du cycle cellulaire. L’apoptose a été induite en utilisant des chocs thermiques, des irradiations gamma, privation de sérum et des inhibiteurs de la synthèse des protéines. Nous avons mis en évidence l'existence d'apoptose spontanée chez la plupart des hybridomes étudiés avec une étude plus approfondie concernant les hybridomes A49 et 12H8. Un hétérohybridome résistant à l'apoptose a été étudié plus en détail (hétérohybridome homme-souris HF2 x 653). Finalement nous avons examiné la toxicité de surfactifs non-ioniques a base de noyau beta-lactame en etudiant respectivement l'apoptose et la necrose sur les hybridomes HF2 x 653 et l'hémolyse sur les globules rouges. Nous avons ensuite débattu des éventuels mécanismes d'action provoquant la mort cellulaire
41
Simmons, William Minnow. "Contributions of viral and cellular gene products to the pathogenesis and prognosis of aggressive lymphomas". Thesis, University of Wolverhampton, 2016. http://hdl.handle.net/2436/609034.
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High grade aggressive lymphomas have high mortality. By their nature, more than 40% of patients die from these diseases even with the improved treatment strategies currently available for oncology patients. The characteristic feature is that they are functionally heterogeneous and therefore have different biological and molecular signatures which make it difficult for all groups to respond to same line of treatment. Based on the above, I set out to look at the impact of viral and cellular gene products on these groups of diseases: In chapter 3 I developed monoclonal antibodies against HERV‐K10. I subsequently investigated their expressions in aggressive lymphomas including Diffuse Large B‐cell lymphoma, Hodgkin’s lymphoma and Primary CNS lymphomas. I showed HERV‐K10 is expressed in cell lines of aggressive lymphomas, but not in paraffin‐embedded tissues. In chapter 4 I showed that the expression of ATM using immune‐histochemistry techniques in aggressive lymphomas does offer a guide to prognosis and treatment. Nearly 30% of Diffuse Large B‐cell lymphomas express ATM, 55% of Hodgkin’s lymphomas and more than 80% of Primary CNS lymphomas. I also showed there is a correlation of ATM expression and EBV‐driven aggressive lymphomas and that this has a poor prognostic significance. Chapter 5 analysed the results obtained by generating, validating and evaluating data base of DLBCL and PCNSL from a retrospective cohort over a 17‐year period. The results confirmed that prognostic indicators including ATM, S1PR2, Autotaxin and EBV using immuno‐histochemistry techniques help with categorising aggressive lymphomas into different prognostic groups and does influence future management. In summary, my results showed there is a critical place for immuno‐histochemistry techniques in convincingly helping understand the expressions of viral and cellular gene products in aggressive lymphomas and in contributing positively to their management.
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McQueen, Anne. "Ammonium ion effects on hybridoma cell physiology". Thesis, 1989. https://thesis.library.caltech.edu/614/1/McQueen_a_1989.pdf.
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NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.
Ammonium ion is an important inhibitor of mammalian cell growth. Measurements of changes in hybridoma intracellular pH upon NH4Cl addition (using a flow cytometric assay in which cells are stained with a pH-sensitive fluorescent dye) indicate that [...] causes a steady-state cytoplasmic acidification. There is a correlation between NH4Cl effects on growth and on intracellular pH, and these effects are independent of the external pH value, suggesting that [...], not NH3 (produced by the dissociation of [...] at higher external pH values), is the species responsible for growth inhibition.
Ammonium ion has similar effects on batch growth in flasks in an incubator and in a bioreactor at controlled pH and dissolved oxygen concentration. Both decreases in the external pH value and increases in the NH4Cl concentration inhibit glucose consumption in batch growth. This is hypothesized to be due to intracellular pH decreases. However, cell growth is not dependent on glucose consumption. Changes in the external pH value and in the NH4Cl concentration, which result in growth inhibition, have no effect on the specific antibody production rate.
A mechanism is proposed and a mathematical model is developed to explain the intracellular pH decreases caused by [...]. This model incorporates a detailed description of the behavior of the Na+/H+ -exchanger, which is assumed to be the main pHi controller of the cell. The model is able to reproduce the salient features of the experimental results.
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McQueen, Anne T. "Ammonium Ion Effects on Hybridoma Cell Physiology". Thesis, 1989. https://thesis.library.caltech.edu/614/3/mcqueen-a_1989.pdf.
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Ammonium ion is an important inhibitor of mammalian cell growth. Measurements of changes in hybridoma intracellular pH upon NH4Cl addition (using a flow cytometric assay in which cells are stained with a pH-sensitive fluorescent dye) indicate that NH+4 causes a steady-state cytoplasmic acidification. There is a correlation between NH4Cl effects on growth and on intracellular pH, and these effects are independent of the external pH value, suggesting that NH+4, not NH3 (produced by the dissociation of NH+4 at higher external pH values), is the species responsible for growth inhibition.
Ammonium ion has similar effects on batch growth in flasks in an incubator and in a bioreactor at controlled pH and dissolved oxygen concentration. Both decreases in the external pH value and increases in the NH4Cl concentration inhibit glucose consumption in batch growth. This is hypothesized to be due to intracellular pH decreases. However, cell growth is not dependent on glucose consumption. Changes in the external pH value and in the NH4Cl concentration, which result in growth inhibition, have no effect on the specific antibody production rate.
A mechanism is proposed and a mathematical model is developed to explain the intracellular pH decreases caused by NH+4. This model incorporates a detailed description of the behavior of the Na+/H+-exchanger, which is assumed to be the main pHi controller of the cell. The model is able to reproduce the salient features of the experimental results.
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Sen, Sucharita. "Production of monoclonal antibody 520C9 by hybridoma cell HB8696 culture". Thesis, 2012. http://localhost:8080/xmlui/handle/12345678/6791.
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Barnabe, Norman C. J. "The relationship between intracellular nucleotides and hybridoma cell culture productivity and viability". 1998. http://hdl.handle.net/1993/1344.
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Monoclonal antibody (Mab) production is dependent upon the metabolic state of hybridomas. The objective of this research project was to determine if the intracellular nucleotide pool could be used as an indicator of specific antibody productivity (qMab) or of cell viability. Serum-free cultures of the murine hybridoma (CC9C10) which secretes Mab against bovine insulin were used as a model in these studies. Changes in qMab were monitored in relation to the measured intracellular nucleotide pool. Under certain culture conditions the qMab increased significantly (65-97%) and was shown to be concomitant with an increase (13-65%) in UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine (UDP-GNac) and concomitant with a decrease in growth rate (22-59%). Hybridomas were then grown in conditions favorable for sugar-nucleotide accumulation. Supplementation of cultures with tunicamycin or NH$\sb4$Cl caused a x5 increase in intracellular UDP-GNac but without significantly affecting the qMab. Tunicamycin, but not NH$\sb4$Cl, inhibited Mab glycosylation. However, non-glycosylated Mab was secreted at the same rate as the glycosylated form. Therefore, neither the availability of UDP-GNac nor glycosylation appear to limit productivity in these cells. The data suggests that a reduction in growth rate rather than UDP-GNac accumulation may cause an increased qMab. From a study of the relationship between cell viability and intracellular nucleotides, it was found that programmed cell death (apoptosis) was preceded by an intracellular decrease in CTP and UTP. These nucleotides may act as mediators of apoptosis and may be used as predictors of imminent cell death. The level of intracellular nucleotides depends on the availability of nutrients. Increasing medium glucose (0-25 mM) resulted in the proportional increase in NTP levels ($>$45%), although further enhancement was not observed at glutamine $>$0.5 mM. The relationships between intracellular nucleotides levels and cell viability are potentially useful for monitoring bioreactor cultures for large scale production processes. Manipulating nucleotide levels by culture supplementation may prove to be an important tool for the control of cell growth and the prevention cell death.
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Dorka, Penny. "Modelling Batch and Fed-batch Mammalian Cell Cultures for Optimizing MAb Productivity". Thesis, 2007. http://hdl.handle.net/10012/3166.
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The large-scale production of monoclonal antibodies (MAb) by mammalian cells in batch
and fed-batch culture systems is limited by the unwanted decline in cell viability and
reduced productivity that may result from changes in culture conditions. Therefore, it
becomes imperative to gain an in-depth knowledge of the factors affecting cell growth and cell viability that in turn determine the antibody production. An attempt has been made to obtain an overall model that predicts the behaviour of both batch and fed-batch systems as a function of the extra-cellular nutrient/metabolite concentrations. Such model formulation will aid in identifying and eventually controlling the dominant factors in play
to optimize monoclonal antibody (MAb) production in the future.
Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was grown in D-MEM medium (Gibco 12100) with 2% FBS. A systematic approach based on Metabolic Flux Analysis (MFA) was applied for the calculation of intracellular fluxes for
metabolites from available extracellular concentration values. Based on the set of
identified significant fluxes (from MFA), the original metabolic network was reduced to
a set of significant reactions. The reactions in the reduced metabolic network were then combined to yield a set of macro-reactions obeying Monod kinetics. Half saturation constants were fixed empirically to avoid computational difficulties that parameter estimation for an over-parameterized system of equations would cause. Using Quadratic Programming, the proposed Dynamic Model was calibrated and model prediction was carried out individually for batch and fed-batch runs. Flux distribution for batch and fedbatch
modes were compared to determine whether the same model structure could be applied to both the feeding profiles. Correlation analysis was performed to formulate a
Biomass Model for predicting cell concentration and viability as a function of the extracellular metabolite concentrations in batch and fed-batch experiments. Quadratic
Programming was applied once again for estimation of growth and death coefficients in the equations for viable and dead cell predictions. The prediction accuracy of these model equations was tested by using experimental data from additional runs. Further, the Dynamic Model was integrated with the Biomass Model to get an Integrated Model capable of predicting concentration values for substrates, extracellular metabolites, and viable and dead cell concentration by utilizing only starting concentrations as input.
It was found that even though the set of significant fluxes was the same for batch and fedbatch operations, the order of these fluxes was different between the two systems. There was a gradual metabolic shift in the fed-batch system with time indicating that under conditions of nutrient limitation, the available energy is channeled towards maintenance rather than growth. Also, available literature with regard to cell kinetics during fed-batch
operation suggests that under nutrient limited conditions, the cells move from a viable, non-apoptotic state to a viable apoptotic state. This is believed to lead to variations in antibody production rates and might explain inaccurate predictions for MAb obtained from the model proposed in the current work. As a result more detailed analysis of the system and in particular, the switch from non-apoptotic to apoptotic state is required.
As a continuation of efforts to study the system in-depth, fluorescence imaging is
currently being applied as a tool to capture the changes in cell morphology along the
course of experimental batch and fed-batch runs. These experiments maybe able to
elucidate the transition from non-apoptotic to apoptotic cells and this information maybe
used in the future to improve the accuracy of the existing mathematical model.
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Huang, Pei-Ru y 黃佩如. "Deciphering the factor(s) in human plasma increasing the yield of hybridoma after cell fusions". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/46743800833852627778.
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碩士輔仁大學
生命科學系碩士班
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Clinically, chemotherapy is one of the most utilized strategies for curing cancers. Howeve, chemodrugs may sometimes inhibit the growth of some normal cells and thus result in adverse effects. Our previous study have shown that for th production of mouse monoclonal antibodies utilizing human plasma (HP) as the nutrient supplement may significantly increase the yield of hybridoma after the cell fusion experiment. Supprisingly, the antifolate sensitive mouse myeloma cells were conferred a drug resistance by human plasma. In this study, proteomic approaches were utilized to dissect the possible mechanism of the observed phenomona. In comparison to the fetal bovine serum (FBS), HP allow mouse myeloma cells (SP2/0) a higher growth rate, but introduce an antifolate, aminopterin, resistance. In the presence of HP, the LD50 of mouse myeloma cells elevate from 0.15 μM to 1.09 μM. Such HP related antifolate resistance was not observed for human hepatocytoma cells (HepG2) and chronic myelogenous leukemia cells (K562), but for human breast cancer cells (MCF7). The possible factors in HP that confers the antifolate resistance were isolated and identified as adenine, a supplement in blood bags. Adenine was found repossible for either the antifolate resistance in some cancer cells or the promotion of hybridoma growth. The results from proteomic analysis implies that through the salvage pathway exogenous supplement of adenine might decrease the metabolic impediment by antifolate, and increase the energy level of cells. Our results suggest the introduction of adenine during blood transfusion may decrease the efficacy of certain chemotherapies.
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Vochyánová, Klára. "Příprava monoklonálních protilátek proti proteinu VP2 lidských polyomavirů". Master's thesis, 2013. http://www.nusl.cz/ntk/nusl-322097.
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Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...
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Villanueva, Alexander Ian. "The influence of Toll-like receptors on murine invariant natural killer T cell activation". Thesis, 2013. http://hdl.handle.net/10214/7252.
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Invariant natural killer T (iNKT) cells are a versatile subclass of T lymphocytes which recognize glycolipid antigens. iNKT cells are capable of rapidly producing a broad array of cytokines in response to stimulation; thus, they play an important role in the early regulation of a variety of immune responses. It was hypothesized that iNKT cells express functional Toll-like receptors (TLRs) and that stimulation of TLRs by their ligands modulates iNKT cells responses. In the first objective, it was revealed that upon stimulation with anti-CD3 monoclonal antibody and interferon (IFN)-α, expression of TLRs was enhanced in iNKT cells. Furthermore, stimulation of iNKT cells with TLR ligands led to a significant increase in the expression of several cytokines. In the second objective, the mechanisms behind the modulatory effects of the TLR9 ligand (CpG-ODN) on iNKT cells were determined. Altogether, these findings suggest a direct role for TLRs in iNKT cell activation.Ontario Graduate Scholarship
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Petersen, Johathan Franklin. "Shear stress effects on cultured hybridoma cells in a rotational couette viscometer". Thesis, 1989. http://hdl.handle.net/1911/16281.
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Cells growing in stirred bioreactors exist in a complex fluid mechanical environment. A number of forces act on the cells in the reactor, including fluid shear stresses. If agitation is sufficiently rapid, these forces may be lethal to the cells. In this study, the effects of well defined fluid shear on cell damage were investigated in a rotational couette viscometer.
The shear sensitivity of the cells was modulated by the age of the culture. For cells that experience a prolonged stationary phase, the cells were quite sensitive to shear for both young and old cultures. If the stationary phase was short, the resistance to shear was higher throughout, and declined slightly with increasing culture age.
The shear sensitivity of the cells was also modulated by specific components of the cytoskeleton. Disruption of the microfilaments made the cells more sensitive to shear, while disruption of the microtubules had no effect on shear sensitivity. Shear sensitivity also depended on energy metabolism in the cells. Inhibiting respiration increased shear sensitivity, and inhibiting glycolysis caused a further increase in shear sensitivity.
Addition of fetal bovine serum to the culture medium made the cells more resistant to shear in a dose dependent manner. Addition of the pluronic polyol F68 to culture media had no effect. Polyethylene glycol increased shear sensitivity.
Subjecting the cells to extreme agitation over an extended period resulted in a population of cells that grew more readily at high agitation rates. This subpopulation had a higher specific growth rate and less shear sensitivity than unselected cells.