PubMed Health⌕ Search

Biomedical subjects

A N Emery

Publications and source records attributed to A N Emery.

At least 19 recordsLinked to original sources
PubMed2001

Polysaccharide synthesis as a carbon dissipation mechanism in metabolically uncoupled Xanthomonas campestris cells.

The utilization of xanthan metabolism as an excess carbon dissipation path in Xanthomonas campestris cells under sub-lethal acid stress was studied. To highlight growth limitation during metabolic uncoupling due to acid toxicity a antibiotic was added. The simultaneous addition of enoxacin and acetic acid showed that the xanthan production per unit of biomass raises with increasing concentrations of enoxacin, which seems to indicate that when the cell is prevented from growing it finds a path to convey the extra carbon. In parallel, although the effect of acetic acid is not very significant, its presence appears to increase xanthan. This tendency seems to be accentuated with increasing concentrations of enoxacin. In fact, in presence of 0.15 mM of acetic acid, 2.88 and 5.76 microM of antibiotic produces xanthan/biomass yields of 8.13 and 9.82 g g(-1) which drop to below half those values (3.55 g g(-1)) when enoxacin is removed. When enoxacin was kept constant, xanthan/biomass yields showed small increments with the increase of acetic acid. Thus, with 1.44, 2.88 and 4.32 microM enoxacin concentrations, the addition of organic acid produces a 6--8% stimulation of xanthan.

Carbon↗
PubMed2001

Scale-down model to simulate spatial pH variations in large-scale bioreactors.

For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.

Bacillus subtilis↗
PubMed1999

Problems in predicting cell damage from bubble bursting.

The question is addressed as to whether observed parameter(s) characterizing single bubble burst (bubble jet height and speed) can be used to predict cell damage in sparged animal cell cultures. Bubble burst profiles are examined in the presence of realistic concentrations of fetal calf serum (FCS) or Pluronic F-68 using a high-speed video technique. The damage to TBC3 hybridoma cells from bubble sparging, characterized as a first-order decline, is reduced by even very small concentrations of both FCS and Pluronic F-68, but neither single bubble burst parameters nor surface properties give usable correlations with death rate constants. &copy 1999 John Wiley & Sons, Inc.

Bioreactors↗
PubMed1999

Bcl-2 over-expression reduces growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells.

Recent studies have suggested that Bcl-2 can affect cell cycle re-entry by inhibiting the transition from G0/G1 to S phase. In this study, we have taken a novel route to the study of the relationship between Bcl-2 expression and cell cycle progression. Continuous cultures of pEF (control) and Bcl-2 transfected murine hybridoma cells were operated at a range of dilution rates from 0.8 day-1 down to 0.2 day-1. The specific growth rate of the pEF cell line was the same as the dilution rate down to a value of 0.6 day-1. However, as the dilution rate was reduced stepwise to 0.2 day-1, the growth rate levelled-off at approximately 0.55 day-1 and this coincided with a fall in culture viability. By contrast, the specific growth rate of the Bcl-2 transfected cell line followed the dilution rate down to a value of 0.3 day-1 with high levels of cell survival. At high dilution rates, the cell cycle distributions were very similar for both cell lines. However, the distributions diverged as the dilution rate was reduced and, at a rate of 0.2 day-1, the percentage of G1 cells in the Bcl-2 culture was 80%, compared to only 56% in the pEF cell population. This corresponded with a greater extension in the duration of the G1 phase in the Bcl-2 cells, which was 1.7 days at the lowest dilution rate tested, compared to only 0.6 day for the pEF cell line. The durations of the G2/M and S phases remained constant throughout the culture. The maximum doubling time was 1.2 days in the pEF culture compared to 2.3 days in the Bcl-2 culture. Analysis of amino acids, ammonia and lactate concentrations indicated that the observed effects on cell cycle dynamics were probably not due to differences in the culture environment. It is suggested that the expression of Bcl-2 can effect G1 to S phase transition in continuously cycling cells, but this is only apparent at suboptimal growth rates.

Amino Acids↗
PubMed1998

A comparison of intensive cell culture bioreactors operating with hybridomas modified for inhibited apoptotic response.

It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by inhibiting their apoptotic response can give improved process performance in terms of cell number and viability in intensive cell culture. Two cell perfusion systems, one using a spin filter and the other an ultrasonic filter, are compared using two cell lines. One cell line is transfected with the bcl-2 gene (TB/C3 bcl-2) which encodes the 'anti-apoptotic' human bcl-2 protein and the other cell line (TB/C3 pEF) with a negative transfection vector. Both reactor systems give similar retention performance for both cell lines. Bcl-2 transfected cells reach higher cell densities than the control cell line, and the percentage of apoptotic cells is clearly lower than with pEF cells. The maximum cell numbers of the bcl-2 cell line are 1.21 x 10(7) ml-1 in the ultrasonic filter culture and 1.58 x 10(7) ml-1 in the spin filter culture, respectively. Using the pEF cell line the maximum cell number reaches 6.0 x 10(6) ml-1 with ultrasonic retention and 5.9 x 10(6) ml-1 in the spin filter. The use of ultrasound in this cell retention system has no apparent influence on cell growth, productivity or viability. Selective retention of viable cells is detectable but the effect of removing non-viable cells is negligible.

Antibodies, Monoclonal↗
PubMed1995

Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus.

Insect cell growth can be significantly improved by close attention to the conditions used in the inoculum stages. Initial cell concentration, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of Spodoptera frugiperda (Sf9) cells. The percentage of cells infected with wild and recombinant baculovirus AcNPV and (in the later case) the beta-galactosidase yield in fresh medium was appreciably affected by the stage of the growth curve that cells were in when infected and by the multiplicity of infection (MOI). However, the cell density at the time of infection and the medium condition showed little direct influence on infectivity. There may, however, be an indirect influence in that these factors determine the relative distribution of cells in the cell cycle. The infectivity is then in turn affected by the relative frequency of cells in the G1, S and G2/M phases. Insect cell specific oxygen uptake rates (1.3-3.4 x 10(-17) mol per cell per s) were essentially similar to or less than those measured for hybridoma cells. However, when Sf9 cells were infected with baculovirus, the specific oxygen uptake rate increased by up to 40%.

Animals↗
PubMed1995

Oxygenation of intensive cell-culture system.

The abilities of various methods of oxygenation to meet the demands of high-cell-density culture were investigated using a spin filter perfusion system in a bench-top bioreactor. Oxygen demand at high cell density could not be met by sparging with air inside a spin filter (oxygen transfer values in this condition were comparable with those for surface aeration). Sparging with air outside a spin filter gave adequate oxygen transfer for the support of cell concentrations above 10(7) ml-1 in fully aerobic conditions but the addition of antifoam to control foaming caused blockage of the spinfilter mesh. Bubble-free aeration through immersed silicone tubing with pure oxygen gave similar oxygen transfer rates to that of sparging with air but without the problems of bubble damage and fouling of the spin filter. A supra-optimal level of dissolved oxygen (478% air saturation) inhibited cell growth. However, cells could recover from this stress and reach high density after reduction of the dissolved oxygen level to 50% air saturation.

Aerobiosis↗
PubMed1994

Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture.

The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1 x MEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3 x 10(7) cells ml-1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.

Animals↗
PubMed1994

Growth and interferon-gamma production in batch culture of CHO cells.

The relationship between growth and interferon-gamma (IFN-gamma) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. The Ks and mu max-values were found to be 0.771% (v/v) serum and 0.031 h-1 respectively. The average specific IFN-gamma production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN-gamma production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates.

Ammonia↗
PubMed1994

Hybridoma growth and monoclonal antibody production in a dialysis perfusion system.

Hybridoma cells were grown in perfusion culture using a stirred reactor within which a tubular membrane was suspended. Nutrient and product flows through the membrane to and from the culture environment occurred by diffusion processes alone. A mathematical model of the transfer and reaction process enabled both the characterization of a membrane mass transfer coefficient and the prediction of the maximum cell number achievable under set conditions. Steady states in cell concentration were observed for a range of perfusion rates and membrane areas. Steady states could be maintained for over 180 h without further addition of serum. Antibody was accumulated within the reactor to high concentrations, and at yields on both basal medium and serum that were many times those achieved in other forms of batch culture.

Animals↗
PubMed1993

A flow cytometric study of hydrodynamic damage to mammalian cells.

Flow cytometry has been used to study the mechanisms of damage to mammalian cells by hydrodynamic forces. Cell damage resulted from the stresses created both by bubble entrainment and by bubble bursting caused by vortex formation in highly agitated culture. Damage to the antigen molecules on the cell surface together with increasing leakage and passive transport were observed. Pluronic F-68 reduced the efflux of fluorescein out of cells suggesting the possibility of plugging damaged parts of the membrane or change in membrane molecular organisation. Surface-associated immunoglobulin molecules were also protected by Pluronic F-68. The loss of plasma membrane integrity was followed by the destruction of cytoplasmic matrix. The nuclei were last to be destroyed. The fraction of metabolically active cells was drastically decreased by the intense hydrodynamic forces. However the mitochondrial transmembrane potential, which is linked to the ATP requirements of cells was well as the mean cell size of the live cell population, remained unaffected by the stressful hydrodynamic conditions. F-Actin content peaked during the early exponential phase and declined during the mid and late exponential and death phases of batch culture. The increase of actin during exponential phase was faster in stationary culture than in agitated culture. The increase was associated with the proliferative rate. Furthermore, the response to hydrodynamic forces was not related either to cell size difference or to the stage in the cell cycle.

Actins↗
PubMed1992

The effect of Pluronic F-68 on hybridoma cells in continuous culture.

The comparative effects of Pluronic on cell growth of hybridomas were studied. The addition of 0.05% Pluronic decreased the steady-state cell number in continuous culture by about 12% compared to a non-supplemented culture. In short-term experiments, results demonstrated a gradual decrease in cell number with increasing concentration of Pluronic. Such growth inhibition was found to be a result of lowering the rate of DNA synthesis.

Animals↗
PubMed1992

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch, continuous and perfusion cultures.

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G1 phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G1 phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.

Animals↗
PubMed1992

Further studies of the culture of mouse hybridomas in an agitated bioreactor with and without continuous sparging.

TB/C3 mouse hybridoma cells have been grown at 2 controlled dO2 conditions by headspace and sparged oxygenation. Also a variety of sparging rates and sparger sizes and positions have been employed. Headspace oxygenation at dO2 levels from 5% to 100% of saturation give essentially the same performance as controls. Sparging is generally damaging to cells, the extent of damage decreasing with reduced sparging rate until at below about 0.02 vvm results equivalent to the unsparged conditions are obtained. Damage is clearly linked with bubble-cell interactions at the air-medium interface where bubbles bursting in clusters and of a size less than 5 mm appear to be the most lethal. When the interaction of air sparging with the agitator flow leads to an increase in the number of smaller bubbles and cluster bursts, cell damage is further increased. Pluronic F-68 reduces damage very significantly. Biological aspects are briefly discussed in the light of various biological tests. The practical implications of this work for large scale, free suspension cell culture are outlined.

Animals↗
PubMed1991

Cell cycle, cell size and mitochondrial activity of hybridoma cells during batch cultivation.

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G2 fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.

Animals↗
PubMed1991

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.

Animals↗
PubMed1990

Electron microscopy of hybridoma cells with special regard to monoclonal antibody production.

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.

Animals↗