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M al-Rubeai

Publications and source records attributed to M al-Rubeai.

At least 19 recordsLinked to original sources
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↗
PubMed1998

Apoptosis and cell culture technology.

The importance of apoptosis in cell culture has now been widely recognised. Recent work has shown that this process is widely manifested during the in vitro cultivation of commercially important mammalian cell lines. In this review I summarise what is now known of the characteristics, significance and regulatory mechanisms of apoptosis. As the process of cell proliferation and cell death are now considered intimately related, particular attention is paid to highlight the progress and opportunities in the field of cell culture engineering. The strategies that have been undertaken to prevent the induction of apoptosis in cell culture and those which have been suggested as possibilities to improve culture productivity through the apoptosis route are discussed with given examples.

Animals↗
PubMed1998

Apoptosis and bioprocess technology.

Optimisation of the production of biopharmaceuticals in animal cell lines has become a key area of research. The identification of apoptosis as the major mechanism of cell death during such processes has raised the importance of studies of cell death when implementing culture optimisation strategies. In this article we present an overview of the studies which have demonstrated the induction of apoptosis during the cultivation of industrially important animal cell lines. We also discuss studies which have shown that deprivation of factors such as amino acids, glucose, serum and oxygen are potent inducers of apoptosis in industrial cultures. The suppression of apoptosis under these conditions has been demonstrated by a number of recent reports, and we describe ways in which this knowledge may be applied in the development of novel solutions to some of the technical problems associated with the development of successful large scale culture process. The article concludes with a discussion of future directions for apoptosis research in bioprocess technology.

Animals↗
PubMed1998

Apoptosis in cell culture.

Most cells can exhibit a biochemical pathway which mediates their own destruction in a highly controlled and genetically defined manner. In animal cells, a morphologically distinct form of this 'programmed cell death' has been identified and extensively characterised. This phenomenon, which has been named apoptosis, accounts for most of the cell deaths that take place during the production of biopharmaceuticals from animal cell lines. In the past few years, the factors responsible for the induction of apoptosis in the bioreactor environment have been identified. Furthermore, a growing number of studies have demonstrated that the suppression of apoptosis by the overexpression of anti-apoptosis genes, most notably bcl-2, result in improved culture productivity.

Animals↗
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↗
PubMed1995

Cell death (apoptosis) in cell culture systems.

In this review we describe the process of cell death known as apoptosis, outlining the key morphological and biochemical features. We also identify the stress elements in bioreactors that drive cells to their death. Finally, we outline methods that might be used to enhance the life span and increase the robustness of cells in culture.

Animals↗
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

Prediction of mechanical damage to animal cells in turbulence.

In previous work a model was proposed for estimation of disruption of animal cells in turbulent capillary flows using information about the hydrodynamics, and cell mechanical properties determined by micromanipulation. The model assumed that the capillary flow consists of a laminar sublayer and a homogeneous turbulent region, and within the latter eddies of sizes similar to or smaller than the cells interact with those cells, causing local surface deformations. The proposed mechanism of cell damage was that such deformations result in an increase in membrane tension and surface energy, and that a cell disrupts when its bursting membrane tension and bursting surface energy are exceeded. The surface energy of the cells was estimated from the kinetic energy of appropriate sized eddies. To test the model, cells were disrupted in turbulent flows in capillaries at mean energy dissipation rates ranging from 800 to 2 x 10(4) Wkg-1. The model assumed that the specific lysis rate is almost independent of the number of passes, which was verified by the experimental data. The implication was that despite the damage the cell mechanical properties did not change markedly during multiple recirculations through the capillaries. On average the model underestimated the cell disruption by about 15%. Although the model gave reasonably good predictions, it lacks proper explanation of the independence of the specific lysis rate on the number of passes. In this paper it is shown that this problem can be resolved in principle by consideration of the localisation of the energy dissipation in turbulent capillary flows. The necessity of further modelling of cell-turbulence interactions is demonstrated.

Animals↗
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↗
PubMed1994

Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.

A novel automated image analysis method is described for characterizing the viability and morphology of animal cells from suspension cultures. With the aid of the exclusion dye Trypan Blue, the total and viable cell counts and the percentage of dead cells present are found. The area, perimeter, equivalent diameter, and circularity of the projected image of each cell are also measured, allowing the estimation of cell volume. The image analysis method is inherently sensitive, precise, consistent (non-operator dependent) and relatively fast, taking approximately 22 min to analyze one sample. The data it gives on individual cellular activity, as characterized by Trypan Blue uptake and cell morphology, are valuable in allowing early diagnosis of even subtle changes in the health of a culture. The method should permit better optimization of culture conditions, and will provide data for the modeling of cell population dynamics. As the conventional manual method is operator dependent, relatively limited in the amount of information it provides, and has a tendency to underestimate Trypan Blue takeup, it is suggested that image analysis be the preferred option for animal cell counting and viability determinations.

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

Effect of Pluronic F-68 on the mechanical properties of mammalian cells.

The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility modulus of the cells were significantly greater (60% and 120%, respectively) in a medium with 0.05% (w/v) Pluronic F-68 compared to that without Pluronic. Pluronic F-68 therefore affected the strength of the membranes when the cells were exposed to it for a long period of time, i.e., in culture. The short-term effect of Pluronic F-68 on cell strength was also tested by its addition at various levels up to 0.2% (w/v) immediately before the mechanical property measurements. The resulting cell strength depended on the Pluronic concentration, but a significant short-term effect could only be detected above a threshold of 0.1% (w/v). Previous reports on the effect of Pluronic F-68 on animal cell culture are evaluated in the light of these observations.

Animals↗