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.

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.

Relationship between cell size, cell cycle and specific recombinant protein productivity.

Centrifugal elutriation was used to produce cell cycle enrichedfractions of four commercially relevant recombinant cell lines,chosen to allow for variation in properties due to construct,expression system and parent cell type, from normally growingheterogeneous batch cultures. As these fractions had identicalculture histories and had not been subjected to any insult orstress which was likely to have adversely affected cellularmetabolism, they were ideal for further study of cellularproperties. Specific productivity, cell size and cell cyclestate of replicate elutriated fractions were measured for eachcell line. Results showed that cell size was the major cellulardeterminant of productivity for all cell lines examined. Productformation was not restricted to any particular cell cycle phaseand in all cases, production occurred irrespective of cell cyclephase. Specific productivity was lowest when the majority ofcells in the fraction were G(1), intermediate when themajority of cells in the fraction were S phase and greater whenthe majority of cells in the fraction were in G(2)/M. However, the evidence suggests that size is the major cellulardeterminant of productivity; the apparent relationship betweencell cycle and productivity is secondary and can simply beascribed to the increasing size of cells as they progress thoughthe cell cycle. Thus, in addition to cell density and viabilitycell size is the cellular parameter which should be incorporatednot only into mathematical models of recombinant mammalian cellproduction processes but also into process monitoring andcontrol strategies.

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. © 1999 John Wiley & Sons, Inc.

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.

The role of the cell cycle in determining gene expression and productivity in CHO cells.

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.

Use of cell cycle analysis to characterise growth and interferon-gamma production in perfusion culture of CHO cells.

The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter perfusion cultures and found a significant increase in the accumulated interferon-gamma over that obtained from the nutrient-based controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures.

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.

Apoptosis and its control in cell culture systems.

Apoptosis is a form of programmed cell death which exhibits highly distinctive morphology. Research activity in this area has increased substantially in recent years, primarily due to the realisation that disregulation of apoptosis is involved in the development of a number of pathological conditions, including cancer and AIDS. However, it is now clear that apoptosis also represents the dominant form of cell death during the culture of industrially important cell lines. This review focuses on the induction of apoptosis during industrial cell cultures as well as the effects of the apoptosis suppresser gene bcl-2 on cell survival in conditions relevant to bioreaction environments. We also present new data which demonstrates that bcl-2 can protect cells from apoptosis induced by oxygen deprivation, a finding which has important implications for large scale and intensive cultivation of cells. We also describe experiments which suggest that bcl-2 can reduce the specific nutrient consumption rate of cells.

A rapid method for evaluation of cell number and viability by flow cytometry.

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light.

Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2.

Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. (c) 1996 John Wiley & Sons, Inc.

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production.

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K( L ) a and of mixing (via pH measurements) in bioreactors up to 8 m(3) at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO(2) has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.

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.

Cell cycle and cell size dependence of susceptibility to hydrodynamic forces.

Exposure of animal cells to intense hydrodynamic forces exerted in turbulent capillary flow, and by controiled agitation and aeration, resulted in preferential destruction of S and G(2) cells and the extent of destruction of these cells was dependent upon the intensity of the action. The loss of these cells was possibly due to their larger size. However, the appearance of large numbers of membrane-bound vesicular structures similar to apoptotic bodies as well as cells with low DNA stainability (in a sub-G(1) peak) suggested that the action of adverse hydrodynamic forces on these large cells may at least in part be to induce an apoptotic response. (c) 1995 John Wiley & Sons, Inc.

Death mechanisms of animal cells in conditions of intensive agitation.

The question is addressed as to whether cells which are subject to high-energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench-scale (1-L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m(-3) there was no apparent damage. At 320 W m(-3) cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m(-3) in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller-sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F-actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate.

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%.

Cell death in bioreactors: a role for apoptosis.

The incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease-mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above-mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity.

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.

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.

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.