Cells and bubbles in sparged bioreactors

Ever since animal cells have been grownin-vitro, various techniques have been used to supply the cells with oxygen. The most simple and commonly used large-scale technique to provide oxygen is through the introduction of gas bubbles. However, almost since the beginning ofin-vitro cell culture, empirical observations have indicated that bubbles can be detrimental to the cells. This review will discuss the background of the problem, review the relevant research on the topic, attempt to provide a coherent summary of what we know from all of this research, and finally outline what still needs to be investigated. Specific topics to be covered include: experimental correlations of cell damage with bubbles, cell attachment to bubbles, the hydrodynamics of bubble repture, bioreactor studies, visualization studies, and computer simulations and qualification of cell death as a result of bubble rupture.     

Scale up aspects of sparged insect-cell bioreactors

Conclusion In this chapter we have attempted to evaluate the most important parameters which can be useful for the pur-pose of design and scale up. Insect cells and animal cells in general can be grown well in large vessels. However, none of the theories and parameters discussed in this chapter have been validated on a larger scale than laboratory and small pilot reactors. Selection of the most suitable design and scale-up method there-fore needs in particular studies in larger vessels. The Kolmogorov theory and the killing-volume model are in this respect the most promising approaches for the optimal design of large-scale animal-cell bioreactors.     

Oxygen gradients in small and big sparged insect-cell bioreactors

Conclusions It should be clear from the above that the calculations described here are at best rough estimations yielding order-of-magnitude values. Even though, the following general conclusions can be drawn. The gradients in stagnant layers surrounding the particles which are characteristic for animal-cell bioreactors are relatively small as compared to the gradients which can be expected in the bulk-liquid phases of the three bioreactors considered, in particular to the gradients in the stagnant layer surrounding the air bubbles. It can be concluded that under almost all circumstances gradients are likely to exist and can be very steep in larger vessels and in particular at high cell densities. The effects of gradients, however, are largely unknown; therefore research on the effects of gradients on specific and volumetric productivities and product quality seems to be an interesting area.     

Analysis of cell-to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives

To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). (c) 1995 John Wiley & Sons Inc.     

Evaluation of the killing volume of gas bubbles in sparged animal cell culture bioreactors

The detrimental effect of direct gas sparging on insect cells was investigated in bubble columns with various gas flow rates and bubble sizes. The first-order cell death rate was shown to be directly proportional to the gas flow rate and inversely proportional to the bubble size. The specific killing volume of a bubble, killing volume per unit volume of bubble, was found to have a linear correlation with the specific interfacial area of a bubble. Based on these experimental results and the analysis of a bursting bubble at the liquid surface, it was concluded that the killing volume of a bubble is in the liquid layer surrounding the bubble before its rupture, and most important, in the liquid layer beneath the bubble cavity. Cell damage in the bubble film cap was relatively insignificant compared to that in the liquid layer underneath the bubble cavity, except for very large bubbles (i.e., bubble diameter over 5 mm).     

Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors.

Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media. Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed. Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media. In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation. Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium. In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging. The mechanism of this damage effect was not clear. Pluronic F-68 provided protective effect to cells but not to the medium. A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth. Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.     

Structural features of nonionic polyglycol polymer molecules responsible for the protective effect in sparged animal cell bioreactors.

The nonionic surfactant Pluronic F-68 polyol is commonly used to protect cultured animal cells from the detrimental effects of sparging. In this study we investigated the structural features of the Pluronic F-68 molecule responsible for this protective behavior. Poly(oxyethylene)-poly(oxypropylene) block copolymer polyols of various molecular weights and percentages of hydrophobe (poly(oxypropylene], including both Pluronic and reverse Pluronic polyols, were considered. The potential toxicity of these agents was examined in the absence of sparging (i.e., in spinner flasks) by using the attachment-independent Sf9 insect cell line as a model system. Each polyol resulted in one of three distinct types of behavior in these spinner flask experiments: (1) cells lysed at an exponential rate, (2) inhibition of cell growth (i.e., no net cell growth), or (3) uninhibited cell growth. It was then shown that all of the Pluronic and reverse Pluronic polyols that did not inhibit cell growth provided protection from sparging in the bioreactors used in this study; thus, finding a polyol that protected cells was synonymous with finding one that did not inhibit cell growth. The ability of these polyols to protect animal cells in sparged bioreactors was found to correlate well with the hydrophilic-lipophilic balance (HLB). Those polyols with the largest HLB values were found to be protective agents. These poly(oxyethylene)-poly(oxypropylene) polyols were also shown to be more effective protective agents than pure poly(oxyethylene); thus, the presence of the hydrophobe (poly(oxypropylene] is important in their ability to serve as protective agents.     

Fluid-mechanical damage of animal cells in bioreactors.

The fluid-mechanical and some biological aspects of damage to animal cells in bioreactors due to agitation and/or aeration are attracting renewed attention. In microcarrier bioreactors, cell damage is due to forces generated by the interaction of microcarrier beads with each other and also with small turbulent eddies. For freely suspended cells grown in mixed bioreactors, cell damage is most frequently due to bubble breakup or fast-draining liquid films around rearranging gas-liquid interfaces.     

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 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ 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.     

Physical mechanisms of cell damage in microcarrier cell culture bioreactors

The negative effects of excessive agitation on tissue cells in microcarrier culture have often been ascribed to "shear." Analysis of the fluid mechanics occurring suggests that there are actually three potential damage mechanisms: collisions of a cell-covered microcarrier with other beads, collisions with parts of the reactor (primarily the impeller), and interaction with turbulent eddies the size of the microcarrier beads. Review of the available quantitative information on agitation effects in cell cultures does not establish which mechanism is predominant; the range of experimental variables reported emphasizes power input over the other reactor and impeller parameters. The bead-bead collision model is tentatively supported by the available data, but the other mechanisms may still be significant in some systems. The formation of bead aggregates by cellular bridging provides a parallel means of damaging cells. Breaking of these bridges by any of the three means identified earlier can cause cell destruction and/or the net transfer of cells to formerly bare beads. High concentrations of bridges are favored by lower agitation rates, presumably because the bridges are not as quickly destroyed after formation.     

Effects of bubble–liquid two‐phase turbulent hydrodynamics on cell damage in sparged bioreactor

According to recent experimental studies on sparged bioreactors, significant cell damage may occur at the gas inlet region near the sparger. Although shear stress was proposed to be one of the potential causes for cell damage, detailed hydrodynamic studies at the gas inlet region of gas–liquid bioreactors have not been performed to date. In this work, a second‐order moment (SOM) bubble–liquid two‐phase turbulent model based on the two‐fluid continuum approach is used to investigate the gas–liquid hydrodynamics in the bubble column reactor and their potential impacts on cell viability, especially at the gas inlet region. By establishing fluctuation velocity and bubble–liquid two‐phase fluctuation velocities correlation transport equations, the anisotropy of two‐phase stresses and the bubble–liquid interactions are fully considered. Simulation results from the SOM model indicate that shear and normal stresses, turbulent energy dissipation rate, and the turbulent kinetic energy are generally smaller at the gas inlet region when compared with those in the fully developed region. In comparison, a newly proposed correlation expression, stress‐induced turbulent energy production (STEP), is found to correlate well with the unusually high cell death rate at the gas inlet region. Therefore, STEP, which represents turbulent energy transfer to a controlled volume induced by a combination of shear and normal stresses, has the potential to provide better explanation for increased cell death at the sparger region. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:48–58, 2014     

Mixing studies in bioreactors

Blend times and power consumptions were determined for different arrangements of two equal diameter impellers, a high efficiency A310 and a “Dumbo Ear” impeller with three large, “elephant ear” blades designed for low shear agitation. A 9 l round-bottomed, unbaffled bioreactor was used in these studies. Blend times were taken as the time for the disappearance of the pink color of a basic solution of phenolphthalein on neutralization by excess acid, and the power consumption was obtained from torque measurements. The mixing results show that the Dumbo Ear impeller gives shorter blend times than the A310?at equal rotational speeds for most of the conditions studied. As expected, the Dumbo Ear impeller consumes more power than the A310?at the same rotational speed, due to its large area blades. However, the Dumbo Ear impeller also gives shorter blend times than the A310?at equal power consumptions.     

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.     

The protective effect of serum against hydrodynamic damage of hybridoma cells in agitated and surface-aerated bioreactors.

The effect of serum on the growth rate and metabolism of CRL-8018 hybridoma cells in an agitated, surface-aerated bioreactor was examined. In the employed well-controlled bioreactors at high agitation rates, hybridoma cells in medium containing 1% fetal bovine serum rapidly die in the presence of a vortex with accompanying gas-bubble entrainment, whereas non-agitated control cultures grow normally in medium containing 1% serum. Serum levels greater than 5% counteract the detrimental hydrodynamic effects due to agitation and bubble entrainment. The protective effect is present after short-term (less than 1 h) exposure to 10% serum concentration, suggesting a protection mechanism which is, at least in part, of a physical nature. The apparent cell yields on glucose, lactate, and glutamine decreased with decreasing growth rate due to low serum concentrations. The results are incorporated into a simple model in which the apparent growth rate is the sum of an invariable growth rate and a changing death rate.     

Surface aeration in a small,agitated, and sparged vessel

In a recent publication, a technique was outlined for measuring surface aeration rates in an agitated vessels while sparging, and it was shown that surface aeration rates fall rapidly with increasing sparge rates. That work was conducted in a 0.61 m diam vessels. The work reported here was done in a small vessel (0.22 m diam) where surface aeration has been reported to be of particular significance. In general, the results obtained in the small vessel confirmed those in the large one and in addition were generally in good agreement with those recently published elsewhere for an almost identical geometry. For typical practical power inputs and sparge rates, the rate of surface aeration was never more than 20% of the sparge rate and generally less than 5%. These results indicate that surface aeration is of considerably less importance than has generally been believed following the findings of workers who estimated its effect by comparing KL a values under unsparged conditions with those when sparging.     

Control in bioreactors showing gradients

In large-scale bioreactors gradients often occur as a result of non-ideal mixing. This phenomenon complicates design and control of large-scale bioreactors. Gradients in the oxygen concentration can be modeled with a two-compartment model of the liquid phase. Application of this model had been suggested for the control of the dissolved oxygen concentration with a batch gluconic acid fermentation process as the model system. The control system consists of a controller, an observer and a parameter estimator. In this work, the controller design is reconsidered and, in simulation experiments, the performance of the control system has been investigated. When the parameter values are known, the controller in combination with the observer works adequately. The parameter estimator, however, yields incorrect parameters, which are caused by a coupling between two parameters. This causes a deviation of the estimated states from the process states. The simulation results suggest that a priori knowledge of the parameters is required for application of the model for control and state estimation.     

Oxygen transfer in slurry bioreactors

The oxygen transfer in bioreactors with slurries having a yield stress was investigated. The volumetric mass transfer coefficients in a 40-L bubble column with simulated fermentation broths, the Theological properties of which were represented by the Casson model, were measured. Experimental data were compared with a theoretical correlation developed on the basis of a combination of Higbie's penetration theory and Kolmogoroff's theory of isotropic turbulence. Comparisons between the proposed correlation and data for the simulated broths show good agreement. The mass transfer data for actual mycelial fermentation broths reported previously by the authors were re-examined. Their Theological data was correlated by the Bingham plastic model. The oxygen transfer rate data in the mycelial fermentation broths fit the predictions of the proposed theoretical correlation.     

Concentric-tube airlift bioreactors

Mass transfer coefficients were measured in three concentric-tube airlift reactors of different scales (RIMP, V _L =0.07 m³;RIS?1,V _L =2.50 m³;RIS?2, V _L =5.20 m³). The effects of top and bottom clearance and flow resistances at downcorner entrance were studied in water-air system. Experimental results show that h _s ,h _B and A _d /A _R ratio affect K _L a values as a result of their influence on gas holdup and liquid velocity. The gas-liquid mass-transfer coefficients for all the geometric variables were successfully correlated as Sherwood number with Froude and Galilei numbers, the bottom spatial ratio (B=h _B /D _R ), the top spatial ratio , the gas separation ratio and the downcomer flow resistance ratio (R=A _d /A _R ). The proposed empirical model satisfactorily fitted the experimental data obtained in large airlift reactors and some data presented in literature.     

Transgenic animal bioreactors

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases. This revised version was published online in August 2006 with corrections to the Cover Date.