Foam separation of microbial cells

Batch foam separation has been employed to separate Saccharomyces carlsbergensis cells from their broth without the use of any external surface-active agent. A model has been developed to predict the foamate cell concentration as well as the variation of cell concentration in the bulk liquid in the foam column as a function of time. The model assumes a linear equilibrium relation between the cell concentrations at the interface and the bulk. The foam has interface as well as interstitial liquid. The interface is assumed to be in equilibrium with the interstitial liquid, which in turn is assumed to have the same concentration as the bulk. The interfacial area is calculated by assuming the foam bubbles to be pentagonal dodecahedral in shape. The model has been found to explain the results of foam separation of cells quite well, particularly with respect to the effect of bubble size and aeration rate.     

An investigation of small-molecule surfactants to potentially replace pluronic F-68 for reducing bubble-associated cell damage

It is well known that bubble rupture has a detrimental effect on mammalian cells. As a result, Pluronic F-68 (PF-68), a nonionic surfactant, is commonly used to reduce bubble-associated cell damage in sparged bioreactors. While PF-68 is currently effective, there is a concern with respect to its decrease in effectiveness as cell concentrations increase (Ma et al., 2004, Biotechnol Prog 20:1183-1191). In addition, having more than one effective surfactant for cell culture is also highly desirable. Given the empirical nature in which PF-68 was initially discovered as a cell culture additive, a structure-performance study of small molecule surfactants, a distinct group which have been previously investigated for other purposes, was performed in an attempt to find a replacement for PF-68. In this study, a generic platform was established to initially screen both the type and concentration of these surfactants for cytotoxicity. Promising candidates where then evaluated for their ability to rapidly lower the surface tension (dynamic surface tension) of culture media and their ability to prevent cell-bubble attachment in a specially developed bubble creation and collection system. Several promising small- molecule surfactants, and their effective concentration, were identified, which can reduce cell-bubble attachment efficiently without being harmful to cells.     

Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface

We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration-dependent and height-dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium-bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. (c) 1994 John Wiley & Sons, Inc.     

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 μm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%–30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 10⁶ cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.     

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).     

Cell-microcarrier adhesion to gas-liquid interfaces and foam

The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied. These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces. Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer. These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces. Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells. No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces. It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium. It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.     

Physical modeling of animal cell damage by hydrodynamic forces in suspension cultures

Physical damage of animal cells in suspension culture, due to stirring and sparging, is coupled with complex metabolic responses. Nylon microcapsules, therefore, were used as a physical model to study the mechanisms of damage in a stirred bioreactor and in a bubble column. Microcapsule breaskage folowed first-order kinetices in all experiments Entrainment of bubbles into the liquid phase in the stirred bioreactor gave more microcapsule breakage. In the bubble column, the bubble bursting zone at gas-liquid interface was primarilu responsible for microcapsule breakage. The forces on the microcapsules were equivalent to an external pressure of approximately 4 x 10(4) N . m(-2), based on the critical microcapsule diameter for survival of 190 mum. A stable foam layer, however, was found to be effective in protecting microcapsules from damage. The microcapsule transport to the gas-liquid interface and entrainment into the foam phase was consistent with flotation by air bubbles. This result implies that additives and operation of bioreactors should be selected to minimize flotation of cells. (c) 1992 John Wiley & Sons, Inc.     

Cultivation of hybridoma cells in an inclined bioreactor

Murine hybridoma cells were grown in a bubble column that was inclined up to 45 degrees from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells by rising bubbles and exposure of cells to bursting bubbles were minimized. Maximum viable cell count and exponential growth of the cells were not affected by inclination, but an inclination of 30 degrees gave an antibody titer of 42 mg/L, which more than doubled the yield of 17 mg/L in the vertical position. By comparison, the culture gave yields of 30 mg/L when grown in spinner flasks. The enhanced antibody production in the inclined bioreactor corresponded to a prolonged stationary phase of 45 h. (c) 1995 John Wiley & Sons, Inc.     

Quantification of damage to suspended insect cells as a result of bubble rupture

It is proposed that when cells are either attached to, or very near, a rupturing bubble, the hydrodynamic forces associated with the rupture are sufficient to kill the cells. Four types of experiments were conducted to quantify the number and location of these killed cells. We determined: (1) the number of cells killed as a result of a single, 3.5-mm bubble rupture; (2) the number and viability of cells in the upward jet that results when a bubble ruptures; (3) the number of cells on the bubble film; and (4) the fate of cells attached to the bubble film after film rupture. All experiments were conducted with Spodoptera frugiperda (SF-9) insect cells, in TNM-FH and SFML medium, with and without Pluronic F-68. Experiments indicate that approximately 1050 cells are killed per single, 3.5-mm bubble rupture in TNM-FH medium and approximately the same number of dead cells are present in the upward jet. It was also observed that the concentration of cells in this upward jet is higher than the cell suspension in TNM-FH medium without Pluronic F-68 by a factor of two. It is believed that this higher concentration is the result of cells adhering to the bubble interface. These cells are swept up into the upward jet during the bubble rupture process. Finally, it is suggested that a thin layer around the bubble containing these absorbed cells is the "hypothetical killing volume" presented by other researchers. (c) 1994 John Wiley & Sons, Inc.     

Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells?

It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. (c) 1996 John Wiley & Sons, Inc.     

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.     

Cell adsorption and local accumulation of extracellular polysaccharide in an immobilized Acinetobacter calcoaceticus system

Acinetobacter calcoaceticus can be immobilized on Celite by adsorption. The salt concentrations suitable for immobilized cell fermentation are between 10 and 50 mM phosphate concentration. Low salt concentrations cause desorption of immobilized cells while high salt concentrations inhibit the adsorption of cells on Celite. It is also found that cell adsorption is better at lower pH than at higher pH. An airlift fermentation using immobilized cells at 300 g/L Celite loading shows that about 70% of the total polymer produced is accumulated in Celite pores at a concentration (15.4 g/L) almost threefold higher than that in the bulk liquid (5.7 g/L).     

Cell death in the thin films of bursting bubbles.

A sparged gas bubble floating at the liquid interface has a liquid film which drains and thins until the film spontaneously ruptures at a point. This causes rapid retraction of the film, forming a rim of collected fluid. This rim moves at a constant velocity of about 3 m/s and any cells in the bubble film are rapidly accelerated to this velocity in the moving rim. Half of the surface energy originally in the thin film is converted to kinetic energy of the rim, while the rest is dissipated in this rim. The rate of energy dissipation per mass of rim fluid is approximately 9000 m2/s3, which corresponds to a Kolmogorov eddy size of 3.2 microns in fully developed turbulence or a shear stress of 95 N/m2 in laminar flow. Either of these limiting cases presents an environment in which rapid cell death would be expected. Experiments with Sf-9 insect cells suggest that the cell concentration in these thin films is 0.6 times the bulk liquid concentration and that about 20% of these cells are killed when the film ruptures. An equation based on this mechanism accurately predicts the death rate.     

The fate of Pluronic F-68 in chondrocytes and CHO cells

The surfactant Pluronic F-68 (PF-68) is widely used in large-scale mammalian cell culture to protect cells from shear stress that arises from agitation and gas sparging. Several studies suggested that PF-68 is incorporated into the cell plasma membrane and could enter the cells, but without providing any direct evidence. The current study has examined this question for two cell types, one of pharmaceutical interest (CHO cells) and the other of biomedical interest (chondrocytes or cartilage cells). A fluorescent derivative of PF-68 was synthesized to detect and localize internalized Pluronic with culture time. PF-68 uptake by the cells was quantified and characterized. We clearly demonstrate that PF-68 enters the cells, and possibly accumulates in the endocytic pathway. CHO cells showed an average uptake of 11.7 +/- 6.7 (SEM) microg PF-68/10(6) cells while the uptake of chondrocytes was 56.0 +/- 10.9 (SEM) microg PF-68/10(6) cells, independently of the initial PF-68 concentration (between 0.01 and 0.2%, w/v) and of cell concentration (from 1 x 10(6) to 4 x 10(6) cells/mL). These uptake values were identical for both static and agitated culture conditions. Finally, we found that CHO cells are able to eliminate intracellular fluorescent PF-68 but chondrocytes are not. These results show that the uptake of PF-68 by the cells can severely affect PF-68 concentration in the culture medium and thus shear protection effect.     

Influence of pluronic F68 on oxygen mass transfer

Pluronic F68 is one of the most used shear protecting additives in cell culture cultivations. It is well known from literature that such surface‐active surfactants lower the surface tension at the gas‐liquid interface, which influences the mass transfer. In this study, the effect of Pluronic F68 on oxygen mass transfer in aqueous solutions was examined. Therefore, the gassing in/gassing out method and bubble size measurements were used. At low concentrations of 0.02 g/L, a 50% reduction on mass transfer was observed for all tested spargers and working conditions. An explanation of the observed effects by means of Higbie's penetration or Dankwerts surface renewal theory was applied. It could be demonstrated that the suppressed movement of the bubble surface layer is the main cause for the significant drop down of the k_La‐values. For Pluronic F68 concentrations above 0.1 g/L, it was observed that it comes to changes in bubble appearance and bubble size strongly dependent on the sparger type. By using the bubble size measurement data, it could be shown that only small changes in mass transfer coefficient (k_L) take place above the critical micelle concentration. Further changes on overall mass transfer at higher Pluronic F68 concentrations are mainly based on increasing of gas holdup and, more importantly, by increasing of the surface area available for mass transfer. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1278–1288, 2013     

Quantitative investigations of cell-bubble interactions using a foam fractionation technique

Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging. As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable. By using this technique, the number of cells adsorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the prevetive effects of serum and Pluronic F68 on these interactions. It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions. The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.Abbreviations NBCS new born calf serum - SFM serum-free medium     

Cell death from bursting bubbles: Role of cell attachment to rising bubbles in sparged reactors

Bursting bubbles are thought to be the dominant cause of cell death in sparged animal or insect cell cultures. Cells that die during the bubble burst can come from three sources: cells suspended near the bubble; cells trapped in the bubble lamella; and cells that attached to the rising bubble. This article examines cell attachment to rising bubbles using a model in which cell attachment depends on cell radius, bubble radius, and cell–bubble attachment time. For bubble columns over 1 m in height and without protective additives, the model predicts significant attachment for 0.5‐ to 3‐mm radius bubbles, but no significant attachment in the presence of protective additives. For bubble columns over 10 cm in height, and without protective additives, the model predicts significant attachment for 50‐ to 100‐μm radius bubbles, but not all protective additives prevent attachment for these bubbles. The model is consistent with three sets of published data and with our experimental results. Using hybridoma cells, serum‐free medium with antifoam, and 1.60 ± 0.05 mm (standard error) radius bubbles, we measured death rates consistent with cell attachment to rising bubbles, as predicted by the model. With 1.40 ± 0.05 mm (SE) radius bubbles and either 0.1% w/v Pluronic‐F68 or 0.1% w/v methylcellulose added to the medium, we measured death rates consistent with no significant cell attachment to rising bubbles, as predicted by the model. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 468–478, 1999.     

Damage mechanisms of suspended animal cells in agitated bioreactors with and without bubble entrainment

We show that when freely suspended hybridoma cells are cultured in an agitated bioreactor, two fluid-mechanical mechanisms can cause cell damage and growth retardation. The first is present only when there is a gas phase, and is associated with vortex formation accompanied by bubble entrainment and breakup. In the absence of a vortex and bubble entrainment, cells can be damaged only at very high agitation rates, above approximately 700 rpm, by stresses in the bulk turbulent liquid. Cell damage then correlates with Kolmogorov eddy sizes similar to or smaller than the cell size. In the absence of a vortex, the entrainment and motion of very fine bubbles cause no growth retardation even at agitation rates as high as 600 rpm.     

Oxygen transfer properties of bubbles in animal cell culture media

Oxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles ( approximately 5 mm diameter) < intermediate bubbles ( approximately 1 mm diameter) < micron-sized bubbles ( approximately 100 mum diameter). Under certain conditions, the micron-sized bubbles were capable of achieving oxygen transfer rate up to 100 h(-1), a 10-20-fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new-born calf serum (NBCS) and/or Pluronic F-68 supplemented media. For the intermediate and micron-sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F-68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron-sized bubbles in Pluronic F-68 supplemented media containing antifoam agent and NBCS.     

Microscopic visualization of insect cell-bubble interactions. II: The bubble film and bubble rupture.

In this paper, the second in the series, the use of a microscopic, high-speed video system to study the interactions of two suspended insect cells strains, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with rupturing bubbles is reported. Events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. On the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a result of sparging, it is proposed that cells are killed by the rapid acceleration of the bubble film after rupture and the high levels of shear stress in the boundary layer flow associated with bubble jet formation.