Biotechnol. Prog. 1003, 9, 504-509
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Effect of Silicone Antifoam on Shear Sensitivity of Hybridoma Cells in Sparged Cultures Leo A. van der Pol,**+ Dick Bonarius,z Geert van de Wouw,S and Johannes Tramper1 Bio-Intermediair, P.O. Box 454, 9700 AL Groningen, The Netherlands, and Food and Bioengineering Group, Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, EV Wageningen, The Netherlands
Addition of high concentrations of silicone antifoam (AF) to a suspension of hybridoma cells in a bubble column reduces the death rate when using medium without a protective component. In contrast, high AF concentrations can cause detrimental effects in a sparged medium with a protective component, such as is applied for large-scale animal cell cultures.
Introduction In the scaling up of in vitro cultures of animal cells, great attention has to be given to the shear sensitivity of the cells and as a consequence to the oxygen transfer in the bioreactor (Van’t Riet and Tramper, 1991). Sparging with air or pure oxygen as a method of oxygen supply is simple and efficient but can cause cell damage. This damage has been attributed to the generation of bubbles in the sparger region (Murhammer and Goochee, 19901, the bursting of bubbles at the surface of a suspension (Tramper et al., 1988;Jobses et al., 1991; Handa-Corrigan et al., 1989),or by film drainage in unstable foams (HandaCorrigan et al., 1989). Sparging in a protein-containing liquid, such as a suspension of animal cells in culture medium, results in foaming. Media used for sparged animal cell cultures contain protective additives, such as fetal calf serum (FCS) and Pluronic F68 (Papoutsakis, 1991). These protective components have a foam-stabilizing effect. The formation of a stable foam has been connected with the protective effect against sparging for hybridoma cells growing in a bubble column (HandaCorrigan et al., 1989). Another beneficial effect is that foaming liquids usually are noncoalescing, resulting in a larger surface for gas exchange (Prins and Van’t Riet, 1987; Kawase and Moo-Young, 1990). Adverse effects of foam include the loss of cells in the foam (Aunins et al., 1986; Ghildyal et al., 1988; Bavarian et al., 1991), reduction of effective reactor volume, and risk of overflow and contamination (Prins and Van’t Riet, 1987). Also, the improvement of oxygen transfer by preventing coalescencecan be reduced by the adsorbance of foam-promoting molecules to the interface of bubbles, creating a more rigid interface (Prins and Van’t Riet, 1987; Kawase and Moo-Young, 1990). A certain amount of foamingis acceptable in fermentations, but excessive foam formation should be prevented (Prins and Van’t Riet, 1987). Therefore, antifoams (AF), especially silicone emulsions,are widely used in animal cell cultures in bubble columns, air-lift loop reactors, and sparged stirred-tank reactors. Drawbacks of the use of AF in cultures of animal cells include toxic effects of certain AF types (Aunins et al., 1986; Murhammer and Goochee, 1988; Griffiths, 1988),
* Author to whom correspondence should be addressed. f
8
Bio-Intermediair. Wageningen Agricultural University.
8758-7938/93/3009-0504$04.00/0
difficult separation from the product (Aunins et al., 1986; Prins and Van’t Riet, 19871, and the possible problem of approval by the authorities for pharmaceutical and food applications (Van’t Riet and Tramper, 1991). The reported toxic effects of certain AF types for different kinds of animal cells seem to be mainly due to the use of industrial grade AF emulsions (Aunins et al., 1986;Murhammer and Goochee, 1988), probably indicating the presence of contaminants. In previous work we have reported the effect of reduction of the serum concentration on the shear sensitivity of hybridoma cells in a bubble column. The first-order death rate constant ( h d ) was quantified in the presence of a minimum amount of silicone AF (Van der Pol et al., 1990, 1992). One of the aims of our research is to quantify the cell death rate constant (kd) under standardized conditions in a bubble column, such that the cell death rate is usually much greater than the defined cell growth rate. This is done to evaluate which factors determine cell death caused by sparging without interference from cell growth. The addition of AF to prevent foam formation in the bubble column causes small fluctuations in the AF concentration during a sparging shear-stress experiment, dependent on which protective component is present at a certain concentration in the culture medium. Therefore, it becomes necessary to evaluate the effect of different concentrations of AF on the shear sensitivity of hybridoma cells in the bubble column. In a recent paper it was stated that the foaming properties of culture media have not been considered adequately and that foaming and cell losses in the foam layer are major problems in stirred and sparged bioreactors (Zhang et al., 1992). In this study, the effects of different concentrations of medical grade silicone AF have been evaluated for hybridoma suspension cells in a bubble column in medium with serum or poly(ethy1eneglycol) (PEG) as the protective component and in serum-freemedium without a protective component.
Materials and Methods Cell Line and Medium. The cell line used, PFU-83, is a ratlmouse hybridoma suspension cell which produces
monoclonal antibodies against ratlhuman corticotropin releasing factor (Van Oers et al., 1989). The culture medium was either RPMI 1640 (Seromed)with 0.6 g/dm3 (4 mM) glutamine, 2 g/dm3 NaHC03,5% FCS (Sanbio),
0 1993 American Chemical Society and American Instkute of Chemical Engineers
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andO.l g/dm3streptomycin (Sigma)or 3:l DMEM (Flow)/ Ham's F12 (Seromed) serum-free [DMEIFlBl medium with 375 pg/dm3 EDTA (Titriplex 111) and further additions as described previously (Van der Pol et al., 1992). This serum-free medium contains 5 mg/dm3 transferrin (human, Sigma) as the only protein component. The poly(ethylene glycol) (PEG) polymers PEG 6000 and PEG 20 000 were always added in a concentration of 1g/dm3, from an autoclaved 100 g/dm3 stock solution of PEG in aqua bidest. PEG has been tested previously in sparged animal cell cultures (Murhammer and Goochee, 1990). Components mentioned without the name of the manufacturer were obtained from Merck. Culture Method and Shear Stress. The cells were cultured as a continuous suspension in a 1dm3continuous stirred-tank reactor (CSTR) with a working volume of 0.5 dm3 and a growth rate of 8.5 X 10-6 s-l [growth rate = dilution ratehiability ratio; viability ratio = number of viable cells/total cell number]. The CSTR culture provided defined samples for sparging shear-stress experiments in the bubble column under standard conditions, as described previously (Van der Pol et al., 1990). The suspension (0.08dm3) in the bubble column was sparged with a volumetric flow rate of 6 dm3/ h, resulting in a gas flow per unit culture volume of 75 h-' (or 1.25 wm). This holdsfor allshear-stress experiments, unless stated otherwise in the Results and Discussion section. Experiments in the bubble column were undertaken to quantify the first-order death rate constant (kd) under circumstances that usually cause a much higher death rate than the known growth rate. This means that the standard conditions in the bubble column result in substantial cell death unless a protective component is present in a sufficiently high concentration. A few seconds before the start of an experiment, a certain amount of nonautoclaved 1:lO diluted AF was added. The AF used was medical grade antifoam B silicone emulsion (J. T. Baker) containing poly(dimethy1siloxane). The generation of a foam layer on the medium surface was prevented by touching the surface with a Pasteur pipet filled with 1:25 diluted AF (tipping) when necessary. The standard experiment in the bubble column is therefore carried out in the absence of foam. The standard bubble column experiment lasted for 3 h with sampling every 30 min, starting with an initial viable cell concentration above lO6/cm3, Sampling and cell counting was stopped within 3 h when the number of viable cells dropped below lo5/ cm3, When the viable cell number decreased rapidly, samples were taken more often. Cell Counting and Analysis. Cell numbers were determined under the microscopewith a Fuchs-Rosenthal hemacytometer by the trypan blue exclusion method. The change in the number of viable or dead cells is expressed as a percentage of the total cell concentration (viable and trypan blue stained) at the beginning of the experiment. This was done to make changes in cell numbers comparable to changes in the activity of lactate dehydrogenase. The first-order death rate constant (kd)was determined from the slope of the plot, In (Cv,t/Cv,o)versus time t , with Cv,t being the viable cell concentration at time t and C,,O the viable cell concentration at t = 0. Biological Oxygen Monitoring. The cell-specific oxygen consumption rate (CSOCR) of PFU-83 cells was determined by biological oxygen monitoring with a Y SI membrane kit (standard) in a Tamson vessel at 37 O C . The 0 and 95% set points for the oxygen electrode were
505
Kd versus [AFI Kd (xE-5
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PEO
6 % FCS
151
I ~
01 0
0.02
0.04
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0.1
0.12
(%)
Figure 1. First-order death rate constant of hybridoma cells in a bubble column at an air flow rate of 6 dms/h as a function of the concentrationof antifoam,added at the beginning of a bubble column experiment, in culture medium with serum or PEG as the protective component.
adjusted with nitrogen and air containing 5% COz. Suspension samples of 10cm3were taken from the standard 1 dm3 CSTR culture and transferred rapidly into the Tamson vessel. This takes about 1min. Shortly after the addition of a certain amount of AF the measurements start. The CSOCR was calculated by dividing the decrease in dissolved oxygen,the oxygen uptake rate (mol of 02m-3 5-9, by the viable cell number. Lactate Dehydrogenase (LDH) Assay. The LDH assay was executed with cell-free supernatant (2 min a t 500g in a DADE Immufuge) using a method based on the Boehringer LDH monotest, similar to that of Racher et al. (1990). The increase in LDH activity, before and after a shear-stress experiment, is expressed as a percentage of the 100% LDH score, which is determined with a 0.1% Triton X-100 (Merck) treated (releasing all LDH) sample of the cell suspension before a bubble column experiment. Cell-free supernatant samples were kept at -20 "C for a maximum of 2 weeks.
Results and Discussion Sparging Death Rate and Antifoam Effect. The effect of varying the concentration of silicone AF on the death rate of PFU-83 hybridoma cells was examined in a bubble column in RPMI medium containing 5 % FCS and serum-free DME/F12 medium with or without PEG. For PFU-83 cells cultured in RPMI medium with 5 % FCS, the death rate constant (kd)increases with a higher AF concentration (Figure 1). This experiment was repeated with PFU-83 cells cultured in the CSTR in serumfree DME/F12 medium. Just before a bubble column experiment in the bubble column, PEG 20000 and a variable concentration of AF were added to the suspension sample. Again, an enhancement in the AF concentration from 0.002 to 0.1 5% causes a 4-5-fold increase in the death rate constant, k d (Figure 1). During these experiments,
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Kd versus 80
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150
.'.
0 0
40
0 100
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20
0' 0
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0.06 0.08 [AFI (%I
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Figure 2. Firsborder death rate constant of hybridoma cella in a bubble column at an air flow rate of 3 dma/has a function of the initial concentrationof added antifoam in DMEIF12 medium without a protective component. The values of the death rate constant for the low antifoam concentrationsare also shown after correction for tipping with 1:25 diluted antifoam solution during the bubbling experiment. The results given in Figure 1(obtained at 6 dmVh) are included for comparison.
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it was occasionally necessary to prevent the formation of foam by tipping with diluted AF emulsion. In serum-free DMEIF12 medium without a protective component, the sparging in the bubble column causes a high death rate. In order to try to reduce this death rate, the volumetric air flow rate was reduced to 3 dm3/h. An increase in AF concentration then resulted in a lower k d (Figure 2). This lower k d is still substantially higher than the increased kdvalues a t high AF concentrations in media with a protective component, despite the difference in the air flow rate. In medium without a protective component, and with the addition of AF to a low concentration at the beginning of a bubble column experiment, tipping with diluted AF was necessary during the experiment to such a degree that the AF concentration had to be corrected for the extra added 1:25 diluted AF. The corrected end values are also shown in Figure 2. At higher initial AF concentrations, the tipping was necessary less often and can be neglected. On the average, stress experiments in medium without a protective component lasted about 1.5 h. Antifoam in Serum-Free Medium with PEG. Because only media with a protective component can be applied for the cultivation of animal cells using sparging, the detrimental effect of AF was further examined in DME/ F12 medium with PEG 6000 or 20 000. Medium supplementad with either PEG 6000 or PEG 20 000 resulted in similar k d values in the standard shear experiments (data not shown). Bubble column experiments with PEG 6000 or 20 000 and a high (0.1% ) AF concentration resulted in an increase in trypan blue stained dead cells (Figure 3a,b), especially in the beginning. Lysis of the cells is indicated by the reduction of the total cell number. The reduction in viable
-A- cell total
0
50
time
100 (minuter)
150
200
Figure 3. Change in cell numbers of hybridoma cella in a bubble column during a bubble column experimentin DME/F12 medium with 0.1% antifoam and 1g/dm8PEG 6000 (a) or PEG 20 000 (b). cells before and after a shear-stress experiment corresponds well with the increase in relative LDH activity (Figure 4). For 0.1% AF, the results of duplicate experiments are shown to give an indication of variance. Addition of 0.1% AF to a control sample of the cell suspension from CSTR, incubated in culture flasks for 3-4 h, did not cause any change in the cell numbers. This shows that AF makes the cells more vulnerable to shear caused by sparging but does not have a short-term toxic effect. The addition of 0.1% autoclaved AF emulsion,
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PEG 6000
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CSOCR (xE-17 moVcel.8)
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4
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5%FCS PEQ
-& DME/F12
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30
20
10
0 0.002
0.05 [AFI
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0.1
(%I
Figure 4. Relative change in the viable cell number (-dC,, %) and LDH activity (dLDH, %) of hybridoma cells in a bubble column in DMEIF12 medium with 1 g/dm3PEG 6000 after a bubble column experiment at various antifoam concentrations (data partly used for Figure 3a,b).
however, did have a long-term effect because the growth of the PFU-83 cells in culture flasks with this addition was strongly reduced after 3 days in an incubator. This indicates that a high AF concentration does have a cytostatic effect on PFU-83 cells. The silicone AF effect of increasing the death rate was further investigated by determining the effect of the AF concentration on the consumption of oxygen of PFU-83 cells. The cell-specific oxygen consumptionrate (CSOCR) decreases with increasingAF concentration in serum-free DMEIF12 medium with PEG (Figure 5). A similar decreasing effect was found using RPMI, 5% FCS, and DMEIF12 medium without a protective component. The reduction is most prominent at the lower AF concentrations; at higher concentrations, saturation occurs. A reduction of the oxygen absorption rate (OAR = OUR) caused by the addition of 0.1% silicone base compound AF has also been reported for yeast cells (Philips et al., 1960). Because AF is a hydrophobic surface-activesubstance, it is likely to exert its effect by changing the properties of either the gas-liquid interfaceor the cell-medium interface (Jarvis, 1966). Since the gas-liquid interface is absent during biological oxygen monitoring, the results of Figure 5 suggest that the detrimental effects of silicone AF on the shear sensitivity of cells might involve an effect of AF on the cell membrane. Antifoam in Media without a Protective Component. As stated before, medium without a protective component cannot be applied for animal cell culturing using sparging. Addition of an increasingAF concentration causes opposite effects in medium with or without a protectivecomponent (Figures1and 2). Therefore, control experimentsconcerning the effect of silicone AF in DME/ F12 medium without a protective component have been executed. As mentioned before, the standard shear experiment in the bubble column in media without a
0
0.05
0.1 IAFI
0.15
0.2
(%I
Figure 5. Cell-specificoxygen consumption rate (CSOCR) of hybridoma cells as a function of antifoam concentration.
protective component is difficult to carry out at low AF concentrations, because of the necessary frequent tipping with diluted AF emulsion (Figure 2). Therefore, a comparison is made between stress experiments completely without AF, allowing foam to develop, or with 0.1 % AF, at an air flow rate of 6 dm3/h. Without AF, an unstable foam with bubbles of variable size developed from the beginning of the standard shearstress experiment. This is possibly due to cellular proteins because the DME/F12 medium itself does not generate foaming. The number of viable cells decreased to a value below 105/cm3within 1 h. Also, the total cell number decreased from the beginningof the experiment,indicating cell lysis or loss of cells in the foam (Figure 6a). In the small deposit developing at the top of the foam layer, very few dead cells could be detected. The relative increase in LDH activity correspondswith the decrease in the fraction of viable cells (Figure 7). In medium without a protective component and without AF, cells are thus rapidly lysed in the suspension or in the unstable foam. The addition of 0.1% AF to a cell suspension in medium without a protective component prevents the development of foam. Comparable to DMEIF12 medium with PEG and 0.1% AF (Figure 3a,b), first the number of trypan blue stained dead cells increases while the total cell number shows a slight reduction (Figure 6b). This is followed by the lysis of cells as shown by the decrease in the total cell number. The increase in relative LDH activity is less than the relative decrease in the viable cell fraction (Figure 7), due to unknown reasons. The death rate constant ( k d ) in the presence of 0.1% AF is about one-half of the k d found without AF (Figure 7). Mechanism of Cell Death. From the experiments using medium with a protective component and with AF, it can be concluded that neither serum nor PEG needs the development of a multilayer stable foam to exert its protective effect. Under similar sparging shear-stress conditions, but using less defined culture media, Pluronic F68 protects mouse LS suspension cells in the absence of
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(&) and relative changes in viable cellnumber (-dC,, % ) and LDH activity (dLDH, % ) of hybridomacells in a bubble column in DME/F12 medium without a protective componentand without and with antifoam. The duration of the bubbling experimentsare 40 and 45 min for duplicate experiments without antifoam and 120 min for the single experiment in the presence of 0.1% antifoam (data also partly used for Figure 6a,b).
[AF] 0.1 (96)
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Figure 7. First-order death rate constant
no protection
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0-dCv
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Figure 6. Cell numbers of hybridoma cells in a bubble column
in DME/F12 medium without a protective component, as a functionof time, without (a)or with (b)additionof 0.1 % antifoam.
a foam layer (Kilburn, 1967). The same finding has also been noted for Pluronic F68 with insect cells (Murhammer and Goochee, 1988). Therefore, it seems unlikely that polymers such as Pluronic and PEG act as cell protective agents only by stabilizingfoams, as suggested as by HandaCorrigan et al. (1989). The experiments using DMEIF12 medium without protective polymers show that without AF' an unstable, nonhomogeneous foam layer causes the rapid lysis of cells. This might be caused by film drainage and bubble breakup in the foam.
In the presence of 0.1 % AF, processes associated with the breakup of single bubbles at the surface of the cell suspension cause first an increase in intact dead cells and later lysis. This might be due to rapid oscillations after bubble breakup (Handa-Corrigan et al., 1989) or to the adherence of cells to bubbles resulting in collective injury after the bubble breakup by vertical jetting and ejection of a liquid drop (Chalmers and Bavarian, 1991;Bavarian et al., 1991). Recently, Cherry and Hulle (1992)suggested that the rupture of thin films accounts for most if not all of the deaths of insect cells within a sparged bioreactor, using medium with either 0.1 % Pluronic F68 or 5 % FCS as the protective component. In the DMEIF12 medium, it also cannot be excluded that cells die in the suspension due to rising bubbles, because experiments that indicate the bubble rising zone as relatively harmless have all been done using media with protective components (Handa-Corrigan et al., 1989; Tramper et al., 1987), which can change the gas-liquid interface properties of bubbles. This underlines the importanceof a standard setup of shear-stress experiments using a fully defined medium.
Conclusions Relatively high concentrations of medical grade silicone
AI? emulsioncause an increase in cell death during sparging of PFU-83 hybridoma cells. The detrimental effect is not substantial a t the low concentrations needed for the subtle handling of an existing foam during batch cultivations, using sparging in a medium with a protective component. However, the excessive use of AF to prevent development of any foam or the repeated use of AF' during a continuous cultivation might cause higher rates cell death or reduced growth. The reducing effect of AF on the cell-specific
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oxygen consumption of PFU-83cells occurs mainly a t the low concentrations.
Acknowledgment The authors are indebted to Mrs.Annelies Leeuwe and Mr. Marco Lip for technical assistance. Literature Cited Aunins, J. G.; Croughan, M. S.; Wang, D. I. C.; Goldstein, J. M. Engineering developments in homogeneous culture of animal cells: oxygenationof reactors and scaleup. Biotechnol.Bioeng. Symp. 1989,17,699-723. Bavarian, B.; Fan, L. S.; Chalmers,J. J. Microscopicvisualization of insect cell-bubbleinteractions 1: risingbubbles,air-medium interface and the foam layer. Biotechnol. Prog. 1991,7,140150. Chalmers, J. J.; Bavarian, F. Microscopic visualization of insect cell-bubbleinteractions 2: the bubble film and bubble rupture. Biotechnol. hog. 1991,7, 151-158. Cherry, R. S.;Hulle, C. T. Cell death in thin films of bursting bubbles. Biotechnol. Prog. 1992,8,11-18. Ghildyal, N. P.; Lonsane, B. K.; Karanth, N. G. Foam control in submerged fermentation: state of the art. Adv. Appl. Microbiol. 1988,33,173-222. Handa-Corrigan, A.; Emery, A. N.; Spier, R. E. Effect of gasliquid interfaces on the growth of suspended mammalian cells: mechanism of cell damage by bubbles. Enzyme Microb. Technol. 1989,11,230-235. Jarvis, N. L.;Surface viscosity of polydimethylsiloxane monolayers. J. Phys. Chem. 1966,70,3027-3033. Jbbses, I.; Martens, D.; Tramper, J. Lethal events during gas sparging in animal cell culture. Biotechnol. Bioeng. 1991,37, 484-490. Kawase, Y.;Moo-Young, M. The effect of antifoam agents on ma98 transfer in bioreactors. Bioprocess Eng. 1990,5,169173. Kilburn, D. G. Some factors affecting the growth of mammalian cells in suspension cultures. Ph.D. Thesis, University of London, London, UK, 1967. Murhammer, D. W.; Goochee, C. F.; Scaleup of insect cell cultures: protective effects of Pluronic F-68.BiolTechnology 1988,6,1411-1418. '
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Murhammer, D. W.; Goochee, C. F. Sparged animal cell bioreactors: mechaniim of celldamageand PluronicF-68protedion. Biotechnol. Prog. 1990,6, 391-397. Papoutsakis, E. T. Media additives for protecting animal cell against agitation and aeration damage in bioreactors. Trends Biotechnol. 1991,9, 316324. Philips, K. L.; Spencer, J. F. T.; Sallans, H. R.; Roxburgh, J. M. Effect of antifoam agents on oxygen transfer in deep tank fermentations. J.Biochem. Microbiol. Technol.Eng. 1960,2, 81-91. Prins, A.; van't Riet, K. Proteins and surface effects in fermentation: foam,antifoam,and mass transfer. TrendsBiotechnol. 1987,5,296301. Racher, A. J.; Looby, D.; Griffiths, J. B. Use of lactate dehydrogenase release to assess changes in culture viability. Cytotechnology 1990,3,301-307. Tramper, J.;Joustra,D.; Vlak,J. M. Bioreactordesignfor growth of shear-sensitive insect cells. In Plant and animal cells;Ellis Horwood Chichester, 1987;pp 125-136. Tramper, J.; Smit, D.; Straatman, J.; Vlak, J. M. Bubble-column design for growthof fragile insect cells. BioprocessEng. 1988, 3,37-41. Van Oers, J. W. A. M.; Tilders, F. J. H.; Berkenbosch, F. Characterization and biological activity of a rat monoclonal to rat/human corticotropinreleasingfactor. Endocrinology1989, 124,1239-1246. Van der Pol, L.; Zijlstra, G.; Thalen, M.; Tramper, J. Effect of serum concentration on production of monoclonalantibodies and on shear sensitivity of a hybridoma. BioprocessEng. 1990, 5,241-245. Van der Pol, L.; Bakker, W. A. M.; Tramper, J. Effect of low serum concentrations (0-2.5% ) on growth, production, and shear sensitivity of hybridomacells. Biotechnol.Bioeng. 1992, 40,179-182. Van't Riet, K.; Tramper, J. Basic Bioreactor Design; Marcel Dekker: New York, 1991. Zhang, S.; Handa-Corrigan, A,; Spier, R. E. Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors. J. Biotechnol. 1992,25, 289-306. Accepted April 21, 1993.
