Mammalian cell culture processes Wei-Shou Hu and James M. Piret University of Minnesota, Minneapolis, Minnesota, USA and University of British Columbia, Vancouver, British Columbia, Canada Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications. Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes. Recent advances have included novel biosensor and bioreactor developments. New technologies have also been applied to investigate high cell density bioreactor and culture conditions. Current Opinion in Biotechnology 1992, 3:110-114
Introduction Mammalian cell culture is being used for the production of an increasing n u m b e r of therapeutic and diagnostic proteins that require post-translational processing that is specific to mammalian ceils. Low cell and product concentrations in mammalian cell culture continue to motivate studies of the cell culture environment. The kinetic models and biosensors that are being developed should provide a basis for implementing process control strategies for fed-batch or semi-continuous operation. An important area of interest is the consistency with which proteins undergo post-translational modification as a function of bioreactor environment. High cell density porous microcarriers, hollow fiber bioreactors and novel bioreactors are being investigated to improve the selection of their design and operating parameters. Recent articles have reviewed animal cell culture bioreactor technology [1], porous microcarriers [2], hollow fiber bioreactors [3] and fluid-mechanical d a m a g e in suspension bioreactors [4"]. This review will focus on the developments in mammalian cell culture over the past year.
W h e n developing a process with the aim of increasing productivity, it is critical that the microstructure and the biological activity of the product molecules are not altered as a result of changing operating conditions. It has b e e n s h o w n that the production rate of g-interferon b y Chinese hamster ovary (CHO) cells in continuous culture remains relatively constant, whereas, the fraction of fully glycosylated product varies u p o n changes in glucose feed concentration and dilution rate [10]. The literature on bioprocess factors that influence glycosylation has recently b e e n reviewed [11"]. Differences in tyrosine sulfation b e t w e e n h u m a n proteins and those produced by recombinant CHO cells have b e e n reported [12,13]. Attempts have b e e n made to express enzymes involved in the synthesis of the carbohydrate moiety of glycoproteins in heterologous host ceils [14,15]. This type of approach could possibly be u s e d to modify the host system to allow for the increased production of correctly post-translationally modified proteins. The effects of varying environmental factors (i.e. employing fed-batch or stress cultivation conditions) on protein glycosylation are still not well understood; rapid methods for oligosaccharide analysis will facilitate such studies [16].
Cell culture environment Researchers have continued to increase mammalian cell culture productivity by adding extraneous compounds to the culture m e d i u m [5-7] and by altering cell culture conditions by, for example, increasing the osmolarity [8]. Increasing the initial concentrations of nutrients, however, will often not increase the final cell concentrations [9]. The continued success of improving productivity b y the manipulation of environmental factors during the cultivation period will largely d e p e n d on identifying the key factors for control and developing strategies to estimate the optimal rate of feeding those factors.
Dy na m ic feeding of nutrients Fed-batch and perfusion supplementing of amino acids that have b e e n identified as deficient, has resulted in increased productivity (D Jain, S Gold, D Dislefona, G Cuca, D Benincasa K Ramasubramanyan, A Lenny, G Mark, E Silberklang, Abstract, General Meeting of the European Society of Animal Cell Technology, 1991, Brighton, UK). Amino acids are the most frequent c o m p o n e n t s of the m e d i u m to be examined, probably because they are relatively easy to analyze. The m e c h a n i s m of their beneficial effects is most likely to
Abbreviations CliO--Chinese hamster ovary cells; HFBR--holIow fiber bioreactor.
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vary with cell line and culture conditions used. The enhanced productivity observed in fed-batch or perfusion cultures may be the result of maintaining the concentration profile of key nutrients in an 'optimal window'. Although the beneficial effect of fed-batch culture is becoming more evident, a strategy for its process optimization has not emerged. Because the concentrations of cells and nutrients as well as the rates of consumption vary with time, online instruments and culture-state estimation would be valuable. Several optical probes have b e c o m e commercially available for on-line m e a s u r e m e n t of turbidity or light scattering to estimate cell concentration. A number of recently developed rapid immunoassay techniques are suitable for semi-continuous monitoring of protein production [17"o,18]. For the analysis of chemical species, such as glucose, lactate and amino acids, on-line sampling devices have b e e n used [19,20]. Other environmental variables, which may or may not b e directly related to cellular state, such as fluorescence [21] and redox potential [22], have also b e e n investigated; viable cell concentration appears to be correlated with redox potential [22]. Despite the potential benefits of on-line measurements, reports of studies employing these more efficient, instrumented bioreactors for mammalian cell culture are still scarce. The most significant potential advantages of a highly instrumented bioreactor are in identifying the physiological state of cells in the bioreactor and for controlling the reaction path for optimal production. A most obvious immediate application of such a bioreactor would be for the control of fed-batch or perfusion culture. For such applications, however, it is necessary to have reliable kinetic models to describe the response of cells to the variables (nutrient perfusion rate etc.) that are being controlled. Kinetic models have b e e n d e v e l o p e d to describe the response- of growth rate to growth-limiting nutrient concentrations [23,24]. Attempts have also b e e n made to describe production of a monoclonal antibody using a structured model [25]. The independent variables investigated in those studies m a y not be the ones that are most important for process control or optimization. In most studies, the average properties of the mammalian cell population are considered. Employing flow cytometry, the distribution of properties within a population can be examined. For instance, in an apparently h o m o g e n e o u s population of hybridoma cells, it was demonstrated that the rate of protein synthesis was cell-cycle dependent [26]. A segregated model of antibody production could describe these kinetics. Bioreactors
Manufacturing processes employing mammalian cells go through seed and inoculum culture before reaching the production stage. In the early stages of seed culture and sometimes inoculum culture, conventional cultivation devices, such as roller bottles, spinners or stacking fiat-plates, are employed. The importance of these stages eannot be overemphasized, although most
engineering research work naturally emphasizes the production stage. Robotics systems are increasingly being used to reduce labor requirements and contamination frequency (R Archer, Abstract, Congress on Cell a n d Tissue Culture, 1991, Anaheim, California) w h e n large numbers of roller bottles are used. In an effort to increase productivity, a n u m b e r of highcell density perfusion bioreactors have b e e n developed. We will discuss not only the current research on porous microcarriers and hollow fiber bioreactors but also some novel bioreactors. Porous microcarriers have b e e n used to culture adherent and suspended cells (e.g. hybridomas). This technology can be used in conventional stirred tank bioreactors. A n u m b e r of hollow fiber bioreactors are also being investigated. These bioreactors have b e e n particularly useful in bench-top scale production of monoclonal antibodies. Microcarrier developments Microcarriers d e v e l o p e d primarily for cultivating anchorage-dependent cells have diameters typically in the range of 150-250b tin; using beads of much smaller diameter (10-50 gm), w e observed that a number of celt lines, including green m o n k e y kidney epithelial cells (vero cells) and swine testicular epithelial ceils, form aggregates of about 20 cell-layers from the perimeter to the centre of the aggregates [27"]. Because the b e a d volumes are relatively small and the concentration of cells in each bead is increased, a high cell concentration can be achieved with a relatively low microcarrier concentration.
Collagen and gelatin porous microcarriers that were developed in the early 1980s allow cells to grow to high densities in the connected pores of the interior. Cellulose porous microcarriers have since b e c o m e commercially available [28]. Recently, porous polystyrene [29] and polyethylene [30"] b e a d s have b e e n e m p l o y e d to cultivate v e r o ceils, hybridomas and CHO cells. Fluidized-bed or stirred-tank bioreactors are used for porous microcarrier culture. Porous microcarriers support a higher cell concentration than conventional solid microcarriers at comparable b e a d concentrations. The presence of ceils in the pores has been confirmed by microscopic observation after thin sectioning [31]. The fraction of internal volume occupied b y a n u m b e r of cell lines in gelatin- and cellulose-based porous microcarriers varied from 5% to over 40% [28,31]. Using confocal microscopy, cells in cross-linked gelatin porous microcarriers w e r e s h o w n to b e present primarily in regions away from the center of the b e a d s (JH Kim, HS Lim, BK Han, MVB Peshwa, WS Hui, Abstract, 4th
Annual Meeting of the Japanese Association for Animal Cell Technology, Fukuoka, Japan, 1991, p 15). It should be noted that, if cells form multilayers and penetrate half the radius into the beads, 87% of the b e a d void volume can b e occupied by ceils. Hollow fiber bioreactors A wide variety of hollow fiber bioreactors (HFBR) are available for cell culture. Key distinctions b e t w e e n vari-
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Biochemicalengineering ous HFBRs include their use of hydrophilic, hydrophobic, microporous or ultrafiltration membranes. The pressure gradient along the inside of the fibers generates a secondary flow, k n o w n as Starling flow, in the extracapillary space where the cells are immobilized. The Starling flow has b e e n studied in cell-free HFBRs using nuclear magnetic resonance flow-imaging techniques [32]. In the case of ultrafiltration bioreactors, high molecular weight protein products (i.e. monoclonal antibodies) are concentrated in the extracapillary space. It has b e e n s h o w n b y sectioning frozen ultrafiltration HFBRs that these flows can polarize high molecular weight proteins to the downstream end of the extracapillary space [33"]. This polarization has b e e n used to increase the harvest concentrations of monoclonal antibodies. The polarization of high molecular weight growth factor proteins in ultrafiltration bioreactors can also polarize cell growth and reduce bioreactor productivities. Cycling of the flow direction distributes cell growth and increases HFBR antibody productivities [33"]. Particularly w h e n using serum-free m e d i u m in HFBRs, it may be important to find ways to minimize both cell lysis and the release of proteases. Data related to this problem have b e e n reported by van Erp et al. [34], w h o assayed acid protease activity and the recovery of immunoglobulin G fragments in serum-free HFBR cultures of one hybridoma cell line. Analysis of extracapillary samples confirmed that oxygen is a critical scale-limiting nutrient [34] and improved effectiveness factor plots were d e v e l o p e d [35].
Perspectives The technology for manufacturing pharmaceuticals employing mammalian cells has rapidly developed over the past decade and b e c o m e part of an important industry. Significant progress was made in 1991 towards the optimization of bioreactors and processes. Investigations of the cell culture environment effects will provide the basis for optimizing conditions throughout the cultivation period. In the past, studies of medium design and optimization focused on the initial conditions. Gradually, fed-batch and continuous perfusion processes are being introduced. The implementation of dynamic feeding of nutrients will most likely create a need for more sophisticated on-line m e a s u r e m e n t and control, which will stimulate the dev e l o p m e n t of meaningful and useful kinetic models for process optimization. Care should be taken to ensure that process control via dynamic manipulation of environmental factors produces proteins with consistent post-translation modifications. Overall, mammalian cell culture technology has enabled the efficient production of a wide range of products. In the near future, some aspects of the technology d e v e l o p e d in the past decade should b e adapted to other biomedical applications, including the cultivation of differentiated ceils and tissues (such as stem cells, hepatocytes, and nerve cells) and the screening, identification and isolation of molecules of pharmaceutical importance. Bioreactors that were scaled u p for protein production in the 1980s may b e scaled d o w n for these n e w applications.
Acknowledgements Novel bioreactors Increasing attention has b e e n paid in the past few years to spin-filter or wire-cage reactors for suspension, microcarrier or aggregate cultures [36,37]. As fouling of the screen poses problems for long-term operation of bioreactors, it is worth noting that the fouling of polyamide screens has b e e n s h o w n to b e less than that of stainless steel screens [38] and that the filter openings can b e larger if cells are cultivated on microcarriers or as aggregates [37]. Three other reports of novel bioreactors have b e e n published. In one, cell aggregates w e r e immobilized on polyurethane foam in a p a c k e d - b e d reactor [39]. In another, a packed glass wool bed was used to cultivate anchorage-dependent ceils in a reactor in which gas sparging w a s used to provide fluid circulation through the packed glass wool in the descending fluid region [40]. In the third, a three-phase bioreactor employing porous polyvinyl formal resin particles was used to cultivate suspension ceils [41]. In most of these bioreactors a relatively high cell concentration was achieved. This has resulted in the n e e d for higher rates of perfusion. An alternative technique developed to avoid increased medium flow rates employs a dialysis device for the removal of metabolites or other inhibitory substances [42]. This approach, however, requires large areas of m e m b r a n e in the bioreactor. As a solution, the dialysis tubing can b e installed in an external vessel [43].
Part of this manuscript was prepared while Wei-Shou Hu was visiting the International Center for Cooperative Research in Biotechnology. Support from Osaka University is gratefully acknowledged. This work w a s supported in part by grants from the National Science Foundation, USA (BCS-8551670) to Wei-Shou Hu and from the National Science and Engineering Research Council of Canada to James Piret.
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W-S Hu, Department of Chemical Engineering and Materials Science, 151 Amundsen Hall, 421 Washington Avenue S.E., University of Minnesota, Minneapolis, Minnesota 55455, USA. JM Piret, Biotechnology Laboratory and Department of Chemical Engineering, University of British Columbia, 237~5174 University Boulevard, Vancouver, British Columbia, V6T 1Z3 Canada.
Introduction Mammalian cell culture is being used for the production of an increasing n u m b e r of therapeutic and diagnostic proteins that require post-translational processing that is specific to mammalian ceils. Low cell and product concentrations in mammalian cell culture continue to motivate studies of the cell culture environment. The kinetic models and biosensors that are being developed should provide a basis for implementing process control strategies for fed-batch or semi-continuous operation. An important area of interest is the consistency with which proteins undergo post-translational modification as a function of bioreactor environment. High cell density porous microcarriers, hollow fiber bioreactors and novel bioreactors are being investigated to improve the selection of their design and operating parameters. Recent articles have reviewed animal cell culture bioreactor technology [1], porous microcarriers [2], hollow fiber bioreactors [3] and fluid-mechanical d a m a g e in suspension bioreactors [4"]. This review will focus on the developments in mammalian cell culture over the past year.
W h e n developing a process with the aim of increasing productivity, it is critical that the microstructure and the biological activity of the product molecules are not altered as a result of changing operating conditions. It has b e e n s h o w n that the production rate of g-interferon b y Chinese hamster ovary (CHO) cells in continuous culture remains relatively constant, whereas, the fraction of fully glycosylated product varies u p o n changes in glucose feed concentration and dilution rate [10]. The literature on bioprocess factors that influence glycosylation has recently b e e n reviewed [11"]. Differences in tyrosine sulfation b e t w e e n h u m a n proteins and those produced by recombinant CHO cells have b e e n reported [12,13]. Attempts have b e e n made to express enzymes involved in the synthesis of the carbohydrate moiety of glycoproteins in heterologous host ceils [14,15]. This type of approach could possibly be u s e d to modify the host system to allow for the increased production of correctly post-translationally modified proteins. The effects of varying environmental factors (i.e. employing fed-batch or stress cultivation conditions) on protein glycosylation are still not well understood; rapid methods for oligosaccharide analysis will facilitate such studies [16].
Cell culture environment Researchers have continued to increase mammalian cell culture productivity by adding extraneous compounds to the culture m e d i u m [5-7] and by altering cell culture conditions by, for example, increasing the osmolarity [8]. Increasing the initial concentrations of nutrients, however, will often not increase the final cell concentrations [9]. The continued success of improving productivity b y the manipulation of environmental factors during the cultivation period will largely d e p e n d on identifying the key factors for control and developing strategies to estimate the optimal rate of feeding those factors.
Dy na m ic feeding of nutrients Fed-batch and perfusion supplementing of amino acids that have b e e n identified as deficient, has resulted in increased productivity (D Jain, S Gold, D Dislefona, G Cuca, D Benincasa K Ramasubramanyan, A Lenny, G Mark, E Silberklang, Abstract, General Meeting of the European Society of Animal Cell Technology, 1991, Brighton, UK). Amino acids are the most frequent c o m p o n e n t s of the m e d i u m to be examined, probably because they are relatively easy to analyze. The m e c h a n i s m of their beneficial effects is most likely to
Abbreviations CliO--Chinese hamster ovary cells; HFBR--holIow fiber bioreactor.
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vary with cell line and culture conditions used. The enhanced productivity observed in fed-batch or perfusion cultures may be the result of maintaining the concentration profile of key nutrients in an 'optimal window'. Although the beneficial effect of fed-batch culture is becoming more evident, a strategy for its process optimization has not emerged. Because the concentrations of cells and nutrients as well as the rates of consumption vary with time, online instruments and culture-state estimation would be valuable. Several optical probes have b e c o m e commercially available for on-line m e a s u r e m e n t of turbidity or light scattering to estimate cell concentration. A number of recently developed rapid immunoassay techniques are suitable for semi-continuous monitoring of protein production [17"o,18]. For the analysis of chemical species, such as glucose, lactate and amino acids, on-line sampling devices have b e e n used [19,20]. Other environmental variables, which may or may not b e directly related to cellular state, such as fluorescence [21] and redox potential [22], have also b e e n investigated; viable cell concentration appears to be correlated with redox potential [22]. Despite the potential benefits of on-line measurements, reports of studies employing these more efficient, instrumented bioreactors for mammalian cell culture are still scarce. The most significant potential advantages of a highly instrumented bioreactor are in identifying the physiological state of cells in the bioreactor and for controlling the reaction path for optimal production. A most obvious immediate application of such a bioreactor would be for the control of fed-batch or perfusion culture. For such applications, however, it is necessary to have reliable kinetic models to describe the response of cells to the variables (nutrient perfusion rate etc.) that are being controlled. Kinetic models have b e e n d e v e l o p e d to describe the response- of growth rate to growth-limiting nutrient concentrations [23,24]. Attempts have also b e e n made to describe production of a monoclonal antibody using a structured model [25]. The independent variables investigated in those studies m a y not be the ones that are most important for process control or optimization. In most studies, the average properties of the mammalian cell population are considered. Employing flow cytometry, the distribution of properties within a population can be examined. For instance, in an apparently h o m o g e n e o u s population of hybridoma cells, it was demonstrated that the rate of protein synthesis was cell-cycle dependent [26]. A segregated model of antibody production could describe these kinetics. Bioreactors
Manufacturing processes employing mammalian cells go through seed and inoculum culture before reaching the production stage. In the early stages of seed culture and sometimes inoculum culture, conventional cultivation devices, such as roller bottles, spinners or stacking fiat-plates, are employed. The importance of these stages eannot be overemphasized, although most
engineering research work naturally emphasizes the production stage. Robotics systems are increasingly being used to reduce labor requirements and contamination frequency (R Archer, Abstract, Congress on Cell a n d Tissue Culture, 1991, Anaheim, California) w h e n large numbers of roller bottles are used. In an effort to increase productivity, a n u m b e r of highcell density perfusion bioreactors have b e e n developed. We will discuss not only the current research on porous microcarriers and hollow fiber bioreactors but also some novel bioreactors. Porous microcarriers have b e e n used to culture adherent and suspended cells (e.g. hybridomas). This technology can be used in conventional stirred tank bioreactors. A n u m b e r of hollow fiber bioreactors are also being investigated. These bioreactors have b e e n particularly useful in bench-top scale production of monoclonal antibodies. Microcarrier developments Microcarriers d e v e l o p e d primarily for cultivating anchorage-dependent cells have diameters typically in the range of 150-250b tin; using beads of much smaller diameter (10-50 gm), w e observed that a number of celt lines, including green m o n k e y kidney epithelial cells (vero cells) and swine testicular epithelial ceils, form aggregates of about 20 cell-layers from the perimeter to the centre of the aggregates [27"]. Because the b e a d volumes are relatively small and the concentration of cells in each bead is increased, a high cell concentration can be achieved with a relatively low microcarrier concentration.
Collagen and gelatin porous microcarriers that were developed in the early 1980s allow cells to grow to high densities in the connected pores of the interior. Cellulose porous microcarriers have since b e c o m e commercially available [28]. Recently, porous polystyrene [29] and polyethylene [30"] b e a d s have b e e n e m p l o y e d to cultivate v e r o ceils, hybridomas and CHO cells. Fluidized-bed or stirred-tank bioreactors are used for porous microcarrier culture. Porous microcarriers support a higher cell concentration than conventional solid microcarriers at comparable b e a d concentrations. The presence of ceils in the pores has been confirmed by microscopic observation after thin sectioning [31]. The fraction of internal volume occupied b y a n u m b e r of cell lines in gelatin- and cellulose-based porous microcarriers varied from 5% to over 40% [28,31]. Using confocal microscopy, cells in cross-linked gelatin porous microcarriers w e r e s h o w n to b e present primarily in regions away from the center of the b e a d s (JH Kim, HS Lim, BK Han, MVB Peshwa, WS Hui, Abstract, 4th
Annual Meeting of the Japanese Association for Animal Cell Technology, Fukuoka, Japan, 1991, p 15). It should be noted that, if cells form multilayers and penetrate half the radius into the beads, 87% of the b e a d void volume can b e occupied by ceils. Hollow fiber bioreactors A wide variety of hollow fiber bioreactors (HFBR) are available for cell culture. Key distinctions b e t w e e n vari-
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Biochemicalengineering ous HFBRs include their use of hydrophilic, hydrophobic, microporous or ultrafiltration membranes. The pressure gradient along the inside of the fibers generates a secondary flow, k n o w n as Starling flow, in the extracapillary space where the cells are immobilized. The Starling flow has b e e n studied in cell-free HFBRs using nuclear magnetic resonance flow-imaging techniques [32]. In the case of ultrafiltration bioreactors, high molecular weight protein products (i.e. monoclonal antibodies) are concentrated in the extracapillary space. It has b e e n s h o w n b y sectioning frozen ultrafiltration HFBRs that these flows can polarize high molecular weight proteins to the downstream end of the extracapillary space [33"]. This polarization has b e e n used to increase the harvest concentrations of monoclonal antibodies. The polarization of high molecular weight growth factor proteins in ultrafiltration bioreactors can also polarize cell growth and reduce bioreactor productivities. Cycling of the flow direction distributes cell growth and increases HFBR antibody productivities [33"]. Particularly w h e n using serum-free m e d i u m in HFBRs, it may be important to find ways to minimize both cell lysis and the release of proteases. Data related to this problem have b e e n reported by van Erp et al. [34], w h o assayed acid protease activity and the recovery of immunoglobulin G fragments in serum-free HFBR cultures of one hybridoma cell line. Analysis of extracapillary samples confirmed that oxygen is a critical scale-limiting nutrient [34] and improved effectiveness factor plots were d e v e l o p e d [35].
Perspectives The technology for manufacturing pharmaceuticals employing mammalian cells has rapidly developed over the past decade and b e c o m e part of an important industry. Significant progress was made in 1991 towards the optimization of bioreactors and processes. Investigations of the cell culture environment effects will provide the basis for optimizing conditions throughout the cultivation period. In the past, studies of medium design and optimization focused on the initial conditions. Gradually, fed-batch and continuous perfusion processes are being introduced. The implementation of dynamic feeding of nutrients will most likely create a need for more sophisticated on-line m e a s u r e m e n t and control, which will stimulate the dev e l o p m e n t of meaningful and useful kinetic models for process optimization. Care should be taken to ensure that process control via dynamic manipulation of environmental factors produces proteins with consistent post-translation modifications. Overall, mammalian cell culture technology has enabled the efficient production of a wide range of products. In the near future, some aspects of the technology d e v e l o p e d in the past decade should b e adapted to other biomedical applications, including the cultivation of differentiated ceils and tissues (such as stem cells, hepatocytes, and nerve cells) and the screening, identification and isolation of molecules of pharmaceutical importance. Bioreactors that were scaled u p for protein production in the 1980s may b e scaled d o w n for these n e w applications.
Acknowledgements Novel bioreactors Increasing attention has b e e n paid in the past few years to spin-filter or wire-cage reactors for suspension, microcarrier or aggregate cultures [36,37]. As fouling of the screen poses problems for long-term operation of bioreactors, it is worth noting that the fouling of polyamide screens has b e e n s h o w n to b e less than that of stainless steel screens [38] and that the filter openings can b e larger if cells are cultivated on microcarriers or as aggregates [37]. Three other reports of novel bioreactors have b e e n published. In one, cell aggregates w e r e immobilized on polyurethane foam in a p a c k e d - b e d reactor [39]. In another, a packed glass wool bed was used to cultivate anchorage-dependent ceils in a reactor in which gas sparging w a s used to provide fluid circulation through the packed glass wool in the descending fluid region [40]. In the third, a three-phase bioreactor employing porous polyvinyl formal resin particles was used to cultivate suspension ceils [41]. In most of these bioreactors a relatively high cell concentration was achieved. This has resulted in the n e e d for higher rates of perfusion. An alternative technique developed to avoid increased medium flow rates employs a dialysis device for the removal of metabolites or other inhibitory substances [42]. This approach, however, requires large areas of m e m b r a n e in the bioreactor. As a solution, the dialysis tubing can b e installed in an external vessel [43].
Part of this manuscript was prepared while Wei-Shou Hu was visiting the International Center for Cooperative Research in Biotechnology. Support from Osaka University is gratefully acknowledged. This work w a s supported in part by grants from the National Science Foundation, USA (BCS-8551670) to Wei-Shou Hu and from the National Science and Engineering Research Council of Canada to James Piret.
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W-S Hu, Department of Chemical Engineering and Materials Science, 151 Amundsen Hall, 421 Washington Avenue S.E., University of Minnesota, Minneapolis, Minnesota 55455, USA. JM Piret, Biotechnology Laboratory and Department of Chemical Engineering, University of British Columbia, 237~5174 University Boulevard, Vancouver, British Columbia, V6T 1Z3 Canada.
