Cytotechnology 3: 39-42, 1990. 9 1990 Kluwer Academic Publishers. Printed in the Netherlands.
High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures
M. Reiter, F. Weigang, W. Ernst and H.W.D. Katinger
Institute of Applied Microbiology, University of Agriculture and Forestry, 1190 Vienna, Austria Received 12 December 1988; accepted in revised form 7 April 1989
Key words: microcarrier culture, oxygenation, immobilisation, perfusion Abstract A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up t o 107 cells per ml were obtained. The described system is scaleable.
Introduction Mammalian cell culture technology is rapidly expanding for the production of various highly specific biologicals. Although suspended cell culture is the favoured technique for mass cultivation and scale-up there is still a requirement for cultivation techniques that allow mass cultivation of anchorage dependent cells. The scale up of the latter techniques is more difficulty and consequently a wide range of cultivation devices have evolved (Griffiths, 1987). The technique of cultivating anchorage dependent cells attached to spherical growth substrata, i.e. to microcarriers, combines both growth of cells on growth supporting surfaces and the advantage of homogenous suspension. More detailed knowledge about the requirements of the various cell lines with respect to their particular morphological and physicochemical nature has led to the development of
more efficient microcarriers during the last decade. Consequently after an initial lag time following van Wezel's pioneering innovation, widespread applications of techniques suitable for cultivation of cells on microcarriers (with cells attached on to their outer surface), or in porous microcarriers (with cells sticking to inner surfaces), have arisen (van Wezel, 1967, 1978; Giard, 1977). Nowadays it is generally accepted that cell retention in the bioreactor (i.e. immobilization) and continuous perfusion or diafiltration is the most useful method of treating cells in vitro for cell propagation and maintenance/production respectively. Simple technical solutions in overcoming 'apparent trivialities' such as oxygen supply to cultured cells as well as cell retention are still a matter of discussion among cell culturists. Spier (1984) first proposed a simple device, essentially a rotating cylindrical cage system ca-
40 pable of cell retention (and for oxygenation). The ideas of Spier were then further developed and used by Reuveny (1985). Here we describe a novel system that allows efficient and bubble free oxygen transfer to cultured cells, as well as microcarrier (cell) retention, in any standard bioreactor configuration. The system basically consists of a vibrating cage. The parameters of the system are described, and their practical application for the mass propagation of VERO-cells, is shown (Katinger, 1987).
5
7 3 4
Description of the system The cultivation system consists of a conventional stirred tank bioreactor equipped with an axial flow impeller (Fig. 1) into which the vibro package (Fig. 2) is installed. The cage of the vibro package is covered with a stainless steel mesh. For microcarrier cultures an absolute pore size of 80-100 micron is used. The vibro package is permanently kept in a vibrating state by means of a vibro mixer (Vibro mixer El, Chemap AG, Switzerland) and thus divides the reactor vessel
2
1
4 5
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\~
2 *--.
f f I ,i
(
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Fig. 1. Vibro cage system. 1) cage 2) vibro mixer 3) slow speed impeller 4) oxygen inlet cage 5) air, CO2 inlet to head space 6) pH control 7) pO2 control 8) fresh medium 9) effluent, harvest 113) flanged membrane seal.
Fig 2. Vibro package. 1) hollow stirrer shaft 2) stainless steel mesh 3) cylindrical screw 4) O-ring 5) gas (02) inlet/outlet 6) spent medium "Mthdrawal tube 7) opening to reactor head space 8) conical holes.
into two sections whereby microcarriers and adherent cells are kept outside the cage within the reactor vessel. For perfusion purposes the fresh media are fed to the reactor through (Fig. 1;8) and spent medium is withdrawn from the inside of the cage via tube (Fig. 1;9). A minimum amplitude and frequency of vibration (e.g. amplitude less than 0.3 mm, frequency 50 Hertz) is necessary for keeping the surface of the cage clean and to prevent plugging. This particular mode of perfusion technique specifically immobilizes microcarriers and cells attached to microcarriers in the
41 100
6o-
l~ 10" 0 11ME (rain)
Fig. 3. Effect of vibration amplitude on the oxygen transfer rate in a marine impeller stirred reactor. Experimental set-up: 3 blade marine impeller, diameter 10 cm, 40 rpm.; working volume, 61. RPMI 1640 medium, 37~ Vibro cage; 100 cmz mesh surface, 80 micron pore size. Amplitude; (1) 0.5 ram; (2) 1.0 mm (50 Hertz). O2-flow rate; 50 cm 3 per minute.
reactor whereas suspended cells (i.e. dead cells or dying off cells respectively) are removed from the reactor. For oxygen supply a simple set point controller is applied. If the oxygen tension as measured by a conventional oxygen probe falls below the set point the controller increases the amplitude of the vibro mixer and simultaneously opens the oxygen inlet valve (Fig. 1;[4]). A minute flow of oxygen gas (or air) is then introduced to the vibro cage and sparged within the cage. Gas bubbles do not pass the stainless steel mesh as long as the pressure within the cage is compensated with that in the reactor head space. Both sparging and diffusion of dissolved oxygen from the cage to the liquid bulk in the reactor vessel are effected by the mode of vibration (for details compare Figs. 2 and 3). Once the desired oxygen tension is reached the controller closes the oxygen inlet valve and reduces the amplitude of the vibro mixer to a certain minimum level that is sufficient to keep the mesh surface of the cage body clean. Due to the different solubilities and diffusivities of oxygen and carbon dioxide respectively, the CO2 gas for controlling the pH-value must be separately introduced together with air into the
reactor head space. Another indicator controller registration (1CR) is used for that purpose. A detailed drawing of the vibro-package is shown in Fig. 2. The vibration of the conically shaped holes in the discs causes a neutral bulk flow within the cage by which the gas bubbles introduced at the bottom of the cage are repeatedly resparged and the residence time of the gas is considerably prolonged. A high fraction of the oxygen gas is therefore dissolved in the liquid phase. The gas flow rate through the cage should be preferentially kept as low as possible in order to achieve efficient sparging and to prevent foaming. At a given frequency the oxygen transfer capacity of the vibro cage system depends on the vibration amplitude applied, as shown in Fig. 3. An amplitude of 0.5 mm leads to modest oxygen transfer (curve 1) whereas an amplitude of 1.0 mm drastically increases the oxygen transfer capacity (curve 2). Any further increase of the amplitude is of minor effect on the oxygen transfer. Slight decreases of the amplitude below 0.5 mm, however, would again greatly reduce the oxygen transfer (results not shown). This particular operational performance of the vibro cage with respect to oxygenation relates to the fact that a certain minimum critical amplitude is necessary for promoting gas sparging conditions at the conical holes within the cage. There are indications that the effect of the amplitude (and/or frequency) is not critical for the oxygen diffusion across the mesh itself. In the latter case the choice of proper mesh material and the type mesh weaving is significant. A transparent mesh type with a maximum open pore surface is the most useful one. The particular performance of the vibro cage described here is advantageous for use in shear sensitive culture systems, because intense vibration is not necessary, neither for oxygenation nor for microcarrier retention. The specific oxygen transfer capacity is in the range of 4 mg OJper cm2 cage surface area in one hour, and therefore approximative 25 times more efficient than gas permeation across membranes (Lehmann, 1987). With proper fluid bulk mixing in a reactor vessel
42
Scale-up studies of microcarrier based systems are currently being carried out and modifications to the system for suspension culture are still in progress.
9 8" 7" 6" 5-
J
Acknowledgements
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We acknowledge the support of technical equipment by Chemap AG, Volketswil, Switzerland. Special thanks go to Immuno AG, Austria for financial support.
Fig. 4. Growth kinetics of monkey kidney cells (VERO) cultivated on gelatin coated microcarriers using different concentrations (5g/l, 10g/l, and 15g/1 Cytodex 3).
the oxygen transfer increases linearly with the area of the cage surface. If we assume an average specific oxygen uptake rate of propagating mammalian cells in the range of approximative 2-3 gg/106 cells h-1 then one 0.1 m2-package would easily supply approximative 1012 cells. Using several package modules would minimize the risk of scale-up ventures. In Fig. 4 we show practical application examples of the vibro cage system for a perfused culture of VERO cells on microcarriers using DMEM (Gibco) containing 5% fetal calf serum in a 6 1 working volume reactor. In all experiments the oxygen tension was maintained at 30% air saturation. One 100 cm2 vibro cage was used. No limitations of the system, either with respect to oxygenation or plugging, were observed.
Conclusion A novel system for aeration and perfusion of microcarrier cultures was established (Fig. 4).
References 1. Criard DJ, Thilly WG, Wang DIC and Levine DW (1977) Virus production with a Newly Developed Microcarrier System. Applied and Environmental Microbiology 34: 668-672. 2. Griffiths JB, Cameron DR and Looby D (1987) A Comparison of Unit Process Systems for Anchorage Dependent Cells. Develop. biol. Standard. 66: 331-338. 3. Katinger HWD (1987) Animal Cell Culture: Biological and technological aspects. Proceedings 4th European Congress on Biotechnology, Amsterdam. 4. Lehmann JH, Piehl GW and Schulz R (1987) Bubble Free Cell Culture Aeration with Porous Moving Membranes. Develop. biol. Standard. 66: 227-240. 5. Reuveny S, Velez D, Macmillan JD and Miller L (1987) Factors affecting monoclonal Antibody production in culture. Develop. biol. Standard. 66: 169-175. 6. Spier RE and Whiteside JP (1984) The Description of a Device which Facilitates the Oxygenation of Microcarrier Cultures. Develop. biol. Standard. 55: 151-152. 7. Van Wezel AL (1967) Growth of Cell Strains and Primary Cells on Microcarriers in Homogeneous Culture. Nature 216: 64-65. 8. Van Wezel AL and Van der Velden-de Groot CAM (1978) Large Scale Cultivation of Animal Cells in Microcarrier Culture. Process Biochemistry 3: 6-8.
Address for offprints: M. Reiter, Institute of Applied Microbiology, University of Agriculture and Forestry, 1190 Vienna, Austria
High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures
M. Reiter, F. Weigang, W. Ernst and H.W.D. Katinger
Institute of Applied Microbiology, University of Agriculture and Forestry, 1190 Vienna, Austria Received 12 December 1988; accepted in revised form 7 April 1989
Key words: microcarrier culture, oxygenation, immobilisation, perfusion Abstract A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up t o 107 cells per ml were obtained. The described system is scaleable.
Introduction Mammalian cell culture technology is rapidly expanding for the production of various highly specific biologicals. Although suspended cell culture is the favoured technique for mass cultivation and scale-up there is still a requirement for cultivation techniques that allow mass cultivation of anchorage dependent cells. The scale up of the latter techniques is more difficulty and consequently a wide range of cultivation devices have evolved (Griffiths, 1987). The technique of cultivating anchorage dependent cells attached to spherical growth substrata, i.e. to microcarriers, combines both growth of cells on growth supporting surfaces and the advantage of homogenous suspension. More detailed knowledge about the requirements of the various cell lines with respect to their particular morphological and physicochemical nature has led to the development of
more efficient microcarriers during the last decade. Consequently after an initial lag time following van Wezel's pioneering innovation, widespread applications of techniques suitable for cultivation of cells on microcarriers (with cells attached on to their outer surface), or in porous microcarriers (with cells sticking to inner surfaces), have arisen (van Wezel, 1967, 1978; Giard, 1977). Nowadays it is generally accepted that cell retention in the bioreactor (i.e. immobilization) and continuous perfusion or diafiltration is the most useful method of treating cells in vitro for cell propagation and maintenance/production respectively. Simple technical solutions in overcoming 'apparent trivialities' such as oxygen supply to cultured cells as well as cell retention are still a matter of discussion among cell culturists. Spier (1984) first proposed a simple device, essentially a rotating cylindrical cage system ca-
40 pable of cell retention (and for oxygenation). The ideas of Spier were then further developed and used by Reuveny (1985). Here we describe a novel system that allows efficient and bubble free oxygen transfer to cultured cells, as well as microcarrier (cell) retention, in any standard bioreactor configuration. The system basically consists of a vibrating cage. The parameters of the system are described, and their practical application for the mass propagation of VERO-cells, is shown (Katinger, 1987).
5
7 3 4
Description of the system The cultivation system consists of a conventional stirred tank bioreactor equipped with an axial flow impeller (Fig. 1) into which the vibro package (Fig. 2) is installed. The cage of the vibro package is covered with a stainless steel mesh. For microcarrier cultures an absolute pore size of 80-100 micron is used. The vibro package is permanently kept in a vibrating state by means of a vibro mixer (Vibro mixer El, Chemap AG, Switzerland) and thus divides the reactor vessel
2
1
4 5
,I/R
0!
\~
2 *--.
f f I ,i
(
J
Fig. 1. Vibro cage system. 1) cage 2) vibro mixer 3) slow speed impeller 4) oxygen inlet cage 5) air, CO2 inlet to head space 6) pH control 7) pO2 control 8) fresh medium 9) effluent, harvest 113) flanged membrane seal.
Fig 2. Vibro package. 1) hollow stirrer shaft 2) stainless steel mesh 3) cylindrical screw 4) O-ring 5) gas (02) inlet/outlet 6) spent medium "Mthdrawal tube 7) opening to reactor head space 8) conical holes.
into two sections whereby microcarriers and adherent cells are kept outside the cage within the reactor vessel. For perfusion purposes the fresh media are fed to the reactor through (Fig. 1;8) and spent medium is withdrawn from the inside of the cage via tube (Fig. 1;9). A minimum amplitude and frequency of vibration (e.g. amplitude less than 0.3 mm, frequency 50 Hertz) is necessary for keeping the surface of the cage clean and to prevent plugging. This particular mode of perfusion technique specifically immobilizes microcarriers and cells attached to microcarriers in the
41 100
6o-
l~ 10" 0 11ME (rain)
Fig. 3. Effect of vibration amplitude on the oxygen transfer rate in a marine impeller stirred reactor. Experimental set-up: 3 blade marine impeller, diameter 10 cm, 40 rpm.; working volume, 61. RPMI 1640 medium, 37~ Vibro cage; 100 cmz mesh surface, 80 micron pore size. Amplitude; (1) 0.5 ram; (2) 1.0 mm (50 Hertz). O2-flow rate; 50 cm 3 per minute.
reactor whereas suspended cells (i.e. dead cells or dying off cells respectively) are removed from the reactor. For oxygen supply a simple set point controller is applied. If the oxygen tension as measured by a conventional oxygen probe falls below the set point the controller increases the amplitude of the vibro mixer and simultaneously opens the oxygen inlet valve (Fig. 1;[4]). A minute flow of oxygen gas (or air) is then introduced to the vibro cage and sparged within the cage. Gas bubbles do not pass the stainless steel mesh as long as the pressure within the cage is compensated with that in the reactor head space. Both sparging and diffusion of dissolved oxygen from the cage to the liquid bulk in the reactor vessel are effected by the mode of vibration (for details compare Figs. 2 and 3). Once the desired oxygen tension is reached the controller closes the oxygen inlet valve and reduces the amplitude of the vibro mixer to a certain minimum level that is sufficient to keep the mesh surface of the cage body clean. Due to the different solubilities and diffusivities of oxygen and carbon dioxide respectively, the CO2 gas for controlling the pH-value must be separately introduced together with air into the
reactor head space. Another indicator controller registration (1CR) is used for that purpose. A detailed drawing of the vibro-package is shown in Fig. 2. The vibration of the conically shaped holes in the discs causes a neutral bulk flow within the cage by which the gas bubbles introduced at the bottom of the cage are repeatedly resparged and the residence time of the gas is considerably prolonged. A high fraction of the oxygen gas is therefore dissolved in the liquid phase. The gas flow rate through the cage should be preferentially kept as low as possible in order to achieve efficient sparging and to prevent foaming. At a given frequency the oxygen transfer capacity of the vibro cage system depends on the vibration amplitude applied, as shown in Fig. 3. An amplitude of 0.5 mm leads to modest oxygen transfer (curve 1) whereas an amplitude of 1.0 mm drastically increases the oxygen transfer capacity (curve 2). Any further increase of the amplitude is of minor effect on the oxygen transfer. Slight decreases of the amplitude below 0.5 mm, however, would again greatly reduce the oxygen transfer (results not shown). This particular operational performance of the vibro cage with respect to oxygenation relates to the fact that a certain minimum critical amplitude is necessary for promoting gas sparging conditions at the conical holes within the cage. There are indications that the effect of the amplitude (and/or frequency) is not critical for the oxygen diffusion across the mesh itself. In the latter case the choice of proper mesh material and the type mesh weaving is significant. A transparent mesh type with a maximum open pore surface is the most useful one. The particular performance of the vibro cage described here is advantageous for use in shear sensitive culture systems, because intense vibration is not necessary, neither for oxygenation nor for microcarrier retention. The specific oxygen transfer capacity is in the range of 4 mg OJper cm2 cage surface area in one hour, and therefore approximative 25 times more efficient than gas permeation across membranes (Lehmann, 1987). With proper fluid bulk mixing in a reactor vessel
42
Scale-up studies of microcarrier based systems are currently being carried out and modifications to the system for suspension culture are still in progress.
9 8" 7" 6" 5-
J
Acknowledgements
43" 2 1 0
'
2;
'
2o
'
/o
'
~
'
,;~
'
,~o
'
,;o
'
,~o
'
,~o
hue (.)
We acknowledge the support of technical equipment by Chemap AG, Volketswil, Switzerland. Special thanks go to Immuno AG, Austria for financial support.
Fig. 4. Growth kinetics of monkey kidney cells (VERO) cultivated on gelatin coated microcarriers using different concentrations (5g/l, 10g/l, and 15g/1 Cytodex 3).
the oxygen transfer increases linearly with the area of the cage surface. If we assume an average specific oxygen uptake rate of propagating mammalian cells in the range of approximative 2-3 gg/106 cells h-1 then one 0.1 m2-package would easily supply approximative 1012 cells. Using several package modules would minimize the risk of scale-up ventures. In Fig. 4 we show practical application examples of the vibro cage system for a perfused culture of VERO cells on microcarriers using DMEM (Gibco) containing 5% fetal calf serum in a 6 1 working volume reactor. In all experiments the oxygen tension was maintained at 30% air saturation. One 100 cm2 vibro cage was used. No limitations of the system, either with respect to oxygenation or plugging, were observed.
Conclusion A novel system for aeration and perfusion of microcarrier cultures was established (Fig. 4).
References 1. Criard DJ, Thilly WG, Wang DIC and Levine DW (1977) Virus production with a Newly Developed Microcarrier System. Applied and Environmental Microbiology 34: 668-672. 2. Griffiths JB, Cameron DR and Looby D (1987) A Comparison of Unit Process Systems for Anchorage Dependent Cells. Develop. biol. Standard. 66: 331-338. 3. Katinger HWD (1987) Animal Cell Culture: Biological and technological aspects. Proceedings 4th European Congress on Biotechnology, Amsterdam. 4. Lehmann JH, Piehl GW and Schulz R (1987) Bubble Free Cell Culture Aeration with Porous Moving Membranes. Develop. biol. Standard. 66: 227-240. 5. Reuveny S, Velez D, Macmillan JD and Miller L (1987) Factors affecting monoclonal Antibody production in culture. Develop. biol. Standard. 66: 169-175. 6. Spier RE and Whiteside JP (1984) The Description of a Device which Facilitates the Oxygenation of Microcarrier Cultures. Develop. biol. Standard. 55: 151-152. 7. Van Wezel AL (1967) Growth of Cell Strains and Primary Cells on Microcarriers in Homogeneous Culture. Nature 216: 64-65. 8. Van Wezel AL and Van der Velden-de Groot CAM (1978) Large Scale Cultivation of Animal Cells in Microcarrier Culture. Process Biochemistry 3: 6-8.
Address for offprints: M. Reiter, Institute of Applied Microbiology, University of Agriculture and Forestry, 1190 Vienna, Austria
