Agitation, aeration and perfusion modules for cell culture bioreactors
For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.
Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.
Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 μm) as well as anchorage dependent cells grown on microcarriers (pore size 75 μm) over six weeks to 3 months.
Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.
Key wordsaeration agitation animal cell bioreactors perfusion
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- Boraston R, Thompson PW, Garland S and Birch JR (1984) Growth and oxygen requirements of antibody producing mouse hybridoma cells in suspension culture. Develop. Biol. Standard. 55: 103–111.Google Scholar
- Büntemeyer H, Bödeker BGD and Lehmann J (1987) Membranestirrer-reactor for bubble-free aeration and perfusion. In: Spier RE and Griffiths JB (eds) Modern approaches to animal cell technology, (pp. 411–419). Butterworth.Google Scholar
- Eisenlauer J and Horn D (1985) Fibre-optic sensor technique for flocculant dose control in flowing suspensions. Colloids and Surfaces 14: 121–134.Google Scholar
- Fenge C, Buzsaky F, Fraune E and Lindner-Olsson E (1991) Evaluation of a spin filter during perfusion culture of recombinant CHO cells. In: Spier RE, Griffiths GB and MacDonald C (eds) Animal cell technology: Development, processes and products. (pp. 429–432). Butterworth-Heinemann, Oxford.Google Scholar
- Fenge C, Fraune E and Schügerl K (1990) Physiological investigations in high density perfusion culture of free suspended animal cells. In: Spier R, Griffiths J and Meignier B (eds) Production of Biologicals from Animal Cells in Culture. (pp. 262–265). Butterworth, Oxford.Google Scholar
- Fenge C, Fraune E and Schügerl K (1992) Perfusion bioreactor performance at different cell bleed rates. In: Spier RE, Griffiths JB and MacDonald C (eds) Animal cell technology: Development, processes and products. (pp. 365–370. Butterworth-Heinemann, Oxford.Google Scholar
- Fleischaker RJ and Sinskey AJ (1981) Oxygen demand and supply in cell culture. Eur. J. Appl. Microbiol. Biotechnol. 12: 193–197.Google Scholar
- Fraune E, Fenge C, Kuhlmann W and Broly H (1990) Development of perfusion bioreactors for high density cultures. In: White M, Reuveny S and Shafferman A (eds) Biologicals from Recombinant Microorganisms and Animal Cells—Production and Recovery. (pp. 159–164). VCH, Weinheim.Google Scholar
- Grönvik K-O, Frieberg H and Malmström U (1989) Centritech Cell — a new separation device for mammalian cells. In: Spier RE, Griffiths JB, Stephenne J and Crooy PJ (ed) Advances in animal cell biology and technology for bioprocesses. (pp. 428–433). Butterworth.Google Scholar
- Himmelfarb P, Thayer PS and Martin HE (1969) Spin filter culture: The propagation of mammalian cells in suspension. Science 164: 555–557.Google Scholar
- Hoffmann J, Tralles S and Hempel DC (1992) Test system zur Untersuchung der mechanischen Beanspruchung von Partikeln in Bioreaktoren. Chem. Ing. Tech., in press.Google Scholar
- Hooker BS, Lee JM and An G (1990) Cultivation of plant cells in a stirred vessel: effect of impeller design. Biotechnol. Bioeng. 35: 296–304.Google Scholar
- Kilburn DG and Webb FC (1968) The cultivation of animal cells at controlled dissolved oxygen partial pressure. Biotechnol. Bioeng. 10: 801–814.Google Scholar
- Kuhlmann W (1987) Optimization of a membrane oxygenation system for cell culture in stirred tank reactors. Develop. Biol. Standard. 66: 263–268.Google Scholar
- Lehmann J, Piehl GW and Schulz R (1987) Bubble free cell culture aeration with porous moving membranes. Develop. Biol. Standard. 66: 227–240.Google Scholar
- Martin N, Brennan A, DeNome L and Shaevitz J (1987) High productivity in mammalian cell culture. Bio/Technology 5: 838–840.Google Scholar
- Miller WM, Blanch HW and Wilke CR (1988) A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH. Biotechnol. Bioeng. 32: 947–965.Google Scholar
- Radlett PJ, Telling RC, Whitside JP and Maskell MA (1972) The supply of oxygen to submerged cultures of BHK 21 cells. Biotechnol. Bioeng., 14: 437–445.Google Scholar
- Rebsamen E, Goldinger W, Scheirer W, Merten O-W and Palfi GE (1987) Use of a dynamic filtration method for separation of animal cells. In: Spier RE and Griffiths GB (eds) Modern approaches to anima cell technology (pp. 548–555). Butterworth.Google Scholar
- Reuveny S, Velez D, Macmillan JD and Miller L (1986) Factors affecting cell growth and monoclonal antibody production in stirred reactors. J. Immunol. Methods 86: 53–59.Google Scholar
- Sato S, Kawamura K and Fujiyoshi N (1983) Animal cell cultivation for production of biological substances with a novel perfusion culture apparatus. J. Tiss. Culture Methods 8: 4, 167–171.Google Scholar
- Siegel U, Fenge C and Fraune E (1991) Spinfilter for continuous perfusion of suspension cells. In: Spier RE, Griffiths GB and MacDonald C (eds) Animal cell technology: Development, processes and products. (pp. 434–436.) Butterworth-Heinemann, Oxford.Google Scholar
- Tolbert WR, Lewis C, White PJ and Feder J (1985) Perfusion culture systems for production of mammalian cell biomolecules. In: Feder J (ed) Large scale mammalian cell culture. (pp. 97–125). Academic Press.Google Scholar