Cell Culture Reactors

The production of flavour substances by cell or tissue cultures is still a dream for the future in most cases. Today the extraction of product from intact living plants is still less expensive than the production by isolated cells and tissues. On the other hand, it is very attractive to make use of the secondary metabolism of plant cells for the synthesis of natural flavours in a controlled way to avoid contaminating by-products and thus considerably simplify downstream processing. Further advantages of such cell culture systems would be the independence from agriculture combined with the risk for possible shortage and variances in product quality, the ability to scale-up the process to create an inexhaustible source of well-defined product. 

Secondary metabolism comprises the side paths of the ordinary metabolism, so-called primary metabolism which are activated in the cell in rest situations or under limiting conditions for nutrient and energy supply. In most cases, secondary metabolism is linked to the building blocks responsible for growth and reproduction which are products of primary metabolism and is hallmarked by a multitude of reactions, intermediates and final products. Starting materials for the secondary metabolism are e.g. amino acids, sugars and the co-enzymes of the primary metabolism. Only a very small fraction of formation mechanisms and the product variants of plant secondary metabolism have been characterized yet. 

Isolated plant cells also tend to de-differentiate with progressing in-vitro cultivation time. Thus product generation can come to an absolute halt. In many cases dedifferentiation can be delayed by the formation of a tissue-like structure of cells. This 

Plant cells or tissues may be fermented like micro-organisms in the submerged fermenter if grown on the surface of carrier beads or are kept in suspension. There is also experience in the operation of special membrane reactors for this purpose [13]. 

Scientific results regarding the generation of flavouring materials from plant cell cultures on laboratory scale are available for quinine, capsaicin, quassin, vanillin, cocoa, citrus oils, peppermint oils, apple etc. [14, 15]. 

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Vorlop J Lehmann J (1989) Oxygen transfer and carrier mixing in large-scale membrane stirrer cell culture reactors. In Spier RE, Griffiths JB, Stephenne J Crooy PJ (eds) Advances in Animal Cell Biology and Technology for Bioprocesses, pp. 366-369. Butterworth, London. 

Scheirer W Merten OW (1991) Instrumentation of animal cell culture reactors. In Ho CS Wang DIC (eds) Animal Cell... 

Mammalian growth usually shows respiratory quotients close to 1, that is, carbon dioxide formation (CER) equals oxygen uptake rate (OUR). With respect to the relatively poor mass transfer conditions installed in cell culture reactors (compared to microbial conditions), dissolved CO2 (dCOj) levels can accumulate during fed-batch processes. While optimum dC02 concentrations appear to be cell-specific, growth inhibiting partial pressures of approximately 100 mbar had been identified for, for example, hybridoma cells [81]. 

Scheirer, W. and Merten, O.-W. (IWl) Instrumentation of animal cell culture reactors, pp. 405-443. In Animal Cell Bioreactors, C.S. Ho, D.I.C. Wang, Eds., Butterworth-Heinemann, Stoneham, MA... 

WORKED CELL CULTURE REACTOR DESIGN EXAMPLE... 

Sieblist, C, Hageholz, O., Aehle, M., Jenzsch, M., Pohlscheidt, M., and Lubbert, A. (2011) Insights into large-scale cell-culture reactors II. Gas-phase mixing and COj stripping. Biotechnol. 

Wu, J., and M. F. A. Gossen (1995). Evaluation of the killing volume of gas bubbles in sparged animal cell culture reactors. Enzyme Microb. Technol, 14, 980-983. 

There is an interior optimum. For this particular numerical example, it occurs when 40% of the reactor volume is in the initial CSTR and 60% is in the downstream PFR. The model reaction is chemically unrealistic but illustrates behavior that can arise with real reactions. An excellent process for the bulk polymerization of styrene consists of a CSTR followed by a tubular post-reactor. The model reaction also demonstrates a phenomenon known as washout which is important in continuous cell culture. If kt is too small, a steady-state reaction cannot be sustained even with initial spiking of component B. A continuous fermentation process will have a maximum flow rate beyond which the initial inoculum of cells will be washed out of the system. At lower flow rates, the cells reproduce fast enough to achieve and hold a steady state. 

Liquid-impelled loop reactor Production of anthraquinones Plant cell culture 124... 

Possible contamination by chemical or biological substances is one of the most important concerns when producing pharmaceutical proteins. Plant cell cultures ensure the production of the desired protein in a controlled, sterile and sealed environment and can be adapted to cGMP conditions. Therefore, the risk of contamination is minimized and the production conditions can be modified more easily in a contained reactor than in the field. Another advantage is the ability to freeze plant suspension cells in liquid nitrogen [66, 67] so that master and working cell banks can be established, a prerequisite for cGMP procedures [68]. 

Among the wide choice of reactor designs, the biofilm reactor is one of the best suited for azo-dye conversion as it meets two important process requisites. The first is related to the hindered growth feature of bacterial metabolism under anaerobic conditions. The second is related to the necessity to increase cell densities (see previous section) with respect to those commonly harvested in liquid broths [55, 56]. Except for bacteria that forms aggregates spontaneously, immobilization of cells on granular carriers and membrane reactor technology are the two common pathways to achieve high-density confined cell cultures in either discontinuous or flow reactors. 

Varley, J. and Birch, J. 1999. Reactor design for large scale suspension animal cell culture. Cytotechnology 29(3), 177-205. 

Incubation temperature and medium pH are also important regarding proteolytic activity of baculovirus infected insect cell cultures. Cruz et al. [25] have shown that the highest proteolytic activity was obtained at the normal culture conditions, 27 °C and pH 6.5. This could then be considered a drawback when the production of protease sensitive particles Hke HIV-CLPs and HIV-VLPs is envisaged [5]. The pH of Sf9 cells has been reported to reach a minimum of 5.9 in serum-free media under uncontrolled pH conditions in stirred tank reactors... 

Varley, J Birch, J. (1999). Reactor design for large scale suspension animal cell culture. Cytotechnology 29(3), 177-205. Wright, G. et al. (1991). High level expression of active human -antitrypsin in the milk of transgenic sheep. Bwj Technology 9, 830-834. 

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