A fundamental research of growth, metabolism and product formation of tobacco suspension cells at different scales

  • Eine grundlegende Untersuchung von Wachstum, Metabolismus und Produktbildung von Tabaksuspensionszellen in unterschiedlichen Maßstäben

Ullisch, David; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2012)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2012


For over two decades, plant cell cultures have been promising hosts for the expression of recombinant proteins such as hormones, growth factors, full-size antibodies and antigens. So far, over 700 different plant cell cultures are stored in the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig. Among these plant cell cultures, the tobacco cell line Nicotiana tabacum Bright Yellow 2 (BY-2) was chosen as a good host cell line for the production of recombinant proteins, as this cell line is well characterized – showing high growth rates and high cell synchrony. Up to now, individual studies have only handled one or two parameters (i.e. biomass, osmolality or conductivity) for studying BY-2 cell growth. Such limited studies, however, do not provide a comprehensive insight into BY-2 cell metabolism. A first objective of this thesis, is to identify the optimal growth conditions for tobacco suspension cultures in shake flasks and to comprehensively characterize the growth of a transgenic BY-2 cell line. Hereby, multiple growth parameters were analyzed offline and online by using a Respiration Activity MOnitoring System (RAMOS). A faster shaking frequency resulted in clearly higher oxygen transfer rates and biomass concentrations. Moreover, a reproducibly observed shift of the oxygen transfer rate (OTR) could be identified to indicate ammonium depletion in the medium. Today, the MS-medium is the preferred medium for the cultivation of tobacco suspension cells, even though it was formulated for an optimal growth and not for the production of recombinant proteins. Here, the fluorescent proteins GFP and YFP are used as model proteins and their expression was elucidated in detail. Based on the correlations between nutrient consumption, cell growth and product formation, it is the intention to improve the standard MS-medium to enhance the expression of the recombinant proteins. The initial ammonium concentration was found to have significant influence on either cell growth and played a pregnant role in protein synthesis. After the MS-medium was improved, the GFP concentration nearly doubled. When this improved ammonium enriched medium was applied to another transgenic tobacco cell line similar improvements to the amount of the glycoprotein influenza hemagglutinin (HA) produced by Nicotiana tabacum NT-1 cells could be achieved. Furthermore, a controlled-release system was successfully applied to plant suspension cultures. Using this controlled-release system where additional ammonium was supplied to the plant cells, an increase of 40% GFP intensity was observed. Plant cells are maintained in suspension by pipetting a certain volume of grown culture into fresh medium. Applying this subcultivation method, results in non-defined growth conditions of plant cells. Due to that problem, plant suspension cultures always have to compete with animal cultures for the production of therapeutically relevant proteins as they have the advantage of an established cell banking system. Moreover, researchers are facing the huge problem of genetic instability of plant cells where growth and recombinant protein production tremendously vary. This well-known problem has been poorly documented so far. This growth variability of plant suspension cells was identified by relating the measured values of 22 oxygen transfer rates in a period of over two years. After the implementation of a new subculturing method a significantly better reproducibility of plant cell growth was obtained. However, the productivity, detected by fluorescence and Western blot, decreased by 80% in the same period. Besides the cultivation of plant cells in shake flasks, in this work, plant suspension cells were also cultivated in stirred tank reactors and microtiter plates (MTPs). As there are no geometric similarities between shake flasks and stirred tank reactors, a scale-up is not a trivial process. Here, the scale-up was carried out under the assumption of a constant volumetric power input. It could be shown, that an increasing viscosity was the key parameter influencing cell growth in the fermenter. According to literature there is only one publication dealing with plant cell cultivation in small scale. In this thesis BY-2 cell growth as well as GFP expression was monitored online using the BioLector technology. In addition, it was demonstrated that medium modifications influenced plant cell growth and the GFP production in the same way as shown in shake flasks. Ultimately, this thesis provides a deep insight into tobacco cultivations in shake flasks, fermenters and MTPs. The combined offline and online analysis of tobacco suspension cultures was used for a detailed growth characterization and media optimization to improve growth and boost target product formation. In conclusion, the RAMOS technology allows the online analysis of oxygen consumption and has been proven to be a useful analytical tool investigating plant suspension cultures.