Entwicklung einer Strategie zur Fed-Batch-Kultivierung von Pflanzenzellen in Schüttelkolben

Abstract:
Most biotechnological productions are operated in the fed-batch mode. Nevertheless, screening is still performed in batch-mode due to its simple handling and highly parallelized processes. To overcome the problem of choosing a wrong production strain or culture medium, the screening process should be conducted in fed-batch mode as well. A new membrane-based fed-batch technique using shake flasks for feeding glucose or other nutrients was developed at the Chair of Biochemical Engineering at RWTH Aachen University. This system uses a dialysis membrane, across which glucose diffuses from a feed reservoir into the culture medium. This fed-batch system has already been tested for microbial expression systems such as Escherichia coli, Corynebacterium glutamicum or Hansenula polymorpha. Recently, the production of recombinant proteins in plant cell suspension cultures has become more and more relevant. Nonetheless, all cultivations using plant-cell suspension cultures are performed in batch-mode. The question is whether fed-batch cultivations using plant cells are possible and if they are advantageous over using the batch-mode. In this study, fed-batch cultivations using the membrane-based fed-batch shake flasks with a transgenic tobacco cell line Nicotiana tabacum NT-1 were performed for the first time. The existing fed-batch system for microbial systems had to be adapted for the use with plant cells. This includes an adjustment of the shaking frequency as well as an increase of both the glucose concentration in the feedreservoir as well as the thickness of the dialysis membrane. During the fed-batch experiments with the transgenic cell line N. tabacum NT-1 worse growth and protein production were observed compared to batch cultivations. In order to identify the reason for these results, the influence of glucose instead of sucrose was examined first. As the use of glucose affects the osmotic pressure in the culture medium, this might lead to a decreased growth of the plant cells. The results did not reveal an inhibition in growth due to the osmotic pressure, nonetheless. To identify the reasons of the decreased growth and product formation, compounds of the fedbatch- system (such as the dye dextrane blue, as well as locking rings and silicone rings for membrane fixation) were tested for biocompatibility. Stainless steal and the dye dextrane blue did not have an influence on the growth of the plant cells. Silicone, however, was found to have an effect on growth of the tobacco plant cells.

Keywords:
fed-batch shake flask, plant cell culture, Nicotiana tabacum NT-1