|Title:||Metabolic profiling of NK92 cells during dynamic small-scale culturing||Language:||English||Authors:||Friedberg, Raoul||Qualification level:||Diploma||Advisor:||Herwig, Christoph||Assisting Advisor:||Kozma, Bence||Issue Date:||2021||Citation:||
Friedberg, R. (2021). Metabolic profiling of NK92 cells during dynamic small-scale culturing [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2021.86571
|Number of Pages:||116||Qualification level:||Diploma||Abstract:||
The Natural Killer cell line NK-92 is a well-established culture of lymphocytes that has been consistently used with high antitumor cytotoxicity and as a suitable transfection host, it is among the few lymphoid cell line derived products that have been successfully infused into patients with advanced cancer with clinical benefit and minimal side effects. However, the therapeutic use of NK cells depends on a precisely tuned cell activation, differentiation, expansion and separation protocols with considerable amounts of serum and exogenous cytokines, additional to the fact that they are very sensitive to process parameters, such as temperature, population density, shear stress and aeration. Current NK-92 cell expansion process is performed primarily through a batch and split strategy that is carried on plates, flasks and bags. This approach tends to generate less efficient cultures than dynamic cell suspension systems. As important as the evaluation of the effect of process parameters on cell growth, the determination of cell cytotoxicity, and its relationship with the metabolic profile of the expanded cells has become a major research topic in the immunotherapy field. Distinguishing the specific nutrient uptake characteristics of functional and non-functional lymphocytes may be the key for establishing suitable on-line culture monitoring strategies, able to predict and adjust cell growth within an integrative approach that includes cell quality.To make NK cell therapy accessible for every patient, a dynamic culturing approach is beneficial, as it provides a higher output of therapeutic cells. Therefore, the goals of this thesis are to explore the effect of different stimulation strategies and process parameters on NK92 cell culturing in dynamic conditions, especially the effect of shear stress on viability, aiming towards scaling it up from traditional flask-based culturing into medium lab scale bioreactors. In order to achieve this transition culturing experiments under different dynamic conditions were performed to find the agitation that leads to optimal growth. The produced cells were tested for their cytotoxic capabilities, to ensure their functionality. Lastly, the concentrations of secondary metabolites and amino acids were quantified to find possible correlations between uptake rates, growth rates and functionality.The results of this thesis show that a dynamic culturing approach is not only possible but leads to an increase of the growth rate, while viability and cytotoxicity did not experience a significant change. Furthermore, concentrations of important metabolites and amino acids were quantified and uptake rates successfully linked to the cells growth rate. The quantification of uptake rates and correlations with cell growth rate are previously unreported in literature. Conclusions towards the composition of the ideal culturing medium can additionally be derived from the collected data, which could lead to a further increase in the growth rate as and cost efficiency. Further experiments need to be conducted regarding correlations between cytotoxicity and uptake rates, using stirred small scale systems and finally transition into a lab scale bioreactor.
|Keywords:||Natural Killer cells; Metabolic profiling; Amino Acid Metabolism; NK92||URI:||https://doi.org/10.34726/hss.2021.86571
|DOI:||10.34726/hss.2021.86571||Library ID:||AC16190823||Organisation:||E166 - Institut für Verfahrenstechnik, Umwelttechnik und technische Biowissenschaften||Publication Type:||Thesis
|Appears in Collections:||Thesis|
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