Browsing by Subject "Cell culture"

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Molekulare Interaktionen von Milchsäurebakterien mit enterohämorrhagischen Escherichia coli und humanen Darmepithelzellen
(2011) Stöber, Helen; Schmidt, Herbert
The interactions of 19 benign strains of lactic acid bacteria, bifidobacteria and staphylococci with five enterohemorrhagic Escherichia coli (EHEC) strains of different serotypes and virulence gene spectrum were investigated using a HT29 cell culture infection model. As a parameter for the infection the secretion of Interleukin 8 (IL-8) of the infected cells was analyzed by ELISA. None of the used benign strains induced an IL-8 secretion, whereas the infection with the EHEC strains leads ? independent of their virulence profile - to high amounts of IL-8. In coinfection assays with the pathogen EDL933 (O157:H7) and different test strains the secretion of IL-8 of the cultured cells was decreased by a few strains. With 12 of 19 tested strains, a weak reduction < 30 % of IL-8 secretion of HT29 cells after coinfection with EHEC O157:H7 strain EDL933 was observed. Six strains reduced the IL-8 secretion up to 60 % and the strain B. breve DSMZ 20083 decreased the IL-8 production about 73 %. Coinfection assays with different strains of one species (B. adolescentis DSMZ 20083 and DSMZ 20086 as well as L. johnsonii BFE 633 and DSMZ 10533) showed the strain specificity of the observed anti-inflammatory effect, due to different capabilities of IL-8 reduction. In further coinfection assays with different EHEC strains of the serotypes O103:H2, O26:H-, 0157:H- and O113:H21 different abilities of the benign strains to influence the infection with the different pathogen strains were noted. Therefore the protective anti-inflammatory effect is strain specific for the tested benign bacteria and also depends on the application of EHEC strains with different sero- and virulence types. Further investigations indicated the imperative of living bacteria for the observed protective effect; neither culture supernatant nor inactivated bacteria showed an effect on the IL-8 secretion of the EDL933 infected HT29 cells. The analysis of the cell culture supernatants 6 h after infection with different bacteria detected the production of lactic and acetic acid. The application of these acids in infection assays with EDL933 did not lead to an reduced IL-8 secretion of the infected cells. Therefore the production of organic acids did not explain the protective effect. The induction of IL-8 could not be traced back to the influence of a single virulence factor. Four PMK5 strains with deletions in different virulence genes induced similar IL-8 secretions in comparison to cells infected with the wild-type strain. Coinfection assays with the mutants and S. pasteuri LTH 5211 showed also similar IL-8 reductions than coinfection assays with the wild-type strain. It is to suppose that the anti-inflammatory effects of the benign bacteria do not influence a single virulence factor of the tested EHEC strains. As a second parameter the activation of the transcription factor ?Nuclear Factor kappa B? (NF-κB) of coinfected HT29 cells was monitored using a reporter-genassay. In comparison to the single EHEC-infection, the NF-κB activation was reduced by all tested lactic acid bacteria, bifidobacteria and S. pasteuri LTH 5211 in coinfection trials significantly. No strain-specificity and no pathogen-specificity could be observed. Interestingly, stimulation of the HT29 cells with benign bacteria led to inhibition of NF-κB activity, the measured values were less than the values of the negative control PBS. A gene expression analysis of toll-like receptors (TLRs), recognizing bacteria on cell surfaces and initiating the immune response, showed no regulation for TLR2. Infection with EDL933 led to down regulation of TLR4 and to up regulation of TLR9. Stimulation with L. rhamnosus GG, L. johnsonii DSMZ 10533 or L. fermentum DSMZ 20052 led neither to regulation of TLR4 nor TLR9. The benign bacteria did not influence the EHEC-induced TLR4 regulation in coinfection trials; in contrast the regulation of TLR9 was reduced significantly. The model described here is useful for screening basic effects of protective bacteria that are able to counteract EHEC-mediated effects on human cells and to study the molecular interaction between bacteria as well as between bacteria and human cultured cells.
Temperaturgesteuerte Zellzahlregelung für Bioreaktoren
(2021) Loges, Karin Martina; Hitzmann, Bernd
Biopharmaceutical cell culture processes are multi-stage processes. Starting with low cell numbers and small culture volumes, the upstream process takes place step by step in a reactor cascade with increasing volume. To achieve constant product quality and high reproducibility, each reactor must be seeded with a defined initial cell concentration and all cultures must show very similar growth rates. However, batch to batch variations of growth rates can occur in biological systems and disrupt the reproducibility of the inoculation with defined cell concentrations. In order to control the growth rate and thus increase the reproducibility from batch to batch, a self-adapting cell number controller was developed. This controller can be rapidly introduced in a typical industrial (biopharma) environment. At the beginning of the process, the user specifies the desired seed cell concentration and the point in time at which this cell concentration should be reached. In the following, the cell number controller automatically regulates the growth rate of the cells during the process and leads it to the specified process end parameters. The regulation of the cell growth rate is achieved with adjustment of the process temperature. In the development phase of the cell number controller, a converted reactor system was adjusted and qualified. The temperature dependency of the biomass probe was examined. After that, the dependence of the cell growth rate on the temperature was analysed, described in the form of a mathematical model and implemented in a control algorithm. The required temperature is calculated with the aid of a numerical optimization process, using the previously derived mathematical model of the temperature-dependent growth rate, the online measured values and the specifications of the user. In order to achieve greater flexibility within the upstream process and to be able to react to disruptions in the process flow, it is also possible to dynamically adjust the target parameter, set by the user at the beginning, during the running process. To optimize the controller, it was analysed how the mathematical algorithm can be adapted to different cell clones and which tests are absolutely necessary to determine the cell specific, temperature-dependent growth rate. In this context, a self-learning algorithm was implemented so that it is now possible to use the controller without preliminary tests and to ensure constant control quality in the event of possible changes in the cell growth of a cell clone. During this research project, the functionality of the cell number controller, such as the changes in the process end parameters during the ongoing process and the iterative-adaptive optimization of the mathematical algorithm on different cell clones, could be verified experimentally. The biological reactions of the cells to the temperature changes within the cell number-controlled precultures and within the subsequent production stage were also analysed. Furthermore, a possible influence on the quantity and quality of the products was examined. All examined biological reactions of the cells during the cell count regulation showed a reversible behaviour, which are normalized in the following process steps at 37 °C within two days. In addition, no negative influence of the cell-number-controlled preculture on product quality and quantity could be determined. In summary, a functionally adaptable cell number controller was developed and tested with two different CHO cell clones for a possible influence on cell metabolism, apoptosis, product quantity and Quality.