Optimizacija postopka priprave celične banke za enkratno uporabo iz celične linije HEK293 z vključkom za človeški protein B-celični maturacijski antigen in NF-κB-luciferazni reporter

This master's thesis presents a comprehensive optimisation of the single-use cell bank (SUCB) preparation process from a genetically modified HEK293 cell line expressing the human B-cell maturation antigen (BCMA) and an NF-κB-luciferase reporter. The aim of the study was to improve the cell viability, growth rate, and functional performance of SUCBs used in bioassays, while also evaluating alternative cell counting methodologies. Using a two-phase design of experiments (DOE) approach, key parameters influencing cell culture performance were identified and optimised. The first DOE experiment focused on cultivation conditions, revealing that lower working volumes, extended passage durations, and high viable cell concentrations negatively impacted cell viability and proliferation. The second DOE experiment refined freezing and thawing parameters, establishing that 5 % DMSO with a 30-minute exposure time and preheated thawing medium yielded optimal results. A comparative analysis between a haemocytometer and the Vi-Cell XR revealed statistically significant differences in cell concentration and viability measurements, highlighting the need for standardisation across analytical platforms. The genomic profiling of optimal and suboptimal cell banks revealed differential expression patterns linked to ER stress and apoptosis in suboptimal conditions, while optimal conditions promoted miRNA regulation and stress resilience. The optimised SUCB met all predefined criteria, including high viability (> 91 %), target viable cell concentration (≥ 1 × 10⁷ viable cells/mL), sterility, and confirmed functionality in luciferase-based bioassays. These findings contribute to improved reproducibility and efficiency in bioanalytical workflows, and offer insights into the molecular mechanisms underlying cell bank quality.