LAUSR

Cell passage number is of high importance in experimental design and can cause biochemical, genetic, and/or morphological changes to a cell. This project aimed to understand the impact of increasing cell passage number on the structure and function of epithelial cells from different organs. Two epithelial cell types were studied, NCI-H441 lung cells (type II alveolar cells), and HepG2 liver cells. Predefined ranges of 11-20, 21-30, 31-40, and 41-50 were studied. Cells were exposed to 1µg/mL tumour necrosis factor α (TNFα) and 15µM hydrogen peroxide (H2O2) via submerged or quasi-air liquid interface, according to GIVIMP guidelines. Endpoints tests included cytotoxicity (lactate dehydrogenase (LDH) release), cell death (BCL2, BAX, FAS), (pro-) inflammatory mediators (IL6 & IL8), oxidative stress (SOD1) and genetic toxicology (DNA damage (p21 & p53)). In addition, confocal microscopy was used to observe any changes to cell morphology. Preliminary data indicates the NCI-H441 epithelial cell passage range has an effect on the secretion of (pro-)inflammatory mediators, with an increase in IL8 and IL6 in higher passage ranges (41-50) compared to lower passage ranges (11-20) when stimulated with TNFα as a positive control. In HepG2 cells there is also an increase in IL8 production across passage range, however, it is considerably less than NCI-H441 cells. In addition, IL6 production in HepG2 cells was negligible across all passage ranges. LDH assay showed minimal variation between cell type and passage number. Data obtained suggests passage range, depending on cell type, has an influence on the effect level of the end point measured.