Cellular Morphology, Proliferation, and Protein Expression in Chemically Defined versus Serum-Based Culture Conditions

Abstract

Cell culture has become a central technique in biomedical research, advancing our knowledge of cellular processes, disease mechanisms and many more. Successful cell culture conditions essentially require few key components. Cells need a constant supply of nutrients and growth promoting factors to mimic their native environment, and replicate their physiological state. Serum supplied to the cells provides this advantage, together with the basal medium. Over the years, as a gold standard the Fetal Bovine Serum is utilized. As much benefits this serum provides, it also comes with numerous concerns. Mainly, it originates from an animal source, and as such, could lead to issues in reproducibility of results. There are numerous efforts worldwide to test naturally derived alternatives, xeno-free and chemically defined serums for cell culture. Positive results have been demonstrated in terms of cellular morphology, proliferation and expression analysis in the alternative scenarios. Despite this, and limitations associated with FBS, there is not a single non-animal/chemically defined serum, which could serve as a standardized replacement for a broad range of cell types. The goal of this thesis was to utilize a chemically defined serum, named Panexin Basic, as an alternative source. Its performance on cellular behavior in terms of morphology, proliferation and protein expression was tested. Firstly, metabolic assay (PrestoBlue) was performed to identify differences in metabolic activity of cells, which served as an indirect indicator of proliferation trends. Statistical analysis revealed significantly higher metabolic activity in FBS-treated cells, compared to ones cultured in Panexin (p ≤ 0.05). Further, microscopic analysis revealed significant difference in morphology between cells cultured in FBS and Panexin. Particularly, FBS-treatedcells exhibited significantly larger areas, and distinct shape descriptors (roundness,circularity, and solidity; p ≤ 0.05). Based on the cell-level analysis, FBS treatment yielded higher protein expression than Panexin, even when comparing cells of similar size. Overall, both serum environments supported a heterogeneous population, with cells differing in shape, size and protein expression.