Cell-Free Mitochondrial DNA in Cell Culture Supernatant: Fragment Size Analysis and FBS Contamination Assessment

Background/Objectives: Circulating cell-free DNA (cfDNA) consists of genomic DNA (cf-nDNA) and mitochondrial DNA (cf-mtDNA) fragments released from cells primarily through apoptosis and necrosis. In healthy individuals, the main source of cfDNA is apoptosis, whereas in cancer patients, necrosis predominates. Considering that in vitro cfDNA models are valuable research tools, this study presents an in vitro characterization of cf-mtDNA patterns released into the culture medium by four human cell lines: normal dermal fibroblasts (Hs27), induced pluripotent stem cells (iPSCs), melanoma cells (BMel), and prostate cancer cells (PC3). Furthermore, as fetal bovine serum (FBS)—a widely used supplement in cell culture media—has been shown to contain bovine cfDNA, species-specific primers were employed to eliminate potential artifacts arising from this contamination in in vitro experiments. Methods: Fragmentation analysis of cf-mtDNA was conducted by amplifying the human MT-CYB gene and the D-loop region in four cell lines using species-specific primers. Two indices, Q and λ, were employed to quantify fragmentation. Results: These indices reveal that cancer cells exhibit the highest degree of fragmentation compared to fibroblasts, whereas stem cells show the lowest degree of fragmentation. This study identified species-specific primers for the human and bovine MT-CYB gene, confirming the presence of bovine cf-mtDNA in cell culture media supplemented with FBS. Conclusions: in vitro cellular models are useful for studying the mechanisms of cfDNA release and fragmentation; designed primers provide a reliable tool for assessing contamination across different growth time points minimizing interference errors and non-specific amplifications.