The use of cell culture models for studying the biological properties of cell-free DNA

The development of clinically meaningful tests based on molecular analyses of cell-free DNA (cfDNA) requires an improved understanding of its (1) molecular and cellular origin, (2) physical properties, and (3) dynamics in blood circulation. However, the in vivo characterization of cfDNA is complicated significantly by the immense heterogeneity of a single blood sample, which in turn is liable to significant intra- and interindividual variation. While it is often difficult to extrapolate in vitro findings in order to predict functioning in the human body, cell culture models have historically proven extremely useful in biology (e.g., elucidating most of the fundamental structures and mechanisms at the molecular level) and many domains of biomedical science (e.g., drug toxicity testing). To this end, we argue that cell culture models can be used to obtain useful knowledge about the biological properties of cfDNA. Based on data compiled from eight characterized cell lines (HepG2, HeLa, HEK-293, RD, A375, 143B, HMEC-1, and PCS201010), a number of interesting findings have been made. First, both the average amount and size of cfDNA present in culture medium varies significantly between different cell types when investigated under normal physiological conditions. Second, both the amount and size of cfDNA in the culture medium of a single cell line fluctuates over different incubation periods (in these experiments the amount of cfDNA has been normalized either in terms of the total cellular protein content or the total number of cells). Finally, excluding the small cfDNA fragments that are derived from apoptosis, these studies indicate the intriguing possibility that a fraction of cfDNA originates from a different mechanism. While the exact mechanisms that may be involved in such an active or regulated release of cfDNA are still unknown, our study suggests that it may be released as a consequence of genomic instability.