Non-disruptive in vitro monitoring of cellular states with cell-free DNA methylation

Regenerative cell therapies and stem-based in vitro models all aspire the approximation of a given start cell to an alternate (often more mature) cell state. However, 'monitoring', hence controlling, such a dynamic process requires disruption of the precious sample, destroying architecture and introducing variation. Here, we establish real time-compatible, fully non-invasive monitoring of cell fates through simple cell-free DNA medium sampling. We demonstrate that cell-free DNA methylation non-disruptively reveals genome-wide DNA methylation transitions during epigenetic drug treatment in stem cells, and reports the gradual maturation of 3D hepatocytes grown in bioreactors. Our cell-free DNA methylation platform enables monitoring a broad variety of in vitro systems, has implications for the real-time evaluation human therapeutic cells, and the potential to be readily integrated into large-scale bioreactor workflows. Overall design: Whole genome bisulfite profiling of cell-free and genomic DNA from KH2 mESCs treated with DNMT1-Inhibitor GSK-3484862 for four days, followed by a two day recovery period. DMSO-treated cells served as a negative control. Whole genome Nanopore-Sequencing of differentiating 3D bioreactor hepatocytes over 20 days.