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

Individual CpG methylation signatures characterize each mammalian cell type, thereby offering a powerful readout for clinical and diagnostic applications. Here, we apply this principle to in vitro cellular systems, using cell-free DNA released into the culture medium to track epigenomic responses to small-molecule treatments and bioreactor-based directed differentiation. Coupled with Oxford Nanopore sequencing, our approach enables rapid assessment of global CpG methylation dynamics and enhancer states. This strategy provides a streamlined method that can be readily applied and customized for non-disruptive monitoring of various in vitro screening modalities.