*Purpose: Despite the potential benefits and expanding use of advanced dynamic perfusion systems in clinical practice, to date there is no robust and non-invasive method to assess graft preservation quality. As it is released from injured, necrotic, and apoptotic cells, cell-free DNA (cfDNA) is a promising biomarker of tissue injury. This study was designed to test whether quantification of cfDNA concentration in the organ perfusate can serve as an accurate and efficient measure of tissue injury and cell death in donation after circulatory death (DCD) kidney allografts undergoing ex-vivo hypothermic machine perfusion.

*Methods: To mimic DCD kidney grafts, 8 porcine kidneys were subjected to 3 hours of warm ischemia, flushed with Plasmalyte, and perfused with non-oxygenated UW at 4^oC for 48 hours on a peristaltic pump. Tissue biopsies and perfusate samples were collected for biochemical and histological analysis and quantification of cfDNA of nuclear origin by qPCR after 1, 12, 24, and 48 hours.

*Results: CfDNA levels increased steadily in the perfusate over the course of hypothermic perfusion, with the highest cfDNA content consistently observed after 48h. The rise of cfDNA content was associated with a progressive rise in histological features of tissue necrosis throughout perfusion. Furthermore, increments in perfusate cfDNA levels were also associated with higher levels of tissue pro-apoptotic caspase-3 proteolytic activation and increased detection of TUNEL positive tubular and glomerular apoptosis.

*Conclusions: Our study provides evidence for the applicability of cfDNA monitoring of the perfusate of ex vivo hypothermic kidneys as an accurate and quantitative marker of tissue integrity and cellular injury in a preclinical pig model.

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