*Purpose: Injury during preservation is a complex process that affects graft function. A deeper understanding and better prediction of liver viability are demanded to improve transplant outcome and the utilization of marginal organs. We propose that liver cells are released from the liver during perfusion which can provide critical information with respect to organ quality and injury mechanisms.

*Methods: The perfusate of fresh, 24hrs cold ischemia (CI) and 72hrs-CI rat livers was collected during 3hrs subnormothermic machine perfusion (SNMP) and analyzed using multi-channel imaging flow cytometry to detect hepatocytes, sinusoidal endothelial, stellate, Kupfer, pit (liver associated NK) and dendritic cells. The experimental CI-groups correspond to the CI limit for 100% transplant survival and 0% transplant survival in rats after SNMP, respectively. The first 100ml and last 100ml that flushed through the liver were collected separately from the 400ml perfusate to study the change.

*Results: On average ~26×10⁶ cells were released in the perfusate. The perfusate cell profiles changed significantly depending on CI time. Hepatocytes, sinusoidal endothelial cells, and stellate cell were nearly absent in the perfusate of fresh livers and increased significantly in the perfusate of both the 24hrs-CI and 72hrs-CI group. Conversely, the fraction of Kupfer cells was highest in the perfusate of fresh livers and decreased significantly with increasing CI time. We also observed that the cell profiles of the 24hrs-CI and 72hrs-CI groups but not the fresh controls changed significantly over time during perfusion, demonstrated by a significant increase in pit and sinusoidal endothelial cells. Although rare, dendritic cells were detected, accounting for 0.14% of the cells and did not change with CI time. Results indicate that over 3500 dendritic cells are released during perfusion.

*Conclusions: Perfusate cell profiles are potential candidates as markers of graft quality and tissue injury and could provide valuable information about organ viability and immunogenicity. Characterization of the different cell types for further evaluation may lead to the discovery of new pathways for therapeutic intervention. Further, this might provide the basis for in vivo studies of IRI and rejection in combination with rare blood cell isolation/detection.

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