Defatting heptocytes under flow

There is a critical shortage of transplantable livers in both the US and the world. One method to increase the donor pool is to develop methodology to recondition extended criteria donor grafts, a large portion of which are moderate to severe macrosteatotic livers. Transplantation of these livers often leads to primary nonfunction caused by an increased susceptibility to the effects of ischemia reperfusion injury that result from the harvesting, transportation, and transplantation of the liver. Our lab has developed a novel cocktail of defatting reagents that can, over a period of two days, render hepatocytes lean and with good cell viability and function. Despite this accomplishment, in order to be feasibly performed in a clinical setting, the defatting process must be completed in a matter of hours. The current project focuses on understanding the differences between defatting in a static versus flow environment and then using this information to develop the ideal parameters for defatting whole organs. Our hypothesis in the current work is that using flow and the appropriate defatting agents, steatotic hepatocytes can be defatted in a clinically relevant time of hours, without decreasing cell viability or function. In order to test this hypothesis, a perfusion reactor was constructed which holds microscope slides that can be seeded with fatty hepatocytes. Perfusion of defatting media was performed at different flow rates over a six hour period. Results showed that within a six hour period, significant defatting (as compared to static cultures) was observed at both 2.0 and 4.0 ml/min as indicated by oil red O staining. Current experiments are focused on further evaluation of hepatocyte viability and function (e.g. albumin, urea, and cytochrome P450 function). These types of parametric experiments will form the basis for future perfusions with whole organs from obese rats.