Person:

Uygun, Basak

Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Hypermetabolism is a significant sequela to severe trauma such as burns, as well as critical illnesses such as cancer. It persists in parallel to, or beyond, the original pathology for many months as an often-fatal comorbidity. Currently, diagnosis is based solely on clinical observations of increased energy expenditure, severe muscle wasting and progressive organ dysfunction. In order to identify the minimum number of necessary variables, and to develop a rat model of burn injury-induced hypermetabolism, we utilized data mining approaches to identify the metabolic variables that strongly correlate to the severity of injury. A clustering-based algorithm was introduced into a regression model of the extent of burn injury. As a result, a neural network model which employs VLDL and acetoacetate levels was demonstrated to predict the extent of burn injury with 88% accuracy in the rat model. The physiological importance of the identified variables in the context of hypermetabolism, and necessary steps in extension of this preliminary model to a clinically utilizable index of severity of burn injury are outlined.

The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.