Ex Vivo Heart Perfusion for 72 Hours Using Plasma Cross Circulation.

Preservation of a donor heart for transplantation is limited to 6-8 hours. Based on our demonstration of 12 hour perfusion with plasma cross circulation, this study aimed to evaluate ex vivo heart perfusion (EVHP) for up to 72 hours using cross plasma circulation (XC-plasma) from a live, awake paracorporeal sheep (PCS). Six ovine hearts were perfused for 72 hours using plasma cross circulation at a rate of 1 L/min with a live, awake PCS. Controls were seven perfused hearts without cross circulation. Experiments were electively ended at 72 hours, and epinephrine (0.1 mg) was delivered to demonstrate hormonal responsiveness. All controls failed at 6-10 hours. All six hearts perfused for 72 hours maintained normal heart function, metabolism, and responsiveness to epinephrine. Blood gases, electrolytes, and lactate levels were normal and stable throughout the study. All hearts appeared suitable for transplantation. We have demonstrated successful normothermic EVHP for 72 hours.

Normothermic Ex Vivo Heart Perfusion: Effects of Live Animal Blood and Plasma Cross Circulation.

Prolonged normothermic ex vivo heart perfusion could transform cardiac transplantation. To help identify perfusate components that might enable long-term perfusion, we evaluated the effects of cross-circulated whole blood and cross-circulated plasma from a live paracorporeal animal on donor porcine hearts preserved via normothermic ex vivo heart perfusion. Standard perfusion (SP; n = 40) utilized red blood cell/plasma perfusate and Langendorff technique for a goal of 12 hours. Cross-circulation groups used a similar circuit with the addition of cross-circulated venous whole blood (XC-blood; n = 6) or cross-circulated filtered plasma (XC-plasma; n = 7) between a live paracorporeal pig under anesthesia and the perfusate reservoir. Data included oxygen metabolism, vascular resistance, lactate production, left ventricular function, myocardial electrical impedance, and histopathologic injury score. All cross-circulation hearts were successfully perfused for 12 hours, compared with 22 of 40 SP hearts (55%; p = 0.002). Both cross-circulation groups demonstrated higher oxygen consumption and vascular resistance than standard hearts from hours 3-12. No significant differences were seen between XC-blood and XC-plasma hearts in any variable, including left ventricular dP/dT after 12 hours (1478 ± 700 mm Hg/s vs. 872 ± 500; p = 0.17). We conclude that cross circulation of whole blood or plasma from a live animal improves preservation of function of perfused hearts, and cross-circulated plasma performs similarly to cross-circulated whole blood.

Quantification of Mesenchymal Stem Cell (MSC) delivery to a target site using in vivo confocal microscopy.

The ability to deliver cells to appropriate target tissues is a prerequisite for successful cell-based therapy. To optimize cell therapy it is therefore necessary to develop a robust method of in vivo cell delivery quantification. Here we examine Mesenchymal Stem Cells (MSCs) labeled with a series of 4 membrane dyes from which we select the optimal dye combination for pair-wise comparisons of delivery to inflamed tissue in the mouse ear using confocal fluorescence imaging. The use of an optimized dye pair for simultaneous tracking of two cell populations in the same animal enables quantification of a test population that is referenced to an internal control population, thereby eliminating intra-subject variations and variations in injected cell numbers. Consistent results were obtained even when the administered cell number varied by more than an order of magnitude, demonstrating an ability to neutralize one of the largest sources of in vivo experimental error and to greatly reduce the number of cells required to evaluate cell delivery. With this method, we are able to show a small but significant increase in the delivery of cytokine pre-treated MSCs (TNF-α & IFN-γ) compared to control MSCs. Our results suggest future directions for screening cell strategies using our in vivo cell delivery assay, which may be useful to develop methods to maximize cell therapeutic potential.