Hypothermic continuous machine perfusion improves metabolic preservation and functional recovery in heart grafts.

The number of heart transplants is decreasing due to organ shortage, yet the donor pool could be enlarged by improving graft preservation. Hypothermic machine perfusion (MP) has been shown to improve kidney, liver, or lung graft preservation. Sixteen pig hearts were recovered following cardioplegia and randomized to two different groups of 4-hour preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems, Itasca, Il). The grafts then underwent reperfusion on a Langendorff for 60 min. Energetic metabolism was quantified at baseline, postpreservation, and postreperfusion by measuring lactate and high-energy phosphates. The contractility index (CI) was assessed both in vivo prior to cardioplegia and during reperfusion. Following reperfusion, the hearts preserved using CS exhibited higher lactate levels (56.63 ± 23.57 vs. 11.25 ± 3.92 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio (0.4 ± 0.23 vs. 0.04 ± 0.04; P < 0.001), and lower phosphocreatine/creatine (PCr/Cr) ratio (33.5 ± 12.6 vs. 55.3 ± 5.8; P <0.001). Coronary flow was similar in both groups during reperfusion (107 ± 9 vs. 125 + /-9 ml/100 g/min heart; P = ns). CI decreased in the CS group, yet being well-preserved in the MP group. Compared with CS, MP resulted in improved preservation of the energy state and more successful functional recovery of heart graft.

Although pig allogeneic mesenchymal stem cells are not immunogenic in vitro, intracardiac injection elicits an immune response in vivo.

BACKGROUND: In vitro, mesenchymal stem cells (MSCs) have demonstrated a low immunogenic profile. In this study, we tested the immune response to allogeneic MSCs in immunocompetent swines both in vitro and in vivo. METHODS: Major histocompatibility complex-controlled swine leukocyte antigen (SLA) and SLA were used as donor and recipient, respectively. In vitro, proliferative responses were tested by mixed lymphocyte reaction (MLR) or cocultures and cytokine profiling by enzyme-linked immunosorbent assay. In vivo, allogeneic MSCs were injected in cardiac infarcted area (n=3) and compared with subcutaneous injections of either MSCs (n=2) or peripheral blood mononuclear cells (PBMCs; n=2). Two additional animals received a skin graft as controls. No immunosuppression was used. Specific antidonor humoral responses were tested by flow cytometry and complement-dependent cytotoxicity assay. RESULTS: In vitro, either unstimulated MSCs or interferon (IFN)-gamma stimulated MSC failed to elicit a proliferative response (stimulation index: 1.23 vs. 1.12 vs. 36.9 for controls, P<.001). Concomitantly to the absence of proliferation to MSCs, low production of IFN-gamma and interleukin-2 was evidenced in supernatants while the production of Th2 cytokines was comparable to controls. In vivo, all animals receiving skin grafts, subcutaneous PBMCs and intracardiac MSCs injections developed donor-specific cellular and humoral responses (immunoglobulins M and G) with antibody-complement-mediated cytotoxicity. Subcutaneous MSCs injection needed a rechallenge to similarly develop a cytotoxic humoral response. CONCLUSIONS: Intracardiac allogeneic porcine mesenchymal stem cells elicit an immune response despite their low immunogenic profile in vitro. This result suggests that in vivo characteristics of allogeneic MSCs might differ and emphasizes the importance of pursuing research both in vitro and in vivo.

Hypothermic continuous machine perfusion enables preservation of energy charge and functional recovery of heart grafts in an ex vivo model of donation following circulatory death.

OBJECTIVES: Cardiac transplantation using hearts from donors after circulatory death (DCD) is critically limited by the unavoidable warm ischaemia and its related unpredictable graft function. Inasmuch as hypothermic machine perfusion (MP) has been shown to improve heart preservation, we hypothesized that MP could enable the use of DCD hearts for transplantation. METHODS: We recovered 16 pig hearts following anoxia-induced cardiac arrest and cardioplegia. Grafts were randomly assigned to two different groups of 4-h preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems©, Itasca, Il). After preservation, the grafts were reperfused ex vivo using the Langendorff method for 60 min. Energetic charge was quantified at baseline, post-preservation and post-reperfusion by measuring lactate and high-energy phosphate levels. Left ventricular contractility parameters were assessed both in vivo prior to ischaemia and ex vivo during reperfusion. RESULTS: Following preservation, the hearts that were preserved using CS exhibited higher lactate levels (57.1 ± 23.7 vs 21.4 ± 12.2 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate ratio (0.53 ± 0.25 vs 0.11 ± 0.11; P < 0.001) and lower phosphocreatine/creatine ratio (9.7 ± 5.3 vs 25.2 ± 11; P < 0.001) in comparison with the MP hearts. Coronary flow was similar in both groups during reperfusion (107 ± 9 vs 125 ± 9 ml/100 g/min heart; P = ns). Contractility decreased in the CS group, yet remained well preserved in the MP group. CONCLUSION: MP preservation of DCD hearts results in improved preservation of the energy and improved functional recovery of heart grafts compared with CS.

Intracardiac allogeneic mesenchymal stem cell transplantation elicits neo-angiogenesis in a fully immunocompetent ischaemic swine model.

OBJECTIVES: Autologous mesenchymal stem cell transplantation has been shown to improve myocardial function in ischaemic cardiomyopathy. We studied one hypothetical mechanism, neo-angiogenesis, using allogeneic mesenchymal stem cell transplantation in an ischaemic swine model. METHODS: Allogeneic mesenchymal stem cells were injected in the peri-infarct area (1×10(6) cells kg(-1)) 2 weeks after myocardial infarction. Myocardial infarction alone (n=3) served as a control group. In the myocardial infarction-mesenchymal stem cells group (n=6), tacrolimus was given from day 0 to day 12. Capillary density and inflammatory/rejection processes (anti-factor VIII and anti-CD3/CD68 monoclonal antibodies, respectively) were compared between groups. RESULTS: In scarred myocardium, capillary density was similar between both ischaemic groups: 15.4 (±15.3) and 14.7 (±15.2) vessel/field in myocardial infarction-mesenchymal stem cells and myocardial infarction-alone groups (non-significant). In viable myocardium adjacent to the infarction, capillary density was significantly increased in the myocardial infarction-mesenchymal stem cells group than in the myocardial infarction-alone group (p=0.002). The number of infiltrating CD3+ cells was equivalent in both myocardial infarction-alone and myocardial infarction-mesenchymal stem cells groups (CD3+: 8.6% vs 9.3%, non-significant). However, CD68+ cell infiltration was more prominent after mesenchymal stem cell transplantation (4.7% vs 2% in myocardial infarction alone, p<0.01). CONCLUSIONS: Allogeneic mesenchymal stem cell transplantation enhances angiogenesis after myocardial infarction. This effect is limited to the viable myocardium. Using a concomitant 12-day course of tacrolimus, no mesenchymal stem cell-specific cellular immune response was demonstrated.

Inhibition of humoral response to allogeneic porcine mesenchymal stem cell with 12 days of tacrolimus.

BACKGROUND: In vivo studies have highlighted allogeneic mesenchymal stem-cell (MSC) immunogenicity. We investigated in vitro MSC-immunosuppressive drugs interaction and further tested in vivo the humoral response to intracardiac allogeneic MSC transplantation in a mini-swine model receiving a short course of immunosuppression. METHODS: For in vitro experiments, long-term culture MSCs were used. Immunosuppressive drugs tested were mycophenolate mofetil, cyclosporin, tacrolimus (TAC), sirolimus (SIR), and everolimus. Cell proliferation/viability was assessed on day 7. For each drug, the C50 was determined, and the agonistic effect between immunosuppressive drugs and MSCs on alloreactivity was measured in proliferation assay of MSC-peripheral blood mononuclear cell cultures. For in vivo experiments, one-haplotype swine leukocyte antigen class I and II mismatch (n=11) were used. Allogeneic MSCs were transplanted into ischemic myocardium. TAC was administered 12 days. Donor-specific antibody response was assessed by flow cytometry and complement-mediated cytotoxicity assay. RESULTS: All drugs except TAC significantly decreased cell proliferation (from 17% to 62%). In MSC-peripheral blood mononuclear cell co-culture assay, MSCs' immunomodulatory properties were maintained when TAC or SIR were used. In vivo experiments showed that only 2 of 11 animals under TAC developed donor-specific antibodies. Importantly, sera from those two animals did not elicit a complement-mediated cytotoxic response. CONCLUSIONS: Immunosuppressive drugs significantly affect proliferation and viability of MSCs, but neither TAC nor SIR had a detrimental impact on MSCs' immunomodulatory properties. In this large-animal model, addition of short course of immunosuppression seems to overcome the immune response to intracardiac allogeneic MSCs, which was recently demonstrated to occur in the absence of immunosuppression.