Bridging the gap between in vitro and in vivo models: a way forward to clinical translation of mitochondrial transplantation in acute disease states.

Mitochondrial transplantation and transfer are being explored as therapeutic options in acute and chronic diseases to restore cellular function in injured tissues. To limit potential immune responses and rejection of donor mitochondria, current clinical applications have focused on delivery of autologous mitochondria. We recently convened a Mitochondrial Transplant Convergent Working Group (CWG), to explore three key issues that limit clinical translation: (1) storage of mitochondria, (2) biomaterials to enhance mitochondrial uptake, and (3) dynamic models to mimic the complex recipient tissue environment. In this review, we present a summary of CWG conclusions related to these three issues and provide an overview of pre-clinical studies aimed at building a more robust toolkit for translational trials.

Cardiac tissue model of immune-induced dysfunction reveals the role of free mitochondrial DNA and the therapeutic effects of exosomes.

Despite tremendous progress in the development of mature heart-on-a-chip models, human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip with circulating immune cells to model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute myocarditis. We observed hallmarks of coronavirus disease (COVID-19)-induced myocardial inflammation, as the presence of immune cells augmented the secretion of proinflammatory cytokines, triggered progressive impairment of contractile function, and altered intracellular calcium transients. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the heart-on-a-chip and then validated in COVID-19 patients with low left ventricular ejection fraction, demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation-induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2-induced myocardial inflammation, we established that administration of endothelial cell-derived exosomes effectively rescued the contractile deficit, normalized calcium handling, elevated the contraction force, and reduced the ccf-mtDNA and cytokine release via Toll-like receptor-nuclear factor κB signaling axis.

Heart-on-a-chip model of immune-induced cardiac dysfunction reveals the role of free mitochondrial DNA and therapeutic effects of endothelial exosomes.

Cardiovascular disease continues to take more human lives than all cancer combined, prompting the need for improved research models and treatment options. Despite a significant progress in development of mature heart-on-a-chip models of fibrosis and cardiomyopathies starting from induced pluripotent stem cells (iPSCs), human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip system with circulating immune cells to model SARS-CoV-2-induced acute myocarditis. Briefly, we observed hallmarks of COVID-19-induced myocardial inflammation in the heart-on-a-chip model, as the presence of immune cells augmented the expression levels of proinflammatory cytokines, triggered progressive impairment of contractile function and altered intracellular calcium transient activities. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the in vitro heart-on-a-chip model and then validated in COVID-19 patients with low left ventricular ejection fraction (LVEF), demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2 induced myocardial inflammation, we established that administration of human umbilical vein-derived EVs effectively rescued the contractile deficit, normalized intracellular calcium handling, elevated the contraction force and reduced the ccf- mtDNA and chemokine release via TLR-NF-kB signaling axis.

Evaluation of 10°C as the optimal storage temperature for aspiration-injured donor lungs in a large animal transplant model.

BACKGROUND: Our recent work has challenged 4°C as an optimal lung preservation temperature by showing storage at 10°C to allow for the extension of preservation periods. Despite these findings, the impact of 10°C storage has not been evaluated in the setting of injured donor lungs. METHODS: Aspiration injury was created through bronchoscopic delivery of gastric juice (pH: 1.8). Injured donor lungs (n = 5/group) were then procured and blindly randomized to storage at 4°C (on ice) or at 10°C (in a thermoelectric cooler) for 12 hours. A third group included immediate transplantation. A left lung transplant was performed thereafter followed by 4 hours of graft evaluation. RESULTS: After transplantation, lungs stored at 10°C showed significantly better oxygenation when compared to 4°C group (343 ± 43 mm Hg vs 128 ± 76 mm Hg, p = 0.03). Active metabolism occurred during the 12 hours storage period at 10°C, producing cytoprotective metabolites within the graft. When compared to lungs undergoing immediate transplant, lungs preserved at 10°C tended to have lower peak airway pressures (p = 0.15) and higher dynamic lung compliances (p = 0.09). Circulating cell-free mitochondrial DNA within the recipient plasma was significantly lower for lungs stored at 10°C in comparison to those underwent immediate transplant (p = 0.048), alongside a tendency of lower levels of tissue apoptotic cell death (p = 0.075). CONCLUSIONS: We demonstrate 10°C as a potentially superior storage temperature for injured donor lungs in a pig model when compared to the current clinical standard (4°C) and immediate transplantation. Continuing protective metabolism at 10°C for donor lungs may result in better transplant outcomes.

Static lung storage at 10°C maintains mitochondrial health and preserves donor organ function.

Cold static preservation on ice (~4°C) remains the clinical standard of donor organ preservation. However, mitochondrial injury develops during prolonged storage, which limits the extent of time that organs can maintain viability. We explored the feasibility of prolonged donor lung storage at 10°C using a large animal model and investigated mechanisms related to mitochondrial protection. Functional assessments performed during ex vivo lung perfusion demonstrated that porcine lungs stored for 36 hours at 10°C had lower airway pressures, higher lung compliances, and better oxygenation capabilities, indicative of better pulmonary physiology, as compared to lungs stored conventionally at 4°C. Mitochondrial protective metabolites including itaconate, glutamine, and N-acetylglutamine were present in greater intensities in lungs stored at 10°C than at 4°C. Analysis of mitochondrial injury markers further confirmed that 10°C storage resulted in greater protection of mitochondrial health. We applied this strategy clinically to prolong preservation of human donor lungs beyond the currently accepted clinical preservation limit of about 6 to 8 hours. Five patients received donor lung transplants after a median preservation time of 10.4 hours (9.92 to 14.8 hours) for the first implanted lung and 12.1 hours (10.9 to 16.5 hours) for the second. All have survived the first 30 days after transplantation. There was no grade 3 primary graft dysfunction at 72 hours after transplantation, and median post-transplant mechanical ventilation time was 1.73 days (0.24 to 6.71 days). Preservation at 10°C could become the standard of care for prolonged pulmonary preservation, providing benefits to both patients and health care teams.