Ischemia-reperfusion injury (IRI) plays a crucial role in the development of primary graft dysfunction (PGD) following lung transplantation. A promising novel approach to optimize donor organs before transplantation and reduce the incidence of PGD is mitochondrial transplantation. In this study, we explored the delivery of isolated mitochondria in 4 hours ex vivo lung perfusion (EVLP) before transplantation as a means to mitigate IRI. To provide a fresh and viable source of mitochondria, as well as to streamline the workflow without the need for donor muscle biopsies, we investigated the impact of autologous, allogeneic, and xenogeneic mitochondrial transplantation. In the xenogeneic settings, isolated mitochondria from mouse liver were utilized while autologous and allogeneic sources came from pig skeletal muscle biopsies. Treatment with mitochondrial transplantation increased the P/F ratio and reduced pulmonary peak pressure of the lungs during EVLP, compared to lungs without any mitochondrial transplantation, indicating IRI mitigation. Extensive investigations using advanced light and scanning electron microscopy did not reveal evidence of acute rejection in any of the groups, indicating safe xenotransplantation of mitochondria. Future work is needed to further explore this novel therapy for combating IRI in lung transplantation, where xenotransplantation of mitochondria may serve as a fresh, viable source to reduce IRI.

The microscopic observation of lung tissue is challenging due to its fragile nature. Xylene and isopropanol are common tissue-clearing reagents used before paraffin embedding, yet no studies have compared these two reagents in lung tissue processing. Due to the well-known health risks xylene could introduce to operators, as well as its environmental hazards, it has long been desired that a less harmful alternative to xylene with the same staining effects be introduced. Thus, we systematically assessed the efficacy of isopropanol as a substitution for xylene. Lung tissue obtained from diseased donors and explanted lungs from recipients were processed simultaneously using either xylene or isopropanol prior to paraffin embedding. Scoring of the overall staining quality after H&E staining, along with the ease of sectioning, was compared systematically. Fluorescent staining was performed to explore alveolar morphology and the overall lectin fluorescence signal between groups. To understand differences in antibody staining, the signal-to-noise ratio (SNR) of smooth muscle actin (SMA) and elastin was examined. No difference was observed with regard to ease of sectioning, staining quality, alveolar circularity, alveolar wall thickness or the SNR between slides processed with xylene or isopropanol. This study demonstrated comparable outcomes of isopropanol and xylene in lung tissue processing, suggesting isopropanol as a more favorable, operator- and environment-friendly substitute for xylene with regards to tissue processing.

Donation after circulatory death (DCD) has significantly increased the number of organs potentially available for transplantation. Livers are more vulnerable than most other solid organs to warm ischemia in the DCD process. The injury induced by warm ischemic time (WIT) is further exacerbated during cold ischemia and reperfusion, resulting in tissue damage caused by the production of reactive oxygen species. These are not only cytotoxic but also perpetuate mitochondrial dysfunction and cell death. To improve the utilization of livers from uncontrolled DCD (uDCD) or controlled DCD (cDCD) donors with prolonged WIT, new strategies to mitigate WIT must be developed.Ex vivo normothermic machine perfusion (NMP) has increased DCD organ utilization and improved the assessment of the viability of organs before transplantation. NMP could also serve as a platform for isolated treatment of organs prior to transplant. An innovative approach to mitigate organ injury is to treat damaged livers with mitochondrial transplantation (MTx). In this study, we tested the efficacy of xenogeneic mitochondrial administration to restore the function of porcine DCD livers with two hours of warm ischemia. DCD livers were explanted and connected to NMP, where mitochondrial transplantation was administered as a bolus dose in the portal vein and hepatic artery at the start of perfusion. After four hours of NMP, treated livers demonstrated a significant increase in bile production, improved bile quality, and restored cytoarchitecture.To further assess the transplantation suitability of these livers post-NMP, they were connected to cross-circulation (CC) with a recipient pig. After six hours of CC, untreated livers became oedematous, exhibited significant aspartate aminotransferase elevations in the bile, and showed cellular degeneration, including near-complete loss of Kupffer cells. In contrast, livers treated with MTx maintained function across CC and were comparable to healthy controls. These data validate the efficacy of MTx in restoring function in DCD livers exposed to prolonged ischaemic times, presenting a promising approach to expand the donor pool.