Successful 3-day lung preservation using a cyclic normothermic ex vivo lung perfusion strategy

Abstract

Background: Cold static preservation (CSP) at higher temperatures (10°C) has been recently shown as an optimal strategy up to 24-36h of preservation. Here, we hypothesized that alternating 10°C static storage with cycles of normothermic ex vivo lung perfusion (EVLP) would provide conditions for cellular "recharge", allowing for multi-day lung preservation.

Methods: Donor lungs from male Yorkshire pigs were preserved using 10°C CSP with two cycles of 4h EVLP. After a total of 3 days of preservation, a left lung transplant was performed followed by 4h of graft evaluation. As controls, 2 lungs were preserved solely with continuous 10°C preservation for 3 days and transplanted.

Findings: For animals receiving lungs preserved using a cyclic EVLP protocol, lung function and histological structures were stable and the recipient systemic partial pressure of oxygen/fraction of inspired oxygen (P/F Ratio) after excluding the contralateral lung was 422 ± 61 mmHg. In contrast, lungs preserved solely in continuous cold static storage at 10°C for 72h developed massive lung failure, resulting in recipient death. Metabolomic analysis revealed that EVLP plays a critical role in the re-vitalization of key central carbon energy metabolites (Glucose, Succinate, N-Acetyl Aspartate) and reducing the expression of the inflammasome activation marker CASP1.

Interpretation: In conclusion, we demonstrate for the first time the feasibility of 3-day lung preservation leading to excellent early post-transplant outcomes. The thoughtful combination of cold storage (10°C) and intermittent EVLP can open new opportunities in organ transplantation.

Funding: This work was supported by the UHN Foundation (Grant#1013612).

Keywords: Ex vivo lung perfusion; Lung preservation; Lung transplant; Metabolomics.

Figure 1

Physiological assessment of lungs stored for 72h using cold static preservation alone.Ex vivo lung perfusion (EVLP) as primary mode of assessment. a) Ventilator tubing attached to lung airway. Arrow shows perfusion solution filling the tube reflecting severe pulmonary edema. b) Representative histology after EVLP examination. (scale bar = 300 µm) c) Indocyanine green imaging (ICG) after the first 10 mins of perfusion. Bright intensities indicate development of pulmonary edema. Post Transplant assessment. d) Post-reperfusion fiberoptic bronchoscopy images alongside explanted lung images of control lungs e) Post reperfusion representative histology of control lungs. EVLP: Ex vivo lung perfusion, Rep: Reperfusion AP: Anterior-Posterior, RUL: Right Upper Lobe, RML: Right Middle Lobe, RLL: Right Lower Lobe, LUL: Left Upper Lobe, LLL: Left Lower Lobe.

Figure 2

Extended 10°C cold storage with Intermittent normothermic ex vivo lung perfusion (EVLP) protocol. a) Toronto EVLP circuit. b) Pig lungs were retrieved and stored for 6h at 4°C to simulate transportation to the transplant center, followed by 18h of 10°C storage (Cold1). Lungs then underwent a period of normothermic EVLP for 4h (EVLP1), followed by 20h of 10°C storage (Cold2), and an additional course of 4h of EVLP (EVLP2). Lungs were then stored at 10°C for 20h (Cold3), and then a single-left lung transplant was performed. The recipient animal was monitored post-transplant for 4h.

Figure 3

Lung assessment results during Intermittent ex vivo lung perfusion (EVLP) periods. a-f) Physiologic results (data expressed as mean ± SEM) g-i) Perfusate biochemistry (data expressed as mean ± SEM) j) Serial Indocyanine green (ICG) images. Day 1 represented EVLP1 and Day 2 represents EVLP2. P/F ratio: ratio of oxygen partial pressure to fraction of inspired oxygen.

Figure 4

Lung function after transplantation. a) Representative histology before preservation, after EVLP1, after EVLP2, and post reperfusion during the intermittent EVLP protocol. (scale bar = 300 µm) b-c) Recipient P/F ratio taken during 4h of reperfusion (data expressed as mean ± SEM). d) P/F Ratio after contralateral (native lung) pulmonary artery clamping in the intermittent EVLP group (Data expressed as mean ± SEM). e) Representative gross lung appearance of lungs before preservation, after EVLP1, after EVLP2, and post reperfusion during the intermittent EVLP protocol. P/F ratio: ratio of oxygen partial pressure to fraction of inspired oxygen, EVLP: Ex vivo lung perfusion, PA: Pulmonary artery.

Figure 5

Metabolic restoration during normothermic ex vivo lung perfusion. a) Tissue biopsy schedule of 72h lung preservation studies. Tissue were collected at 0h, 28h, 52h, and 72h for lung undergoing the intermittent EVLP protocol (n=4) or lungs continuously stored at 10°C (n=3). Tissue was then subjected to targeted quantitative analyses using Liquid Chromatography with tandem mass spectrometry (LC-MS-MS). b) Quantitative measurement of central carbon metabolites (2-ketoglutarate, Succinate, N-Acetyl aspartate, glucose, lactate, pyruvate) during 3 day lung preservation with and without intermittent EVLP. The ratio of lactate/pyruvate concentration was quantified and expressed as L/P ratio. (Two-way ANOVA followed by a Bonferroni correction for multiple comparisons. Data expressed as mean ± SEM). L/P: Lactate/pyruvate, (* p<0.05).