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Nature Communications volume 14, Numéro d'article : 4755 (2023) Citer cet article

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La technologie actuelle de perfusion mécanique permet de conserver les foies ex situ pendant de courtes périodes afin d'évaluer leur viabilité avant la transplantation. La perfusion normothermique à long terme du foie est un domaine émergent doté d'un énorme potentiel pour l'évaluation, la récupération et la modification des organes. Dans cette étude, nous avions pour objectif de développer un modèle à long terme de perfusion ex situ comprenant une division chirurgicale et une perfusion simultanée des deux organes partiels. Les foies humains refusés pour la transplantation ont été perfusés à l'aide d'un perfusat à base de globules rouges dans des conditions normothermiques (36 ° C), puis divisés et perfusés simultanément sur des machines distinctes. Dix foies humains ont été divisés, ce qui a donné lieu à 20 foies partiels. La viabilité médiane ex situ était de 125 h et la survie médiane ex situ était de 165 h. La survie à long terme a été démontrée par la clairance du lactate, la production de bile, la production de facteur V et le stockage de l'adénosine triphosphate. Nous rapportons ici la perfusion ex situ à long terme de foies humains et démontrons la capacité de diviser et de perfuser ces organes à l’aide d’un protocole standardisé.

La technologie de perfusion normothermique offre de nombreux avantages par rapport aux techniques traditionnelles de préservation des organes avant la transplantation1. La perfusion d'un foie humain donné avant la transplantation peut prolonger la durée de conservation ex situ à court terme et permettre simultanément une certaine évaluation de la viabilité de l'organe en tant que prédicteur de la fonction du greffon après la transplantation2,3. À ce jour, l’objectif principal de cette technologie a été d’augmenter l’utilité des organes marginaux en utilisant une perfusion à court terme de l’ordre de quelques heures. Cependant, une perfusion de quelques jours à quelques semaines pourrait faciliter une évaluation plus sophistiquée de ces organes avec un potentiel de récupération ou de modification avant la transplantation4,5. Non seulement cela pourrait augmenter le nombre d’organes disponibles pour la transplantation, mais cela pourrait également améliorer la qualité des greffons actuellement utilisés.

À cette fin, une perfusion de foies jusqu’à 7 jours a été rapportée à l’aide d’un système intégré sur mesure dans des conditions subnormothermiques (34 °C)4. La perfusion à cette température a des effets métaboliques protecteurs mais ne simule pas les conditions physiologiques réelles6,7. Le même groupe a également rapporté la transplantation réussie et le suivi d'un an d'un foie perfusé par conservation normothermique ex situ pendant 3 jours8. La perfusion à long terme de foies humains au-delà de 7 jours dans des conditions normothermiques (36 °C) n'a jamais été rapportée et pourrait libérer le potentiel de régénération et de modification des organes avant la transplantation.

La perfusion ex situ à long terme de foies humains dans des conditions normothermiques représente également une opportunité unique pour étudier les tissus humains vivants ex situ. En divisant des foies humains entiers lors d’une perfusion normothermique comme nous l’avons décrit précédemment9,10,11, cette technologie peut être appliquée à deux foies partiels. Cela pourrait fournir un environnement simulé pour tester des produits thérapeutiques avec un contrôle apparié et pour étudier les lésions et la régénération du foie.

Dans cette étude, nous avions pour objectif de développer un modèle de validation de principe de perfusion ex situ normothermique à long terme de foies humains divisés afin de repousser les limites de la perfusion ex situ en prolongeant la survie au-delà de 7 jours et en perfusant simultanément deux organes partiels. De cette manière, nous avons cherché à développer un modèle pour étudier la perfusion hépatique à long terme avec des applications potentielles dans la recherche translationnelle et au-delà.

Tous les foies de donneurs de Nouvelle-Galles du Sud ont consenti à la recherche et ont refusé une transplantation clinique entre février et décembre 2021 et ont été pris en compte pour l'inclusion. Un foie a été décliné en raison d'antécédents connus d'hypertension portale et un second en raison d'une cirrhose. Pour développer le protocole, trois foies entiers ont été perfusés sans fractionnement. En utilisant notre protocole, 10 foies humains donnés ont été divisés, ce qui a donné lieu à 10 LLSG et 10 ERG qui ont été perfusés sur des machines de perfusion distinctes.

50 years) in 3/6, and the remaining due to a prolonged time to the cessation of circulation (>30 min), morbid obesity, and acuity of transplant activity. The median cold ischaemic time (CIT, defined as the time from cold perfusion to reperfusion using the ex situ machine) was 295 min (interquartile range [IQR] 273–430 min) (Supplementary Table 1). For DCD livers, the median time to death (withdrawal of cardiorespiratory support to cessation of circulation) was 20 min (IQR 19–29 min) (Supplementary Table 1)./p>7 days with evidence of lactate clearance and bile production (Supplementary Fig. 1B). Once lactate started to rise beyond 2.5 mmol/L, we observed an irreversible deterioration in organ function which ultimately ended in organ failure in all cases./p>2.5 mmol/L and viability criteria were no longer fulfilled, perfusion was continued for all partial livers in an exploratory fashion to characterise changes relating to organ failure. The time from being non-viable to complete organ failure (lactate >10 mmol/L and exponentially rising with a lack of bile production or unresponsive hypoglycaemia) was typically <48 h (16/20 grafts). The overall median survival was 165 h (IQR 113–224 h), with 9/20 livers surviving for >7 days and 4/20 livers surviving >10 days (Fig. 1B, Supplementary Table 2). The maximum overall survival was 327.5 h. Hepatobiliary viability was assessed using criteria from the DHOPE-COR-NMP trial12. The same two livers that failed due to a technical error were also not viable by these criteria, but all other partial livers met these hepatobiliary viability criteria for up to 48 h of perfusion (Supplementary Table 3). Notably, these livers also all produced bile with a pH >7.40, indicating preserved cholangiocyte function (Supplementary Table 3)./p>10 mmol/L with a lack of bile production or unresponsive hypoglycaemia. All livers demonstrated lactate clearance (C), bile production (D), production of Factor-V (E), and evidence of oxygen consumption (F) until the point of organ failure. Perfusate pH and glucose were typically stable during perfusion until organ failure, which resulted in refractory acidosis and unresponsive hypoglycaemia (G, H). Bile pH was typically alkalotic and bile glucose was typically in the hypoglycaemic range during perfusion (I, J). *Viability according to the criteria proposed by the VITTAL clinical trial (≤2.5 mmol/L, and two or more of: bile production, pH ≥ 7.30, glucose metabolism, hepatic arterial flow ≥150 ml/min and portal vein flow ≥500 ml/min, or homogeneous perfusion)2./p>7 days or ≤7 days, we examined the factors that predicted long-term survival. In total, 9/20 partial livers survived >7 days. This included 4 LLSGs and 5 ERGs, and these partial livers were derived from six different whole livers. Donor characteristics were not significantly different between the two groups. The mean donor age for livers that survived >7 days and ≤7 days was 52.8 ± 13.3 and 53.6 ± 15.4 (p = 0.908), respectively. Donors for all organs were more commonly male (7/9 for livers surviving >7 days and 7/11 for livers surviving ≤7 days) and more commonly retrieved through the DCD pathway (6/9 vs 6/11 respectively) (Supplementary Table 4)./p>7 days at 24 h, 60 h and 72 h after splitting (median 3.674 ml/h/kg liver [IQR 2.247–4.576 ml/h/kg liver] vs 1.714 ml/h/kg liver [IQR 0.478–2.516 ml/h/kg liver], p = 0.008 at 24 h) (Fig. 4B). The perfusate level of Factor-V was significantly higher in the livers that survived >7 days immediately before splitting and at every time point up until 72 h after splitting (mean 47.3 ± 19.9% vs 15.4 ± 12.7%, p < 0.001 at 24 h) (Fig. 4C). Perfusate PT was significantly shorter in livers that survived >7 days immediately before splitting and 4 h after splitting (Fig. 4D). Perfusate urea, albumin, total protein, bile pH, and bile glucose did not demonstrate significant differences between the two groups (Fig. 4, Supplementary Fig. 4)./p>7 days or ≤7 days (A). Bile production and Factor-V levels were significantly higher in the livers that survived >7 days (bile: median 3.674 ml/h/kg liver [IQR 2.247–4.576 ml/h/kg liver] vs 1.714 ml/h/kg liver [IQR 0.478–2.516 ml/h/kg liver], p = 0.008 at 24 h, Mann–Whitney U Test; Factor-V: mean 47.3 ± 19.9% vs 15.4 ± 12.7%, p < 0.001 at 24 h, unpaired two-sided t-test) (B, C). Prothrombin time was significantly shorter for livers that survived >7 days immediately before and 4 h after splitting (median 54 s [IQR 38–48 s] vs 150 s [IQR 55–91 s] at 4 h, p = 0.015, Mann–Whitney U Test) (D). Oxygen consumption, perfusate urea, bile pH and bile glucose did not demonstrate significant differences between the two groups (E–H). Hepatic artery flow was significantly higher in the livers that survived >7 days for the same hepatic artery pressure (median 615 ml/min [IQR 530–674 ml/min] vs 342 ml/min [IQR 308–405 ml/min], p = 0.002, just before splitting, Mann–Whitney U Test) (I, J). Portal venous pressure was not significantly different between the two groups (K). Portal venous flow was significantly higher in the livers that survived >7 days between days 1–3 after splitting (median 1.030 ml/min [IQR 0.320–1.310 ml/min] vs 0.280 ml/min [IQR 0.220–0.970 ml/min], p = 0.049, 1 day after splitting, Mann–Whitney U Test) (L). All grouped data are presented as median (IQR) except for Factor-V, which was normally distributed and presented as mean (standard deviation), n = 20 partial livers, 9 survived >7 days, 11 survived ≤7 days. Normally distributed data and non-normally distributed data were compared at each grouped time point using an unpaired two-sided t-test and a Mann–Whitney U Test, respectively. *p < 0.05./p>7 days both before and after splitting (median 615 ml/min [IQR 530–674 ml/min] vs 342 ml/min [IQR 308–405 ml/min], p = 0.002, just before splitting) (Fig. 4J). This difference was evident using pressure control targets that were only modified to meet minimum flow requirements. After adjusting for the weight of each liver, this difference was still present but less pronounced (Supplementary Fig. 4). The portal venous flows were significantly higher for livers that survived >7 days between days 1 and 3 after splitting (median 1.030 ml/min [IQR 0.320–1.310 ml/min] vs 0.280 ml/min [IQR 0.220–0.970 ml/min], p = 0.049, 1 day after splitting) (Fig. 4L)./p>7 days (median 5% [IQR 0–7.5%] vs 20% [IQR 5–35%], p = 0.041 at 0 h) (Fig. 5A). However, the severity of macrovesicular steatosis, coagulative necrosis, and hepatocyte detachment was not significantly different between the two groups (Figs. 3E, F and 5B)./p>7 days (median 5% [IQR 0–7.5%] vs 20% [IQR 5–35%], p = 0.041 at 0 h, Mann–Whitney U Test) (A). All grouped data are presented as median (IQR), n = 20 partial livers, 9 survived >7 days, 11 survived ≤7 days, *p < 0.05./p>7 days were LLSGs. The machine perfusion revolution has yet to be realised in the field of paediatrics13, perhaps due to technical challenges. Still, the adaptations and modifications achieved in this study pave the way for these advances./p>

7 days and ≤7 days in this study, we were able to identify predictors of long-term survival using liver biochemistry, markers of synthetic liver function, liver haemodynamics, and histopathology. Organs that survived >7 days had significantly higher rates of bile production, higher levels of Factor-V, higher hepatic artery flows, and lower amounts of microvesicular steatosis. These changes were noticeable within the first 48–72 h of perfusion and represented potential targets for defining a signature for long-term survival. Not only does this have implications for the assessment of inherent organ quality, but this signature can be re-evaluated in real-time and guide us in the resuscitation and recovery of these livers in the long term./p>7 days. This model represents the longest-ever perfusion of human livers ex situ under normothermic conditions and has provided new information about how these organs can be evaluated for clinical use and why they fail in the long term. We describe a model suitable for ex situ perfusion of paediatric-sized organs and for expanding the applications of ex situ perfusion technology. Moreover, this technique has tremendous potential in the testing of therapeutics and paves the way for collaboration in the fields of transplantation, basic sciences and beyond./p>400 ml/min and a portal vein flow of >1.2 L/min. Controlled rewarming was performed with a 1° increase in temperature per hour for 4 h (from the initial 32 °C to 36 °C) to maintain perfusion in a temperature range conducive to red blood cell survival and minimise the effects of ischaemia reperfusion injury12,19./p>10 mmol/L or exponentially rising, and there was a cessation of bile production and unresponsive hypoglycaemia. Liver viability according to the DHOPE-COR-NMP trial (lactate <1.7 mmol/L, pH 7.35–7.45, bile production >10 ml and bile pH >7.45) was also assessed during perfusion to include an evaluation of biliary viability12. Our long-term perfusion protocol for split human livers is summarised in Fig. 7./p>

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