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Nature Communications volume 14, numero articolo: 4755 (2023) Citare questo articolo

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L'attuale tecnologia di perfusione meccanica consente di conservare i fegati ex situ per brevi periodi per valutarne la vitalità prima del trapianto. La perfusione normotermica a lungo termine dei fegati è un campo emergente con un enorme potenziale per la valutazione, il recupero e la modifica degli organi. In questo studio, abbiamo mirato a sviluppare un modello a lungo termine di perfusione ex situ comprendente una suddivisione chirurgica e una perfusione simultanea di entrambi gli organi parziali. I fegati umani rifiutati per il trapianto sono stati perfusi utilizzando un perfusato a base di globuli rossi in condizioni normotermiche (36 °C) e quindi divisi e simultaneamente perfusi su macchine separate. Dieci fegati umani sono stati divisi, risultando in 20 fegati parziali. La vitalità mediana ex situ era di 125 ore e la sopravvivenza mediana ex situ era di 165 ore. La sopravvivenza a lungo termine è stata dimostrata dalla clearance del lattato, dalla produzione della bile, dalla produzione del Fattore V e dalla conservazione dell'adenosina trifosfato. Qui riportiamo la perfusione ex situ a lungo termine di fegati umani e dimostriamo la capacità di dividere e perfondere questi organi utilizzando un protocollo standardizzato.

La tecnologia di perfusione meccanica normotermica offre numerosi vantaggi rispetto alle tecniche tradizionali per la conservazione degli organi prima del trapianto1. La perfusione di un fegato umano donato prima del trapianto può prolungare il tempo di conservazione ex situ a breve termine e contemporaneamente consentire una certa valutazione della vitalità dell'organo come predittore della funzione del trapianto post-trapianto2,3. L'obiettivo principale di questa tecnologia fino ad oggi è stato quello di aumentare l'utilità degli organi marginali utilizzando la perfusione a breve termine nell'arco di poche ore. Tuttavia, la perfusione nell'arco di giorni o settimane potrebbe facilitare una valutazione più sofisticata di questi organi con il potenziale di recupero o modificazione prima del trapianto4,5. Ciò non solo potrebbe aumentare il numero di organi disponibili per il trapianto, ma potrebbe anche migliorare la qualità degli innesti attualmente utilizzati.

A tal fine, è stata segnalata la perfusione dei fegati fino a 7 giorni utilizzando un sistema integrato personalizzato in condizioni subnormotermiche (34 °C)4. La perfusione a questa temperatura ha effetti metabolici protettivi ma non simula le condizioni fisiologiche reali6,7. Lo stesso gruppo ha anche riportato il successo del trapianto e il follow-up a 1 anno di un fegato che era stato perfuso utilizzando la conservazione normotermica ex situ per 3 giorni8. Non è mai stata segnalata una perfusione a lungo termine di fegati umani oltre i 7 giorni in condizioni normotermiche (36 °C) e potrebbe sbloccare il potenziale di rigenerazione e modifica degli organi prima del trapianto.

La perfusione ex situ a lungo termine di fegati umani utilizzando condizioni normotermiche rappresenta anche un'opportunità unica per studiare il tessuto umano vivente ex situ. Dividendo interi fegati umani durante la perfusione meccanica normotermica come abbiamo precedentemente descritto9,10,11, questa tecnologia può essere applicata a due fegati parziali. Ciò potrebbe fornire un ambiente simulato per la sperimentazione di agenti terapeutici con un controllo corrispondente e lo studio del danno epatico e della rigenerazione.

In questo studio, abbiamo mirato a sviluppare un modello proof-of-concept di perfusione ex situ normotermica a lungo termine di fegati umani divisi per spingere i confini della perfusione ex situ estendendo la sopravvivenza oltre i 7 giorni e perfondendo contemporaneamente due organi parziali. In questo modo, abbiamo cercato di sviluppare un modello per studiare la perfusione epatica a lungo termine con potenziali applicazioni nella ricerca traslazionale e oltre.

Tutti i fegati dei donatori nel Nuovo Galles del Sud hanno acconsentito alla ricerca e hanno rifiutato il trapianto clinico tra febbraio e dicembre 2021 sono stati presi in considerazione per l'inclusione. Un fegato era compromesso a causa di una storia nota di ipertensione portale e un secondo a causa di cirrosi. Per sviluppare il protocollo, tre fegati interi sono stati perfusi senza dividersi. Utilizzando il nostro protocollo, 10 fegati umani donati sono stati divisi, ottenendo 10 LLSG e 10 ERG che sono stati perfusi su macchine di perfusione separate.

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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