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Nature Communications volume 14, número do artigo: 4755 (2023) Citar este artigo

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Detalhes das métricas

A tecnologia atual de perfusão mecânica permite que os fígados sejam preservados ex situ por curtos períodos para avaliar a viabilidade antes do transplante. A perfusão normotérmica de longo prazo de fígados é um campo emergente com enorme potencial para avaliação, recuperação e modificação de órgãos. Neste estudo, pretendemos desenvolver um modelo de perfusão ex situ a longo prazo, incluindo uma divisão cirúrgica e perfusão simultânea de ambos os órgãos parciais. Os fígados humanos recusados para transplante foram perfundidos utilizando um perfusato à base de glóbulos vermelhos sob condições normotérmicas (36 ° C) e depois divididos e simultaneamente perfundidos em máquinas separadas. Dez fígados humanos foram divididos, resultando em 20 fígados parciais. A viabilidade ex situ mediana foi de 125 horas e a sobrevivência ex situ mediana foi de 165 horas. A sobrevivência a longo prazo foi demonstrada pela depuração de lactato, produção de bile, produção de Fator-V e armazenamento de trifosfato de adenosina. Aqui, relatamos a perfusão ex situ a longo prazo de fígados humanos e demonstramos a capacidade de dividir e perfundir esses órgãos usando um protocolo padronizado.

A tecnologia de perfusão mecânica normotérmica oferece uma série de vantagens sobre as técnicas tradicionais de preservação de órgãos antes do transplante1. A perfusão de um fígado humano doado antes do transplante pode estender o tempo de preservação ex situ no curto prazo e, simultaneamente, permitir alguma avaliação da viabilidade do órgão como um preditor da função do enxerto pós-transplante2,3. O foco principal desta tecnologia até o momento tem sido aumentar a utilidade dos órgãos marginais usando perfusão de curto prazo no intervalo de horas. Entretanto, a perfusão na faixa de dias a semanas poderia facilitar uma avaliação mais sofisticada desses órgãos com potencial de recuperação ou modificação antes do transplante4,5. Isto não só poderia aumentar o número de órgãos disponíveis para transplante, mas também poderia melhorar a qualidade dos enxertos actualmente utilizados.

Para este fim, foi relatada perfusão de fígados por até 7 dias usando um sistema integrado e personalizado sob condições subnormotérmicas (34 °C)4. A perfusão nesta temperatura tem efeitos metabólicos protetores, mas não simula condições fisiológicas reais6,7. O mesmo grupo também relatou o sucesso do transplante e acompanhamento de 1 ano de um fígado que foi perfundido com preservação normotérmica ex situ por 3 dias8. A perfusão a longo prazo de fígados humanos além de 7 dias utilizando condições normotérmicas (36 °C) nunca foi relatada e poderia desbloquear o potencial de regeneração e modificação de órgãos antes do transplante.

A perfusão ex situ de fígados humanos a longo prazo utilizando condições normotérmicas também representa uma oportunidade única para estudar tecidos humanos vivos ex situ. Ao dividir fígados humanos inteiros durante a perfusão normotérmica, como descrevemos anteriormente9,10,11, esta tecnologia pode ser aplicada a dois fígados parciais. Isto poderia fornecer um ambiente simulado para o teste de terapêutica com um controle correspondente e o estudo de lesão e regeneração hepática.

Neste estudo, nosso objetivo foi desenvolver um modelo de prova de conceito de perfusão ex situ normotérmica de longo prazo de fígados divididos humanos para ultrapassar os limites da perfusão ex situ, estendendo a sobrevivência além de 7 dias e perfundindo simultaneamente dois órgãos parciais. Desta forma, procuramos desenvolver um modelo para investigar a perfusão hepática a longo prazo com potenciais aplicações em pesquisa translacional e além.

Todos os fígados de doadores em Nova Gales do Sul consentiram para pesquisa e recusaram transplante clínico entre fevereiro e dezembro de 2021 foram considerados para inclusão. Um fígado foi diminuído devido a uma história conhecida de hipertensão portal e um segundo devido a cirrose. Para desenvolver o protocolo, três fígados inteiros foram perfundidos sem divisão. Utilizando o nosso protocolo, 10 fígados humanos doados foram divididos, resultando em 10 LLSG e 10 ERGs que foram perfundidos em máquinas de perfusão separadas.

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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