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Nature Communications 14권, 기사 번호: 4755(2023) 이 기사 인용

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현재 기계 관류 기술을 사용하면 간을 이식 전 생존 가능성을 평가하기 위해 짧은 기간 동안 현장 외 보존할 수 있습니다. 간의 장기 정상 체온 관류는 장기의 평가, 회복 및 변형을 위한 엄청난 잠재력을 지닌 새로운 분야입니다. 이 연구에서 우리는 수술 분할 및 두 부분 장기의 동시 관류를 포함하는 현장 외 관류의 장기 모델을 개발하는 것을 목표로 했습니다. 이식이 거부된 인간의 간은 정상 체온 조건(36°C)에서 적혈구 기반 관류액을 사용하여 관류된 후 분할되어 별도의 기계에서 동시에 관류되었습니다. 10개의 인간 간을 분할하여 20개의 부분 간을 만들었습니다. 현장 외 생존율 중앙값은 125시간이었고, 현장 외 생존 중앙값은 165시간이었습니다. 장기 생존은 젖산 제거, 담즙 생산, Factor-V 생산 및 아데노신 삼인산 저장을 통해 입증되었습니다. 여기에서 우리는 인간 간의 장기간의 현장 관류를 보고하고 표준화된 프로토콜을 사용하여 이러한 장기를 분할하고 관류하는 능력을 보여줍니다.

정상 체온 기계 관류 기술은 이식 전 장기 보존을 위한 기존 기술에 비해 많은 이점을 제공합니다1. 이식 전에 기증된 인간 간을 관류하면 단기적으로 현장 외 보존 시간을 연장할 수 있으며 동시에 이식 후 이식 기능의 예측 인자로서 장기 생존 가능성을 일부 평가할 수 있습니다2,3. 현재까지 이 기술의 주요 초점은 몇 시간 범위의 단기 관류를 사용하여 주변 장기의 유용성을 높이는 것이었습니다. 그러나 며칠에서 몇 주까지의 관류는 이식 전 회복 또는 수정 가능성이 있는 이들 장기에 대한 보다 정교한 평가를 용이하게 할 수 있습니다4,5. 이는 이식에 사용할 수 있는 장기의 수를 늘릴 수 있을 뿐만 아니라 현재 사용되는 이식편의 품질도 향상시킬 수 있습니다.

이를 위해, 온도 이하의 조건(34°C)에서 맞춤형 통합 시스템을 사용하여 최대 7일 동안 간의 관류가 보고되었습니다. 이 온도에서의 관류는 대사 보호 효과가 있지만 실제 생리학적 조건을 시뮬레이션하지는 않습니다6,7. 같은 그룹은 또한 3일간 정상 체온 보존을 사용하여 관류된 간의 성공적인 이식 및 1년 추적 관찰을 보고했습니다8. 정상 체온 조건(36°C)을 사용하여 7일을 초과하는 인간 간의 장기 관류는 보고된 적이 없으며 이식 전 장기의 재생 및 변형 가능성을 열어줄 수 있습니다.

정상 체온 조건을 사용하여 인간 간을 장기적으로 현장에서 관류하는 것은 현장에서 살아있는 인간 조직을 연구할 수 있는 독특한 기회를 나타냅니다. 이전에 설명한 대로 정상 체온 기계 관류 중에 전체 인간 간을 분할함으로써 이 기술은 두 개의 부분 간에 적용될 수 있습니다. 이는 일치하는 대조군을 사용한 치료제 테스트와 간 손상 및 재생 연구를 위한 시뮬레이션 환경을 제공할 수 있습니다.

이 연구에서 우리는 생존 기간을 7일 이상으로 연장하고 두 개의 부분 장기를 동시에 관류함으로써 현장 외 관류의 경계를 넓히기 위해 인간 분할 간의 장기 정상 체온 외부 관류에 대한 개념 증명 모델을 개발하는 것을 목표로 했습니다. 이러한 방식으로 우리는 중개 연구 및 그 이상 분야에 잠재적으로 적용할 수 있는 장기 간 관류를 조사하기 위한 모델을 개발하려고 했습니다.

뉴사우스웨일스의 모든 기증자 간은 연구에 동의했고 2021년 2월부터 12월 사이에 임상 이식을 거부한 간을 포함 대상으로 고려했습니다. 한 개의 간은 알려진 문맥압항진증의 병력으로 인해 거부되었고 두 번째는 간경변으로 인해 거부되었습니다. 프로토콜을 개발하기 위해 세 개의 전체 간을 분할하지 않고 관류했습니다. 우리의 프로토콜을 사용하여 기증된 인간 간 10개를 분할하여 별도의 관류 기계에 관류된 10개의 LLSG와 10개의 ERG를 생성했습니다.

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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