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Nature Communications volume 14、記事番号: 4755 (2023) この記事を引用

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現在の機械灌流技術では、肝臓を移植前に生存率を評価するために短期間保存することができます。肝臓の長期正常温灌流は、臓器の評価、回復、および修正に多大な可能性を秘めた新興分野です。この研究では、外科的分割と両方の部分臓器の同時灌流を含む、ex situ 灌流の長期モデルを開発することを目的としました。移植を拒否されたヒト肝臓は、正常温度条件 (36 °C) で赤血球ベースの灌流液を使用して灌流され、その後分割され、別の機械で同時に灌流されました。 10 個の人間の肝臓が分割され、20 個の部分肝臓ができました。生息地外生存率の中央値は 125 時間、生息地外生存率の中央値は 165 時間でした。長期生存は、乳酸クリアランス、胆汁産生、第 V 因子産生、およびアデノシン三リン酸の貯蔵によって実証されました。ここでは、ヒト肝臓の長期間の体外灌流を報告し、標準化されたプロトコルを使用してこれらの臓器を分割して灌流する能力を実証します。

常温機械灌流技術は、移植前の臓器保存における従来の技術に比べて多くの利点をもたらします1。移植前に提供されたヒト肝臓を灌流すると、短期間で生息域外保存期間が延長され、同時に移植後の移植片機能の予測因子として臓器生存率のある程度の評価が可能になります2,3。これまでのこの技術の主な焦点は、数時間の範囲の短期間の灌流を使用して辺縁臓器の有用性を高めることでした。しかし、数日から数週間の範囲の灌流は、移植前の回復または修正の可能性を伴うこれらの臓器のより高度な評価を容易にする可能性があります4,5。これにより、移植に利用できる臓器の数が増えるだけでなく、現在使用されている移植片の品質も向上する可能性があります。

この目的のために、標準温度以下の条件 (34 °C) で特注の統合システムを使用して、最長 7 日間の肝臓の灌流が報告されています。この温度での灌流には代謝保護効果がありますが、実際の生理学的状態をシミュレートするものではありません6,7。同じグループはまた、移植の成功と、現場外の常温保存で3日間灌流した肝臓の1年間の追跡調査も報告した8。正常温度 (36 °C) を使用したヒト肝臓の 7 日を超える長期灌流はこれまでに報告されておらず、移植前の臓器の再生と改変の可能性を解き放つ可能性があります。

正常温度条件を使用した人間の肝臓の長期的な現場外灌流もまた、現場外で生きているヒト組織を研究するためのユニークな機会を表します。以前に説明した9、10、11のように、正常温度の機械灌流中にヒト肝臓全体を分割することにより、この技術を2つの部分肝臓に適用できます。これにより、対応する対照を用いた治療薬の試験や肝損傷と再生の研究のための模擬環境が提供される可能性があります。

この研究では、生存期間を 7 日を超えて延長し、同時に 2 つの部分臓器を灌流することにより、ヒト分割肝臓の長期正常温体外灌流の概念実証モデルを開発することを目的としました。このようにして、トランスレーショナルリサーチやそれ以降の分野での応用が期待できる、長期肝灌流を調査するモデルの開発を目指しました。

2021年2月から12月までに研究に同意し、臨床移植を拒否したニューサウスウェールズ州のすべてのドナー肝臓が対象として考慮された。 1 つの肝臓は門脈圧亢進症の既知の病歴により機能低下し、もう 1 つは肝硬変により機能低下しました。プロトコールを開発するために、3 つの肝臓全体を分割せずに灌流しました。私たちのプロトコールを使用して、10 個の提供されたヒト肝臓を分割し、10 個の LLSG と 10 個の ERG を生成し、それらを別々の灌流装置で灌流しました。

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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