器官保存——历史、发展与展望

Engineering ›› 2025, Vol. 44 ›› Issue (1) : 120 -141. DOI: 10.1016/j.eng.2024.12.020

研究论文

器官保存——历史、发展与展望

刘欣萌 ^a^,^b ,
舒志全 ^c ,
张黎明 ^a^,^b ,
李皓玥 ^a^,^b ,
杨静 ^a^,^b ,
张雷 ^a^,^b^,^d^,^*

作者信息 +

Organ Preservation: History, Advancements, and Perspectives

Xinmeng Liu ^a^,^b^,^# ,
Zhiquan Shu ^c^,^# ,
Liming Zhang ^a^,^b ,
Haoyue Li ^a^,^b ,
Jing Yang ^a^,^b ,
Lei Zhang ^a^,^b^,^d^,^*

Author information +

文章历史 +

Received	Accepted	Published
2024-05-08	2024-12-22
Issue Date
2025-01-23

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摘要

随着器官移植、再生医学以及药物研发等领域的快速发展，器官保存技术的重要性日益凸显。尽管相关技术已经取得了一定进展，但现有保存方法在长期维持器官活性和功能方面仍然存在明显局限。因此，迫切需要制定更加可靠与高效的器官保存策略。目前，临床上主要采用静态冷保存和器官机械灌注两种保存方式，这些方法能够维持器官短期生存，但其保存时间通常仅为数天甚至数小时。近年来，低温生物学的迅速发展为该领域带来了全新的视角。本文全面回顾了器官保存技术的发展历程，重点分析了传统保存方法的局限性，并讨论了肾脏、肝脏、心脏、肺和肠道等器官的先进保存技术。最后，本文展望了器官保存领域的未来发展方向，旨在实现长期高效的器官保存，从而应对全球日益严峻的器官短缺问题。

Abstract

Recent advances in organ transplantation, regenerative medicine, and drug discovery have emphasized the critical importance of effective preservation techniques for organs. Despite these advances, current preservation techniques have significant limitations in maintaining the viability and functional efficacy of organs over the long term. As a result, there is a pressing need to develop reliable and efficient preservation strategies for organs. Currently, the clinical standard for organ preservation involves the use of static cold storage and organ machine perfusion, but these methods can only preserve organs for a couple of days or even a few hours. Notably, the development of cryobiology has yielded promising alternatives. In this review, we aim to provide a comprehensive overview of the progression of organ preservation methods, while emphasizing the limitations of traditional approaches. Moreover, we evaluate advanced preservation techniques for organs, including kidneys, livers, hearts, lungs, and intestines. Furthermore, we share a progress perspective on the future of organ preservation, with the ultimate goal of achieving viable long-term preservation to address the pressing issue of organ shortage.

关键词

器官保存 / 器官机械灌注 / 静态冷保存 / 玻璃化 / 保存液

Key words

Organ preservation / Organ machine perfusion / Static cold storage / Vitrification / Preservation solution

引用本文

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刘欣萌,舒志全,张黎明,李皓玥,杨静,张雷. 器官保存——历史、发展与展望[J]. 工程（英文）, 2025, 44(1): 120-141 DOI:10.1016/j.eng.2024.12.020

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1 引言

器官移植作为终末期器官衰竭的核心治疗手段，其临床应用正面临供体短缺的严峻挑战[1]。当前临床采用的器官保存技术存在时效限制，导致器官转运、评估及移植环节存在多重瓶颈，这一现状严重制约着移植医学与再生医学的协同发展。

目前临床主流保存技术包含静态冷保存（SCS）与机械灌注（MP）两类[2‒3]。其中SCS技术可将肾脏、肝脏等器官的保存时间延长至24 h左右，而MP技术通过抑制缺血再灌注损伤（IRI）可将保存时限进一步延长至数日[4‒6]。然而，现有技术体系在实现器官长期高质量保存方面仍存在显著局限性。

近年来，低温保存技术的突破为生物样本长期存储提供了新路径[7‒8]。理论上，当保存温度低于-140 ℃时，器官将进入代谢近乎停滞的“休眠”状态，有望实现生理结构的永久保存[9]。但该技术在实际应用中面临多重挑战，如冷冻/复温过程中产生的机械应力、渗透压波动及热应力积累易导致器官不可逆损伤。针对这些技术瓶颈，研究人员致力于从保存方案优化、新型冻存保护剂（CPAs）开发、快速复温技术探索等维度进行系统性研究[4,10‒14]。

本文系统梳理了器官保存的临床需求与技术发展脉络，深入解析传统保存技术的局限性，论证低温保存技术解决器官短缺问题的可行性。通过调研肾脏、肝脏、心脏等主要移植器官的低温保存研究进展，重点讨论了不同器官保存的特异性技术难点。最后，从跨学科技术融合角度展望器官长期保存的发展方向，为突破器官移植时空限制提供理论支撑。

2 器官保存的需求

器官移植作为终末期器官衰竭最有效的治疗手段，每年挽救了全球数十万患者的生命。其公共卫生效益与癌症治愈相当，但供体的全球性短缺已成为制约医学发展的重大挑战[15]。据世界卫生组织统计，当前移植需求满足率不足10% [16‒17]。新型冠状病毒肺炎（COVID-19）疫情加剧了这一供需失衡，全球成功移植案例从2019年的15万例骤降约18%，至2022年才恢复至15.75万例[18‒21]。以美国为例，2022年全美约11万等待移植患者中仅有4万余例成功获得供体[图1（a）和（b）]，平均每日约20例患者在等待过程中死亡[22‒23]。

值得关注的是，现行器官保存技术的时间局限性加剧了器官资源浪费。2020年统计数据显示，肾脏、肝脏和心脏的弃用率分别达24%、29%和58%，而肺与肠道器官的弃用率更攀升至72%和98% [24‒26]。其核心矛盾在于当前临床保存技术的时间窗口仅以小时计算[27]，导致器官转运时效受限、跨区域分配困难，同时供受体匹配的黄金时间过短[1]。此外，供体摘除后需依赖24 h应急转运体系，这也显著增加了医疗成本[28]。

器官离体后的持续代谢活动会引发双重病理机制：一方面，三磷酸腺苷（ATP）的快速耗竭与代谢物蓄积导致能量危机；另一方面，氧化应激与炎症反应触发IRI [29‒31]。这些级联反应不仅降低移植器官的功能恢复效率，更是导致移植失败的主要诱因。因此，建立能够实现器官长期高质量保存的技术体系，对降低器官弃用率、提升移植成功率具有重大临床意义。

3 冻存保护剂的发展历史

器官保存技术经过数十年研究已取得阶段性进展。1949年，甘油作为首个CPA成功应用于禽类精子保存[32]。随后Lovelock等[33]在牛精子低温保存研究中发现二甲基亚砜（DMSO）具有显著保护效应，但后续研究表明当浓度超过2 wt%时，该物质可能引发遗传突变及小鼠受精卵表观遗传修饰异常[34]。由于器官组织结构的复杂性和细胞间连接对有机小分子的高度敏感性，传统有机CPAs难以满足保存需求。在此背景下，科研人员通过多组分协同作用的创新思路，成功开发出多种新型器官保存液（表1 [35‒40]）。

1966年，Keeler研究团队[41]通过生理盐水灌注大鼠肾脏实验，首次揭示组织损伤与K⁺、Mg²⁺等离子流失的关联性，为CPAs的开发奠定了理论基础。基于此，Collins等[35]于1969年成功研制首款商用保存液（Collins液），不仅实现犬肾30 h保存，更被拓展应用于心脏、肝脏、肺等多器官保存[42]。但该溶液仍存在保存时间短、移植后器官功能延迟恢复等问题。1976年推出的Euro-Collins（EC）液作为低温肾脏灌注液，主导临床应用达15年，直至UW（University of Wisconsin）溶液的问世[36‒37,43‒45]。

1986年开发的UW液因含乳糖酸和棉子糖等非渗透性物质，可有效抑制组织水肿，成为腹内器官保存的“金标准”[37,46‒47]。2020年，Que等[48]将UW液与-8 ℃过冷保存技术相结合，成功实现小鼠心脏保存时间延长至144 h，并在96 h保存后成功移植，标志着保存技术的重大突破。

20世纪80年代，Bretschneider等[49‒50]开发了以低钾、低黏度为特点的组氨酸-色氨酸-酮戊二酸（HTK）液，其组氨酸缓冲系统可维持细胞外环境稳定，酮戊二酸促进ATP储存，甘露醇则抑制细胞肿胀和自由基损伤[38,51]。HTK液凭借优异的流动性优势，在肝肾移植领域获得广泛应用，目前与UW液共同占据美国95%的器官保存市场份额[52]。为优化心脏保护效果，HTK-N液通过添加甘氨酸、丙氨酸等氨基酸及铁螯合剂，抑制缺氧性膜孔形成和冷损伤[39,53‒54]。临床研究表明，HTK-N液可有效改善心脏保存后的收缩功能[39]，并降低冠状动脉搭桥术中的缺血再灌注损伤[55]。

IGL-1（Institute Georges Lopez）液创新性地以聚乙二醇（PEG）替代UW液的羟乙基淀粉[56‒57]，利用其高渗透压特性与生物惰性优势[58]，减少分化簇（CD）4⁺/CD8⁺炎性T细胞浸润[59‒60]，同时降低脂质过氧化等氧化应激损伤[60‒62]。

而Celsior液则通过复合谷胱甘肽、甘露醇、乳糖酸盐及组氨酸实现多机制保护：渗透调节控制细胞肿胀，谷胱甘肽清除自由基，高镁含量拮抗钙超载[40]。研究显示其对肝细胞保护优于HTK/UW液[63]，在胰腺和肾脏移植中与UW液效能相当[64‒65]，且能显著降低延迟移植肾功能（DGF）发生率[44]。

4 低温保存

器官移植前的完全缺血缺氧状态会破坏细胞内外离子与渗透压平衡调节系统[66]，此时ATP依赖型跨膜酶复合体因能量供应中断而失活，导致细胞膜去极化及电压门控钙通道异常开放，引发钙离子内流激增[67]。这种细胞内钙超载会进一步激活钙依赖性磷脂酶和蛋白酶，形成细胞膜损伤-钙内流恶性循环，最终引发不可逆的细胞肿胀和死亡[68]。

针对这一过程，SCS技术通过4 ℃低温环境抑制代谢、降低ATP酶能耗、维持跨膜电化学梯度并阻断凋亡通路激活，目前已成为临床主流保存方案[69‒72]。

SCS的主要作用机制是通过降低ATP的利用率和氧消耗来减少细胞代谢和缺血性损伤[75‒76]，该技术采用专用保存液（如UW溶液）灌洗器官后冰浴贮存[70,73‒74]，如图2（a）所示。在日本，SCS是肾脏保存的唯一指定技术[77]。其实现了肾脏12~24 h的保存，肺和心脏的临床安全时限则分别为6~8 h和4~6 h [73,78]。近年来，SCS技术不断优化，2019年一项研究通过在UW液中加入线粒体靶向硫化氢供体，增强了其抗凋亡与抗氧化性能[79]；2021年临床数据显示，心脏SCS保存时间达到283 min，缺血时长延长至330 min [80]。

然而，4 ℃低温环境仍无法完全避免ATP耗竭与代谢物蓄积的问题，这会导致IRI及移植物延迟功能恢复（DGF）。这些局限性促使机械灌注（MP）等新技术的研发与发展[29‒30]。

5 MP

MP技术的理论基础可追溯至1813年Cesar Le Gallois 提出的体外循环概念，即通过动脉血灌注维持器官活性[81]。19世纪60年代，Bernard等[82‒83]确立了MP的基本原理。1963年，Marchioro等[84]首次实现犬器官在自然血流停止后的体外灌注。之后肾脏可在12~15 ℃低温条件下离体保存[85]，成功维持器官功能达6 h，肝脏可维持器官2 h。这些突破促使研究者提出低温可降低细胞代谢，从而减轻器官保存损伤的假说。20世纪60年代，多项研究聚焦于低温条件下稀释血清或肝素化血液对肾脏保存的影响[86‒87]。这些研究表明，采用低温机械灌注（HMP）技术可将保存时间从数小时延长至数天。HMP的技术原理及设备如图2（b）和（d）[70‒71]所示。1967年，持续灌注和低温储存的结合将器官保存时长提高到一个新的水平，Belzer等[88]将8~12 ℃脉动式氧合血浆灌注技术应用于犬肾保存，成功实现72 h的有效保存[88]。HMP可维持线粒体电子传递能力并提高存活率[71,89]，2016年，荷兰将其列为死亡供肾的标准方案，但仍面临血管痉挛等技术瓶颈[5,90]。

常温机械灌注（NMP）技术在约38 ℃生理环境下，通过含氧灌流液实现器官保护[70]，如图2（c）所示。欧洲肝移植临床试验显示，与SCS组相比，NMP组患者生存率显著提高[72]，如图2（e）所示。实验结果显示，NMP组血清中天门冬氨酸转氨酶峰值降低50%，但仍存在术后胆道狭窄等并发症[91]。最新临床数据表明，NMP技术可缩短43%的住院时间，并显著降低移植肝无功能风险[92]。目前，便携式灌注设备已成功应用于心脏、肺、肾脏等器官的转运过程，实现实时功能监测与评估[93‒96]。

6 过冷保存

过冷保存是一种将温度维持在冰点以下而不形成冰晶的保存技术，如图3（a）[97]所示。该技术的核心原理基于水的过冷却特性，即当温度低于冰点但未触发成核过程时，液态水可维持亚稳态[97]。自然界中，鱼类和昆虫等生物通过合成碳水化合物来稳定细胞膜，并分泌冰晶抑制剂以阻止晶体生长，这一现象为过冷保存技术提供了重要启示[98‒100]。

1996年，Scotte等[101]在UW液中添加2,3-丁二醇，成功在-4 ℃条件下保存了大鼠肝脏。研究发现，肝脏在0 ℃以下保存具有可行性，但需要通过移植模型进行效果评估。后续研究证实了器官过冷保存的优势，即与静态低温保存（SCS）相比，大鼠肺在过冷保存条件下可显著降低IRI [102]。2015年，Bruinsma等[103]将离体灌注与过冷保存技术相结合，成功将大鼠肝脏保存时间延长至4天。2019年，de Vries等[4]通过气液相密封、UW液冰点降低以及灌注方法优化等创新技术，突破了大体积器官（如人肝脏）的低温保存瓶颈，如图3（b）和（c）。

近期，Rubinsky等[104]开创了一种用于生物样本的等容保存方法。该方法将生物样品储存在刚性容器中以保持恒定体积。该技术的独特之处在于过冷过程中形成的低密度冰晶会提升系统压力，从而通过热力学约束有效抑制冰晶生长[105‒108]。2021年，Powell-Palm等[109]利用等容低温保存技术，成功实现人源心肌微组织的无CPA保存，并保留了心肌细胞的自主收缩功能。此后，Năstase等[110‒111]在猪肝脏中实现了48 h无CPA的等容保存，但该技术尚未完成移植验证。

7 部分冷冻保存

过冷保存技术通常采用-6~-4 ℃的温度区间，但为了进一步实现代谢抑制效果，需要探索更低的保存温度。近期，一种采用部分冷冻保存的策略被开发出来，该策略通过维持足够的未冻部分，在促进热力学稳定的同时减轻冰损伤和过度脱水[112]。这项技术的研发灵感来源于自然界中的耐冻生物，Da Silveira Cavalcante等[113]通过-10 ℃冷冻保存斑马鱼心脏的实验，证实冻存后的心脏仍能保持基础心功能与循环指标，并进一步在啮齿类动物的肝脏与心脏中实现了-15 ℃下保存5天的突破。2022年的最新研究通过创新性地结合冰核剂与CPAs，首次实现了人肝脏在-15~-10 ℃条件下的部分冷冻保存，将保存时限延长至5天[114]。

8 长期超低温冷冻保存

器官保存时效的延长显著提升了器官评估、分配及移植的整体效率[1]。其核心优势主要体现在：将紧急移植手术转变为择期手术[115]，既降低医疗成本又提高人类白细胞抗原（HLA）配型精度[116]；突破供受体之间的地理限制；显著减少器官废弃率[22]。

器官超低温保存被视为该领域的终极目标，理论上在液氮温度（-196 ℃）条件下可实现生理代谢完全停滞[117‒118]。在现有技术中，程序化慢速冷冻与玻璃化冷冻[图4（a）]已在细胞和组织保存方面取得重要突破[119]，二者的热力学路径差异如图4（b）[8]所示。下文将重点分析实现器官长期超低温冷冻保存的关键技术要素。

8.1 玻璃化冷冻保存

玻璃化冷冻被视为器官长期保存领域最具前景的技术，其核心优势在于完全避免了传统冷冻过程中的冰晶损伤问题。该技术的发展可追溯至20世纪30年代，研究人员通过快速冷冻实现了蛙类精子、苔藓和果蝇胚胎等生物材料的玻璃化保存[120‒125]。超快冷却速率下的玻璃化对小体积样品效果显著。Fahy等[126]开创性地提出了新思路，即利用高浓度玻璃化溶液置换器官内部分水分。这类溶液通过增强水分子间氢键作用，显著提高介质黏度，从而有效抑制冰晶形成，在快速降温或复温过程中为细胞提供保护[127]。这使得在较低冷却速率下实现玻璃化成为可能。随着技术不断进步，2000年成功实现动脉血管的玻璃化保存[128‒129]。目前，该技术已成为临床卵母细胞和胚胎冷冻的首选方法，并有望突破器官冷冻保存的技术瓶颈[119,130‒132]。

实现玻璃化通常需要高浓度渗透性CPAs [如DMSO、甘油、乙二醇（EG）、丙二醇]与非渗透性CPAs（糖类、聚合物等）的协同作用[133]。Faltus等[134]于2021年系统研究了EG、DMSO、甘油等物质的玻璃化能力，为CPAs的设计提供理论依据。然而该技术仍面临两大挑战：高浓度CPAs的细胞毒性问题[图4（c）]与复温过程中冰核形成导致的脱玻璃化损伤[8,135‒137]。

近年来，研究人员提出了多项创新性解决方案：等容保存技术可显著降低CPAs需求[138‒139]；梯度降温灌注技术有效减轻毒性影响[140]；磁性纳米颗粒结合交变磁场实现快速均匀复温，抑制冰晶再生并防止器官破裂[12,141]。

8.2 血管灌注

通常，仅靠局部暴露和扩散无法实现CPAs向离体器官内部的渗透。将CPAs灌注到血管网络是一种潜在有效的方法[142]。该技术能动态提高器官内CPAs浓度，并且由于灌注过程中器官的代谢活性，允许细胞主动摄取CPAs [66]。实践表明，采用HMP技术已成功延长人肝脏的离体保存时间[4]。最新研究通过灌注磁性纳米颗粒，进一步提高了心脏、肾脏等器官的复温效率[119,132]。

用于CPAs灌注的载体溶液成分至关重要，需要兼顾CPAs的细胞毒性与渗透压变化的影响。CPAs跨膜转运引发的细胞体积变化会改变灌注阻力和器官重量，甚至造成血管损伤[66]。通过选择特定灌注剂、添加渗透缓冲液及低温灌注可部分缓解此类损伤。Karow等[143]通过系统研究DMSO在不同温度下的渗透动力学特性，为冻存策略优化提供了重要依据。

8.3 毒性

高浓度CPAs引发的毒性问题是玻璃化冷冻技术面临的核心挑战，需要深入理解其作用机制并开发相应减毒方案。

目前研究发现，细胞和组织对CPAs中某些成分表现出不同的敏感性，导致特定的毒性。例如，EG的毒性与器官代谢途径相关，而非冷冻保存过程本身[144]。EG在肝脏中通过醛脱氢酶代谢，形成羟乙醛[145‒146]。随后，羟乙醛被醛脱氢酶进一步代谢生成羟基乙酸[147]。此代谢过程可能导致代谢性酸中毒。此外，羟基乙酸可在酶促作用下转化为草酸。在钙离子存在下，草酸可形成草酸钙晶体，进而导致胃肠道刺激、肺水肿和广泛的肺部系统性炎症[148‒149]。

甘油的使用是精子冷冻保存领域的重大突破[32]。然而，它与精子形态、线粒体和活力损伤等副作用相关[150]。此外，当甘油被肾脏中的半胱天冬酶代谢时，会消耗还原型谷胱甘肽[151‒152]。这种消耗进而引发氧化应激、炎症和细胞凋亡，导致肾衰竭[153]。

在渗透性CPAs中，DMSO被认为是细胞的理想CPA。然而，在30 °C下对大鼠的研究发现，浓度超过30%的DMSO会导致心肌超微结构的不可逆改变[154]。将豚鼠心肌暴露于10% DMSO中30 min（室温），会导致与心肌细胞收缩相关的电位持续升高[155]。此效应可能源于DMSO分子与蛋白质的不可逆结合，导致蛋白质构象破坏和膜通道蛋白阻断[156‒158]。DMSO也会影响细胞膜特性[159]。研究发现，细胞在-77 °C下暴露于低浓度DMSO（2.5%~7.5%）会降低细胞膜厚度[160]；在中等浓度（10%~20%）下，DMSO诱导瞬时水孔形成；而在较高浓度（25%~30%）下，还可能导致膜破裂。DMSO还能影响线粒体呼吸和细胞内钙水平[161]。暴露于1% DMSO的成纤维细胞表现出细胞内钙水平快速升高，导致细胞凋亡[162‒163]。

为了降低传统CPAs的毒性，研究人员开发了新型冷冻保护技术。Sui等[164]利用两性离子甜菜碱复合物替代传统CPAs，使红细胞冻存后存活率大于80%；其团队还探索了两性离子聚合物在冷冻保存中的潜力，在冷冻保存的软骨细胞、个旧肺腺癌-82（GLC-82）细胞和HeLa细胞中实现了90%的细胞存活率[165‒166]，如图4（d）所示。Zhu等[167]采用金属有机框架纳米颗粒冷冻红细胞，实现了存活率大于40%。但此类技术在大体积器官中的应用仍需进一步验证。

8.4 玻璃化溶液的加载与卸载

玻璃化冷冻需要高浓度CPAs以防止冰晶形成，但高浓度CPAs可能引发细胞毒性和渗透压损伤。传统多步骤加载/卸载CPAs的流程耗时且复杂[168]。为此，微流控装置应运而生，通过利用层流和精准分子扩散原理，该装置可优化CPAs加载/卸载速度，减少长时间暴露的细胞毒性，同时降低渗透压引起的细胞体积变化[169‒170]。2022年，Zhan等[10]开发了一种高通量、高存活率的胰岛玻璃化冷冻平台，通过程序化控制CPAs加载/卸载流程，显著减少渗透损伤，为胰岛保存技术提供了新方向。

8.5 冷却过程

玻璃化冷冻成功的关键在于实现快速冷却[119]，如图5（a）~（b）所示。其核心原理是当冷却速率达到临界值时，水分子动能降低至无法形成规则冰晶的阈值，从而实现无定形态的玻璃化转变[171‒172]。临界冷却速率（CCR）主要取决于CPAs的配方和浓度，较低的CPAs浓度需要较高的CCR来抑止冰晶形成。例如，DP6（6 mol∙L^-1）、VS55（8.4 mol∙L^-1）和M22（9.3 mol∙L^-1）的CCR分别为40 °C∙min^-1、2.5 °C∙min^-1和0.1 °C∙min^-1 [173‒176]。

基于Johnson-Mehl-Avrami（JMA）动力学理论，可通过结晶速率评估玻璃化能力[177‒180]。Uhlmann等[181‒182]提出利用时间-温度-转变（TTT）曲线确定CCR，但该模型仅适用于等温条件。MacFarlane [183]通过连续冷却转变（CCT）曲线改进非等温结晶过程的CCR预测，但因低温下成核速率等参数难以获取，实际应用仍受限。

Boutron模型提出半经验结晶动力学公式（式1），表明冰晶生成量与冷却速率呈S型关系[184‒187]，如图5（c）所示。

k 4 v = - l n 1 - x 1 / 3 + 12 l n 1 + x 1 / 3 + x 2 / 3 + 3 t a n - 1 3 x 1 / 3 2 + x 1 / 3

（1）

式中，x为冰晶生成比例（0 ≤ x ≤1）；v为冷却速率；k₄为常数。当x = 0.2时，冰晶量可忽略，对应速率即为CCR [188]。

8.6 复温过程

玻璃化溶液在复温过程中面临的主要挑战是脱玻璃化现象[187,189]，如图5（d）所示。因此临界复温速率（CWR）通常比CCR高1~2个数量级[190‒191]。CWR被定义为脱玻璃化可忽略的最小速率，如DP6的CWR为189 °C∙min^-1 [187]。复温终点温度需略高于熔点以避免热损伤[10,192]。为满足样本快速均匀复温的需求，目前已开发出多项创新技术，如纳米加热与射频、电磁加热及高强度聚焦超声（HIFU）[193]。

8.6.1 纳米加热与射频技术

近年来，纳米材料的应用为生物样本冷冻保存复温技术开辟了新途径。基于磁热与光热效应的纳米加热技术，相比传统热传导方法，能够实现更快速、更均匀的复温过程[194]。以碳黑、金纳米棒和液态金属纳米颗粒为代表的光热材料，凭借其优异的光热转换性能，可实现高达107 °C∙min^-1的复温速率，在小体积样本复温中展现出显著优势[195‒198]。然而，这类材料受限于激光穿透深度，难以满足大体积样本的复温需求。磁性纳米材料为解决大样本复温难题提供了新思路。氧化铁纳米颗粒等材料在交变磁场作用下，不仅能显著提升加热速率，还能有效消除温度梯度[12,132]，减少热应力损伤[132]，如图6所示。2017年，Manuchehrabadi等[12]将射频激活的介孔二氧化硅包覆氧化铁纳米颗粒应用于80 mL体系的玻璃化冷冻。此后，该技术被成功拓展至肾脏和心脏等器官的冷冻保存[119,132,141,199‒200]，并在2023年实现了大鼠肾脏长期玻璃化保存及移植成功的重要突破[192]。这些研究成果充分证明了纳米加热技术在器官冷冻保存领域的巨大潜力，为复杂器官的长期保存提供了新的技术路径。

8.6.2 磁热复温

虽然纳米粒子的磁热复温技术为大器官冷冻保存提供了技术支持，但磁性纳米颗粒渗入细胞可能导致的毒性问题仍需解决。针对这一问题，研究人员基于麦克斯韦介电加热理论开发了单模电磁共振（SMER）技术[201]，该技术通过高频电磁场使生物体内水分子摩擦生热，无需引入纳米材料即可在1 s内实现120 ℃的快速均匀升温[13,202‒203]。目前，这项技术已成功用于大体积细胞和动脉血管的玻璃化冷冻复温[204‒205]。

8.6.3 高强度聚焦超声

HIFU技术利用压电效应将电能转化为机械振动，通过产生聚焦压力波实现局部加热[206‒207]。这项已在临床成熟应用于温热组织治疗的技术，近年来被拓展至冷冻复温领域[208‒209]。2023年，Alcalá等[210]利用HIFU技术复温-80 ℃冷冻的秀丽隐杆线虫，可通过超声衰减特性有效抑制了冰晶的重结晶[211‒212]；2024年，Encabo等[213]进一步实现了-6 ℃冷冻小鼠心脏的功能性恢复。相比受线圈位置限制的电磁复温技术，HIFU具有更高的空间灵活性，但其在深低温大器官复温中的应用可行性仍有待系统验证。

9 器官保存质量评估

器官保存质量的评估需要从形态、生理和功能等多个维度进行综合验证，评估结果对于确保器官的质量和适用性起着关键作用，直接影响后续移植手术的成功率以及患者的生存率。建立完善的评估体系能够有效提升保存质量控制水平，为移植患者提供更好的安全保障。

9.1 CPAs加载/卸载评估

CPAs加载/卸载评估主要基于质量传递模型和毒性模型两大理论体系。在质量传递模型方面，包括Krogh圆柱模型[214]、多维模型[215]和网络热力学模型[216]等；在毒性评估方面，则涵盖溶液效应模型[217‒219]、CPA相互作用模型[220]和毒性成本函数模型[184]等。2023年Han团队[186]通过灌注实验推导出Boyle-van’t Hoff方程，将Krogh模型与毒性成本函数相结合，优化了CPA加载/卸载策略。基于Kedem-Katchalsky方程描述毛细血管膜物质传输过程如下：

J v = S · L p P f - P t - R g T C i s, f - C i s, t + σ C c p a, f - C c p a, t

（2）

J c p a = S ω R g T C c p a, f - C c p a, t + J v 1 - σ C c p a, f + C c p a, t 2

（3）

式中，J_v为总流量，包含水和CPA；J_cpa为CPA流量；S为传输表面积；L_p为膜导水性；P代表作用于膜上的液压压力；R_g为通用气体常数；T代表系统温度；C表示物质浓度。ω代表膜对CPA的渗透性，σ为反射系数，两者均与膜的特性相关。下标“t”和“f”分别代表组织与毛细血管内浓度，下标“cpa”和“is”分别代表低温保护剂总浓度及不可渗透溶质（如糖类、糖醇类及聚合物）的浓度。

毒性成本函数（J_tox）模型量化CPA浓度、暴露时间（t）和温度的关系[184]：

k = β · C c p a, t α d N d t = - k · N J t o x = ∫ 0 t f k d t = ∫ 0 t f β · C c p a, t α d t N N 0 = e x p (- J t o x)

（4）

式中，k为毒性比率；常数α、β为CPA特异性参数；C_cpa为组织内CPAs浓度；N₀为组织初始活性；N为活细胞数量；t_f为CPA暴露时长。

9.2 玻璃化/结晶评估

在实际评估中，通常采用视觉观察和热力学分析两种方法。视觉评估主要依据透明度差异进行判断，完全玻璃化的器官呈现均匀透明状态，而脱玻璃化区域则因冰晶生长而呈现浑浊特征[12,119,132]，如图4（a）所示。热力学分析则采用差示扫描量热法（DSC）监测相变特征，当冷却速率低于CCR时会出现结晶峰[187,221]，而复温速率未达到CWR时则会出现去玻璃化峰和冰晶再生峰[图5（d）]。当冷却和复温速率均达到临界阈值后，这些特征峰完全消失，证明实现了理想的无冰晶相变过程。

9.3 器官活力与功能评估

在当前临床实践中，由于供体器官质量风险的增加，大多数器官被归类为“边缘性器官”，这使得器官功能质量的评估和预测尤为重要。冷冻保存后器官的活力和功能性评价是判断保存质量的关键指标，当前研究集中于开发创新的体外和体内评估方法，以提高器官筛选精度和移植成功率。

9.3.1 体外评估

体外评估主要聚焦于细胞活力、组织结构和生物力学性能三个维度。在生物学中，活力涵盖了一系列专门用于单个细胞或生物体生存、生长和发育的细胞功能[222]。细胞功能被认为是活力状态的关键，包括细胞形态的完整性、具有功能完整性的完整膜屏障、维持生命功能和酶活性的能量产生、DNA转录和RNA翻译过程、有效pH梯度的维持以及细胞呼吸和繁殖能力。细胞活力的评估情况是器官活性的指示。2021年，Sharma等[119]测试了玻璃化冷冻保存后大鼠肾脏的活力，发现其表现出比传统冷冻组具有更高的活性。2023年，Lau等[223]研究了NMP对人肝脏保存的效果，他们的研究侧重于评估肝脏的乳酸清除能力、胆汁生成能力以及因子V合成和ATP的储存能力，证明这种保存技术有效延长了肝脏的保存时间，维持时间长达7天。

组织结构评估通常采用苏木精-伊红（H&E）染色技术，Gao等[132]通过该方法证明低温保存后的心脏组织仍能保持完整的肌纤维分支结构。在生物力学性能评估方面，血管拉伸模量等指标能有效反映器官功能状态，Manuchehrabadi等[12]的研究显示玻璃化保存的猪血管的力学性能与新鲜样本无显著差异[12]。

9.3.2 体内评估

体内评估则主要依赖于移植后的器官功能恢复情况。对于肝脏移植，临床常用的评估指标包括天冬氨酸氨基转移酶（AST）等酶释放量和凝血因子合成能力等[72]。Berendsen等[224]开发的超低温联合机械灌注技术能有效维持肝脏功能，使移植后转氨酶等水平恢复正常。2018年的一项随机试验评估了用于肝移植的NMP [72]。该研究通过肝细胞酶释放减少证明，与SCS相比，NMP使移植物损伤减少了50%。肾脏功能的评估则主要依赖血清肌酐和尿素氮浓度等生化指标[3]，研究表明UW和HTK等保存液能较好地维持肾小管功能。此外，Wilson等[225]开发的基因表达分析技术通过解析供受体基因表达谱，为肺移植评估提供了新的技术途径。

10 器官保存策略

器官保存需要根据器官的细胞组成和功能特性差异化采用针对性的保存方法。温度控制是保存过程中的核心要素，低温保存（包括冷保存、过冷保存和冷冻保存）通过抑制分子热运动减缓代谢速率，但需要权衡低温损伤的风险。此外，CPAs的选择与剂量优化同样至关重要，需要在抑制冻存损伤效果与毒性控制之间取得平衡。以下将重点探讨各种主要器官保存技术的最新进展。

10.1 肾脏保存

Collins液、UW液和HTK液等保存液技术的发展，使得SCS已成为当前肾脏保存的临床标准方案[226‒228]。SCS技术可实现肾脏的24~36 h离体保存[229‒231]，主要通过低温抑制细胞代谢来维持器官功能[232]。然而，长时间的SCS保存会导致组织缺氧、酸中毒以及肾小管细胞坏死等问题[233]，同时还会缩短供受体匹配时间，影响手术准备[1,229]，最终降低器官活性和患者生存率[234]。

为了突破SCS的技术局限，MP技术通过持续供氧和营养补充来修复肾脏能量储备，逆转热缺血损伤造成的ATP耗竭[235‒238]。1973年，Sacks团队[239]首次采用高渗电解液作为CPAs，成功实现犬肾72 h的HMP保存，且移植后1周血清肌酐水平恢复正常。2005年，Monzen等 [240]通过HMP技术降低器官代谢率，同样实现了72 h的肾脏保存。

玻璃化冷冻技术为器官长期保存提供了新方向[175]。在一项初步的概念验证研究中，兔肾经M22溶液灌注后成功实现9天的冷冻保存并完成移植[241]，后续研究表明VS4溶液的保存效果优于VS41A [242]，如图7（a）所示。2021年，Sharma等[119]结合纳米粒子磁热复温技术成功实现肾脏的玻璃化冷冻，并通过显微结构分析证明该技术的可行性[图7（b）]。2023年，该团队进一步突破技术瓶颈，将大鼠肾脏玻璃化冷冻保存100天后成功移植，术后血清肌酐、静脉pH值等关键指标均恢复正常[图7（c）~（d）]，这些开创性的研究为器官长期保存奠定了重要的技术基础[192]。

10.2 肝脏保存

肝脏作为一个依赖持续氧气和能量供应的重要器官，在保存过程中极易遭受IRI的影响，导致大量待移植肝脏被弃用[243‒245]。在缺血过程中，肝细胞损伤会激活Kupffer细胞，进而引发炎症因子级联释放和氧化应激反应[246‒248]。

当前临床实践中，SCS因其操作简捷仍是肝脏保存的主流方案[1,249]。然而健康供肝在SCS条件下的最佳保存时间仅为12~18 h。低温缺氧环境会导致肝细胞ATP快速耗竭，并产生大量黄嘌呤代谢产物[250]，这些变化会显著损伤肝窦内皮细胞功能，使移植物功能障碍的发生风险升高至25%~44% [91,251‒253]。针对这一临床挑战，近年来MP技术在肝脏保存领域的应用受到广泛关注[254]。

目前，NMP技术的灌注时间有限，约为9 h，这限制了对肝脏功能的全面评估[72]。为解决这一问题，Eshmuminov等[255]开发了集成式NMP系统，成功将人肝脏灌注时间延长至24 h，并实现长达1周的保存[图8（a）~（b）]。2022年，Clavien等[256]取得重要突破，成功移植了经过3天非原位灌注保存的肝脏[图8（c）]，这项研究为将肝脏保存窗口延长至10天提供了可能性。Tessier等[114]从耐冻动物的生理机制获得启发，结合冰核剂与CPAs来维持液态组分，使大鼠肝脏保存时间达到5天[图8（d）]，但该技术在大型动物模型中的验证仍需进一步深入研究。

10.3 心脏保存

当前心脏移植临床保存主要采用UW液、EC液等SCS方案[257]，然而此类方法存在明显的局限性，包括ATP过度消耗及酸中毒等问题[258‒260]。这些问题会导致细胞凋亡、坏死以及IRI，显著增加移植物功能障碍风险[261‒262]。目前SCS方案的心脏保存时间仅限于4~6 h [263]，而更长时间的保存会加剧冠状动脉内皮损伤，可能引发移植后血管病变甚至移植物衰竭[264]。因此，如何减轻IRI、有效保护心肌细胞成为心脏保存研究的核心目标[265]。近年来，MP技术通过氧合营养液持续灌注，为降低心脏IRI提供了新的解决方案[266‒269]。

早在1968年，研究人员首次对离体犬心实施MP保存，保存时间达到72 h [270]，但移植后心脏仅存活6 h。Wicomb等 [271]实现了人心脏MP保存6~15 h，其中一例移植心脏存活时间长达16个月。McLeod团队[272]通过NMP评估了羊心功能及氧代谢指标，成功实现72 h的保存[图9（a）~（b）]。研究人员采用改良UW液[Columbia University（CU）液]的方案，使大鼠和狒狒心脏保存时间延长至24 h，且狒狒移植后实现长期存活[273]。2019年Kaliyev等[274]通过NMP技术将人心脏保存时间进一步延长至16 h，其开发的器官维护系统（OCS）还能安全评估边缘供体[图9（c）]，为心脏保存开辟了新方向[269,272,275‒276]。除灌注技术外，玻璃化冷冻联合磁性纳米颗粒复温技术也展现出良好前景。Chiu-Lam等[141]利用VS55保存液结合磁性纳米颗粒成功实现心脏保存1周[图9（d）]，但该技术在移植中的实际效果仍需更多实验验证。

10.4 肺保存

当前临床肺移植主要采用SCS技术，其保存时间通常仅有4~8 h [277‒279]。虽然低温环境能够抑制代谢活动，但会显著降低钠钾泵活性，导致细胞内钠、钙离子积累及细胞水肿[68,280‒282]。在这个保存过程中，无氧代谢产生的乳酸会加重组织酸中毒[283]，而钙离子超载则会促使线粒体释放细胞色素C，激活凋亡酶[284‒285]，最终引发细胞功能障碍[286]。

离体肺灌注（EVLP）技术的出现为改善肺保存条件提供了新的解决方案。2001年，Steen等[287]首次报道了EVLP技术的应用，而后，研究人员逐步对EVLP进行优化，该系统如图10（a）[288]所示。随后2008年多伦多团队引入NMP技术，开发出Toronto系统[289]。目前临床上主要采用三种EVLP系统：Toronto、Lund和OCS [290‒291]。其中，Toronto系统采用无细胞Steen灌注液[292‒294]，Lund系统则采用匹配全心输出量的灌注方案，而OCS系统具有便携运输优势，能显著缩短缺血时间，降低严重移植物功能障碍风险[296]。值得一提的是，左心房开放设计可以避免微血管塌陷，从而改善移植预后[295]。

在冷冻保存技术方面，虽然全肺冷冻保存的研究相对较少，但已展现出突破时间限制的潜力[297‒301]。Okamoto等[102]采用-2 ℃过冷保存大鼠肺17 h，其功能与新鲜肺组织相当[图10（b）]。Ali团队[302]通过EVLP技术成功保存人肺10~16 h并完成5例移植，证实该技术能有效维持肺组织的代谢和线粒体功能[图10（c）~（d）]。虽然玻璃化或部分冷冻技术在肺保存中展现出应用前景，但其在全肺保存中的应用仍需进一步深入研究。

10.5 肠道保存

肠道保存技术是决定移植成功与否的关键因素。目前临床小肠保存主要采用SCS结合UW液、HTK液等保存液的技术方案[303‒304]，但该方法的最大保存时间仅为6~8 h [305‒307]。由于小肠是人体内细菌和内毒素最主要的储存场所，其黏膜屏障极易受到IRI的影响，这种损伤会导致微生物移位和内毒素入血[247,309]，进而引发受体免疫排斥反应和菌血症等严重并发症[310‒311]。

为了降低损伤风险，研究人员开始探索MP技术，通过持续输送代谢底物并清除代谢废物来改善保存效果[312]。虽然相关研究相对较少，但已取得一些突破性进展：2003年Zhu等[313]采用UW液灌注法保存小肠，发现能显著改善黏膜完整性；2016年耶鲁大学Munoz-Abraham等[314]首次提出血管-管腔双通路HMP技术用于人小肠保存，其组织病理学结果明显优于传统SCS方法；近年来，NMP技术也备受关注，2021年Ludwig等[315]利用NMP保存猪全长小肠，发现其细胞增殖能力和活性均优于冷保存方法，这表明NMP可能有助于提升移植后小肠的再生能力。目前关于小肠低温冷冻保存的研究尚未见报道。考虑到小肠的特殊生理结构和功能特点，未来需要针对性地开发新型保存技术，并深入探索NMP等机械灌注方法的应用潜力，以提高小肠移植的成功率和患者的长期预后。

11 结果与讨论

当前离体器官保存技术尚难以完全模拟理想的生理微环境，这直接影响了移植后的器官功能恢复。尽管传统SCS技术已广泛应用，但其在维持器官活力和延长保存时限方面仍面临显著局限。MP技术通过持续提供代谢底物并清除代谢废物，展现出更优的保存效能，现已成为研究热点。基于此，研究者已开发出包含不同温度的MP、低体温保存、过冷保存及长期冷冻保存体系。

器官冷冻保存需解决两大核心问题：如何减轻保存过程中的IRI，以及如何在低温条件下抑制器官代谢并避免冷损伤。长期冷冻保存可使器官在超低温下进入生理代谢暂停状态，类似暂停“生命时钟”，但传统慢速冷冻易引发冰晶成核与生长，需精准控制冰晶形成。新型无冰玻璃化保存等技术正在探索中，其关键在于实现无结晶的玻璃态转化，同时降低CPAs毒性。此外，冷冻保存后的复温过程因可能发生冰重结晶而需要更多关注。未来需重点研发低浓度玻璃化CPAs，结合均匀快速复温技术，以突破器官冷冻保存瓶颈。

针对大体积器官的传热传质特性，现有技术通过射频加热与纳米材料协同作用提升复温效率并降低CPAs浓度；而电磁复温技术采用非接触式能量传递机制，在避免外源性纳米颗粒引入的同时实现均匀快速升温，但其对移植器官代谢功能的影响仍需通过离体灌注模型进行系统性评估。

器官质量评估是移植成功的核心保障。对于玻璃化保存器官，需建立包含形态学特征、代谢活性、细胞膜完整性及关键生理功能指标的综合评价系统，重点检测玻璃化转化程度和结晶残留情况，为移植可行性提供科学依据。

总体而言，离体器官保存技术仍面临严峻挑战，需在代谢底物受限、血流灌注中断的离体环境中维持器官活力与功能。未来的技术突破依赖于MP与新型冷冻技术的有机整合，以及个性化保存策略的开发。值得注意的是，不同器官的低温损伤机制存在显著差异，需要针对器官特异性设计保存方案。该领域的持续突破将推动器官保存技术的革新，为建立标准化器官库奠定理论基础，最终实现移植医学的跨越式发展。

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