Progress of mechanical perfusion in split liver transplantation

SUN Jiang-bo, PAN Sheng-hui, CHEN Wei-jia, XU Jian

Department of Organ Transplantation, the Second Affiliated Hospital of Hainan Medical University, Haikou 570105, China

Keywords:Split liver transplantation Mechanical perfusion Cold ischemia time Ischemia reperfusion

ABSTRACT

1.Introduction

1.1 The History of Split-Liver Transplantation (SLT)

Pichlmayr et al.pioneered cleavage liver transplantation beginning in 1989[1], Initially, SLT was offered to one adult and one pediatric recipient for the treatment of their end-stage liver disease, and the splitting of the donor liver was performed ex vivo after acquisition.In the early stages of development, most transplant centers believed that SLT would lead to poor outcomes in the enlarged right hemihepatic portion after surgery and was slow to develop.Later,Goss et al.began to use in vivo cleavage to significantly reduce postoperative complications of SLT (e.g., biliary complications,vascular complications, etc.), while shortening the patient’s CIT(approximately 1.5 h) and improving cross-sectional biliary leakage and bleeding[2].In 2 000, Gundlach et al.began performing left and right hemihepatic splits[3], SLT is now well established in the surgical techniques of splitting and transplantation, but the preservation of the two marginal donor livers obtained by splitting has become a major limiting factor in its development.The preservation of the two marginal donor livers obtained by splitting has become a major limiting factor.

1.2 Current Status of Cleavage Liver Transplantation

At present, SLT with splitting at the left outer lobe boundary is the same as whole liver transplantation in terms of long-term survival,but other studies such as early survival (1-year survival), incidence of re-transplantation, biliary complications, vascular complications and bile leakage from the S4 segment show possible differences between SLT and other whole liver transplants[4].In contrast, the combined prognosis is worse in the right and left hemihepatic types of SLT due to their smaller GRWR and more severe crosssectional damage, with only 1/3 of studies showing no significant difference in survival after hemihepatic transplantation versus total liver transplantation[5].Although in vivo splitting improves the outcome of SLT, for example: (1) shorter cold ischemia time.(2)better identification of the bile ducts and blood supply, allowing more accurate cross-sections of the split; (3) less damage during rewarming (due to the reduction of time for liver repair and in vitro splitting); (4) the ability to observe the blood supply in each segment,which facilitates the proper distribution of vessels[6]; However, the whole process is still problematic, mainly concerning the damage and repair of the liver after cleavage.

1.3 Current Problems with Cleavage Liver Transplantation

SLT is in most cases a combination of larger graft centers (those that can do pediatric liver transplantation) and smaller graft centers(those that primarily perform adult liver transplantation), and most SLT allocations require long-distance transport, which increases the complications associated with CIT prolongation, which makes the actual physiologically available liver volume of the already small GRWR even smaller.A study by Ishii et al.concluded that high levels of ALT and AST after SLT are not suitable for static cold storage (SCS), which increases the occurrence of their early allograft dysfunction (EAD) and severe ischemic injury[7], Angelico et al.found that SCS affects micro-thrombosis in the transplanted liver, which is particularly pronounced in the split cross-section of the S4 segment end vessels, affecting their liver function, and may also cause peribiliary micro-vascular injury, further leading to bile duct epithelial cell damage and the development of distant diffuse bile duct ischemic stenosis[8].In addition, cross-sectional damage is inevitable in SLT, but prolonged SCS increases oxidative stress and immune response in the cross-section, which is also more likely to cause ischemic necrosis or bile leak in the S4 segment[9].Lau et al.concluded that the above factors were more influential in highrisk recipients and pediatric recipients[10].The ability of mechanical perfusion to maintain the donor circulation and to perform therapeutic interventions may be a better way to change the above situation.

2.Mechanical perfusion and cleavage liver transplantation complement each other

The first liver transplant performed by Starzl in 1968 was preserved by mechanical perfusion[11], It can be said that mechanical perfusion arose along with liver transplantation, but due to the imperfect technical conditions at that time, mechanical perfusion did not take advantage of its ideal.Later on, with the emergence of UW fluid and HTK fluid, the status of mechanical perfusion was once reduced[12], It wasn’t until 2009 that Guarrera and colleagues at Columbia University Medical Center conducted the first prospective clinical trial of mechanical perfusion for human liver preservation that the use of mechanical perfusion was “reawakened”[13], This has led to renewed anticipation for mechanical perfusion.With the development of technology and the gradual increase of marginal donors in recent years, especially in the last decade, the clinical studies of mechanical perfusion have increased dramatically[14-16],Mechanical perfusion has once again become a hot topic in the field of organ transplantation.Currently, there are many clinical studies on mechanical perfusion in various types of marginal donor livers such as DCD, elderly donor livers and fatty liver transplantation,and most of them have achieved good results, while the application of mechanical perfusion in SLT still has a large potential for development and unsolved problems.[17].

3.Classification of mechanical infusion

The current study classified machine perfusion for liver maintenance into 3 categories according to temperature,ranging from hypothermic (4℃-10℃), sub-cold (20℃-25℃)to normothermic (35℃-37℃)[18].The current classification of temperature is a broader concept and does not have a strict cutoff value.The three different types of mechanical perfusion are very different, not only in terms of temperature, but also in terms of the molecular biological mechanisms involved, the range of appropriate uses, and the type of perfusion fluid.Each type of mechanical perfusion has its own specific advantages, and there is no clear evidence as to the circumstances in which perfusion is most effective, and there are many studies that have begun to combine different types of mechanical.

3.1 Hypothermic mechanical perfusion (HOMP) in split liver transplantation

3.1.1 Characteristics and mechanism of action of cryogenic mechanical perfusion

The use and development of low-temperature mechanical infusion is the earliest and currently the most detailed related research,given its high safety, simple technical equipment requirements, low technical costs, and easy to carry[19], Cryo-mechanical perfusion is currently considered to be the most likely candidate for widespread clinical use of SLT, especially for transport over long distances in the left outer lobe or the enlarged right hemisphere.cross-sectional release of various factors that increase injury through damageassociated molecular patterns (DAMPs) in SLT plays a key role in early (4- 6 h) ischemic reperfusion, and the damaging stress may be transformed into a potentially lethal innate immune response.Ischemia-reperfusion plays a key role, and injurious stress may be transformed into a potentially lethal innate immune response[20].In contrast, Ishii et al.showed that HOMP was able to significantly reduce the levels of DAMPs (e.g., TNF-α, interferon-γ,interleukin-1β, and interleukin-10).Cryo-mechanical perfusion was able to combine with oxygenating devices, and the delivery of oxygen ions during the synthesis of sufficient ATP caused only slight oxidative stress[21], and can reduce the level of reactive oxygen species and DAMPs[22].And even if the mechanical system fails,it will only be converted to ordinary SCS, and there will be no extremely serious consequences.[23], This is unmatched by other types of mechanical perfusion systems.The above study provides a theoretical basis for the application of cryo-mechanical perfusion in SLT.

3.1.2 Clinical aspects of cryo-mechanical perfusion in cleavage liver transplantation

The current clinical results of cryomechanical perfusion have shown that it improves the poor prognosis of SLT in several ways.van Rijn et al.showed in a multi-center randomized controlled trial(78 HOMP, 78 SCS) that 6% of patients in the HOMP group and 18% of patients in the SCS group developed nonanastomotic biliary strictures, 12% of recipients in the HOMP and 27% in the SCS group developed re post-perfusion syndrome, and early allograft dysfunction occurred in 26% of the HPMP group compared to 40%of the SCS group.The cumulative number of treatments for nonanastomotic biliary strictures after machine perfusion was reduced by almost a quarter compared to the control group[24].This suggests that cryo-mechanical perfusion plays a crucial role in maintaining microvascular perfusion of the biliary tract and reducing graft dysfunction.Spada et al.used dual hypothermic oxygenated machine perfusion (D-HOMP) for perfusion preservation of two portions of the liver in SLT, in which The left outer lobe was preserved for 11 h and the enlarged right hemisphere for 14 h.Despite the significantly prolonged ischemia time, no post-reperfusion syndrome or PNF occurred, the peak maximum lactate monitored before transplantation was ＜5 mmol/L, and all indices recovered rapidly in both recipients after placement into the recipient.Histologically, both partial grafts showed mild ischemia-reperfusion injury at 7 d after SLT[25].D-HOMP is an emerging technique that increases arterial perfusion compared to portal perfusion alone D-HOMP minimizes IRI effects by restoring mitochondrial function, mitigating free radical oxygen radical production during reperfusion, reducing inflammatory responses, and reducing the occurrence of arterial thrombosis and bile duct ischemic necrosis.Thorne et al.showed that by perfusing two donor livers after cleavage with D HOPE for perfusion preservation, the total cold ischemia time was 205 and 468 minutes for LLS and ERL, respectively, and the peak AST and ALT in the left outer lobe after transplantation were 172 IU/L and 107 IU/L, respectively, and 839 IU/L and 502 IU/L in the left outer lobe,respectively, which were significantly less than the level of liver injury in the same type of liver transplantation[26].This suggests that D-HOPE can improve the hepatic utilization of SLT, reduce crosssectional damage, and promote recovery of liver function.

3.2 Normothermic perfusion (NMP) versus split liver transplantation

3.2.1 Characteristics of room temperature mechanical infusion

The theory behind NMP is that the liver organ maintains its metabolic function while maintaining its physiological temperature in vitro.Its perfusion device differs in the way it delivers perfusion solution to the liver in cryo-mechanical perfusion, in order to provide the oxygen required for graft metabolism and to allow longer preservation of the transplanted liver.NMP currently requires the use of oxygen carriers for the isolated perfusion solution, and currently uses red blood cells as oxygen transporters in all published clinical trials.The world’s first study related to normothermic perfusion liver transplantation was first performed in the United States in 2016 by Ravikumar et al.20 patients underwent liver transplantation after NMP.The median NMP time and range was 9.3 (3.5-18.5) h and the median cold ischemia time and range was 8.9 (4.2-11.4) h.The 30-day graft survival rate in the NMP group was similar to that in the SCS group (NMP 100% vs SCS 97.5%, P = 1.00), with a peak AST of 417 (84-4681) U/L in the NMP group compared to the control group 902 (218-8786) U/L was significantly lower compared to the control group (P = 0.03)[27].This study demonstrated the safety and efficacy of splitting under mechanical perfusion during non-transport or upon arrival or transport to the recipient’s transplant center.

3.2.2 Special advantages of normothermic mechanical perfusion in cleavage liver transplantation

Ambient mechanical perfusion has evolved rapidly in the last 10 years, and the perfusion process under ambient mechanical perfusion for splitting, therapeutic interventions, and longer preservation times(even up to several days) may revolutionize SLT.There are already multicenter randomized controlled clinical studies using portable normothermic mechanical perfusion to reduce the incidence of EAD and biliary complications after transplantation, with a sample size of 153 cases using portable normothermic mechanical perfusion(NMP) compared to 147 cases with cryopreservation (ICS), and NMP in particular showed the most significant improvement in EAD (18% vs.31%), while in ischemia-reperfusion There was also a better improvement in terms of ischemia-reperfusion (e.g., there was less moderate to severe lobular inflammation 6% vs.13%),and most importantly, there was a significant improvement in the percentage of DCD donations, with 28 of the 55 DCDs in the NMP group ending up being used, while only 13 of the 51 DCD donor livers in the SCS group ended up being successfully used.This fully demonstrates the significant clinical effect of normothermic mechanical perfusion[14].A study by He et al.showed that the incidence of EAD was significantly lower in the ischemia-free liver transplantation group (sample size 38:130) with normothermic mechanical perfusion, and that AST levels were significantly lower in the recipients, while total bilirubin levels were also significantly lower than in the normal group at 7 d.[28].NMP requires a high oxygen supply for preservation and a dedicated dialysis system for waste removal, making it extremely expensive and complex,with serious consequences in the event of failure.[15].This is the reason why NMP is not widely available in SLT for now, but as the technology matures, NMP may be the most efficient way to preserve organs in a non-transportable state.

3.2.3 Comparison of the characteristics of normal temperature mechanical infusion and low temperature mechanical infusion

The difference between room temperature mechanical infusion and low temperature mechanical infusion is that it can be kept for a longer period of time, even more than 24 h[29].Although the current use of NMP in SLT has not been as widely promoted as HOMP,its advantages in the management of transected bile leaks and exudates, therapeutic interventions (e.g.providing access to RNA gene silencing, cell regulatory therapies, extracellular vesicles of human hepatic stem cells, vasodilators and deglutition therapies,opioid agonists, and many others) are unmatched by other types of preservation methods.However, the NMP technique is complex,may require adjustments and monitoring throughout the process,and may not be suitable during liver transport, whereas ambient perfusion can be used if both recipients are in the same center for SLT, or may work well after cryogenic transport with ambient mechanical perfusion.Another problem with normothermic mechanical perfusion is the two repeated ischemic reperfusions from normothermia to hypothermia, which, despite the short duration of the process, are critical for the liver, but it has been suggested that this problem may be solved by a controlled oxygenated rewarming(COR) type of perfusion under subcritical conditions.

3.3 Sub-nomorthermic machine perfusion (SNMP) and split liver transplantation

Compared with HMP, SNMP consumes less oxygen, restores donor liver function faster, reduces the incidence of endothelial cell injury,decreases portal vein resistance, and increases bile production.[30], It is good for left and right hemihepatic cleavage (the smaller GRWR hemihepatic obtained), expanding the donor pool.SNMP has some advantages over NMP in maintaining liver metabolic activity, and a study by Yoshida et al.found that lactate metabolism was more active and ammonia metabolism was stronger than HOMP in mechanical perfusion under subfreezing conditions[31].When NMP and HOMP are interconverted, there is a significant delay between vascular to complete perfusion of the surrounding liver tissue, and although the time is short, the delay exposes the underperfused region directly to room temperature, which greatly affects the transplanted hepatic bile duct cells.In recent years, the introduction of the new concept of controlled oxygenation rewarming (COR)in SNMP has led to major advances in SNMP research[32], Hoyer et al.showed that COR reduced peak serum transaminases after DCD liver transplantation, and the postoperative survival rates were 84.5%, 82.0%, and 75.8% at 1, 3, and 5 years, respectively.[33].COR was found to significantly reduce hepatic enzyme production, TNFα-related gene expression and activity, free radical-mediated lipid peroxidation (LPO) and portal vascular perfusion resistance during reperfusion, compensating for the problems in HOMP and NMP conversion.

4.Effect of mechanical perfusion on the application criteria of split liver transplantation

The remarkable efficacy of mechanical perfusion in fatty liver,DCD donor liver, and elderly donor liver has further expanded the range of donors for SLT.Mechanical perfusion has a good reversal effect in fatty liver[34], Boteon et al.used a special drug combination perfusion solution that resulted in a 38% reduction in triglycerides and a 40% reduction in macrovesicular steatosis in tissue during 6 hours of mechanical perfusion in donor livers with fatty liver[35].In addition, it may also enable the use of more elderly donors, raising the criteria for SLT to 55 years of age.With increasing aging, several countries such as Italy and Argentina have now increased the age limit for SLT, and mechanical perfusion plays a crucial role in this regard[36], Dutkowski et al.showed that the use of cryo-mechanical perfusion for DCD preservation resulted in a significant reduction in post-transplant ALT, a reduction in biliary complications, and a reduction in early post-transplant survival (1-year survival rate).The same results were achieved compared to brain death, and even with prolonged thermal ischemia time (the greatest effect of ischemiareperfusion) the 5-year graft survival rate of postoperative DCD was 94% compared to 78% for untreated DCD grafts (P = 0.024)[37].The study by Dondossola et al.using dual hypothermic oxygenated perfusion (D-HOPE) for the DCD study showed better survival rates at 6 months and 1 year (both 100%) compared to 80% and 67% in the control group, and the study also found a significantly higher ATP profile after perfusion, suggesting that mechanical perfusion can greatly improve DCD outcomes[38].In conclusion, mechanical perfusion ameliorates changes in the inflammatory response,endoplasmic reticulum stress, mitochondrial damage and apoptosis in marginal donors, thereby improving the energy metabolism of bile duct endothelial cells and hepatocytes.

5.Therapeutic intervention of mechanical perfusion in cleavage liver transplantation

In the future, mechanical perfusion plays an important role in the maintenance of the donor liver and may even play a therapeutic role.For example, studies have already started to apply tissue-type plasminogen activator (t-PA) to treat hepatic arterial thrombosis to improve peribiliary blood supply and reduce bile duct injury[39].In addition, many relevant studies are currently using cleavage to obtain homogeneous two hemispheres, thus reducing heterogeneity in mechanical perfusion studies[40], The reverse facilitates mechanical perfusion in a way that is not possible with other types of marginal donor livers.Early studies have shown that small interfering RNA(siRNA) has a unique advantage in post-transcriptional gene regulation of ischemia-reperfusion in the liver, but this approach has significant limitations in vivo or under static cold storage.The advantage of mechanical perfusion can provide a better platform for gene regulation by dissolving the siRNA into the perfusion fluid,which has a better effect on other organs with fewer side effects and better improvement of ischemia-reperfusion[41].There are many similar examples, such as the reduction of graft-versus-host disease through Treg cell therapy[42], Inhibition of cytokine production by macrophages using human mesenchymal stromal cells to improve hypoxia in the microcirculation[43], Improvement of ischemiareperfusion injury in DCD using prostaglandin E1[44], Application of antibiotics, Miravirsen for perfusion of the liver to reduce recurrence of the original disease and infection[45], In conclusion, mechanical perfusion is not only a method of organ preservation, but can provide a “bridge” to interventional therapy for SLT deficiencies.

6.Summary

SLT significantly increases the source of donors by securing adult liver transplantation while using additional donor livers obtained for pediatric liver transplantation, which is hampered by the poor early prognosis of SLT and postoperative complications associated with splitting.Mechanical perfusion has revolutionized SLT, and the use of cryo-mechanical perfusion in SLT is now starting to enter the clinic.The special advantages of normothermic mechanical perfusion may lead to a radical change in SLT, and subcold mechanical perfusion makes the combined use of all types of mechanical perfusion possible.Combined mechanical perfusion can expand the criteria for SLT in terms of fatty liver, age, and DCD, and in the future It may even be possible to therapeutically intervene in SLT by mechanical perfusion and become a bridge between other interventions and SLT.

Author Contribution and Conflict of Interest

First author: Jiangbo Sun (collected relevant literature and wrote the paper); Corresponding author: Jian Xu (responsible for article conception and review); The rest of the authors participated in article touch-up and formatting correction.There is no conflict of interest related to the content of the article.