Advancing small-animal perfusion models: Normothermic dual-vessel ex vivo rat liver machine perfusion based on a clinical setup

doi:10.21203/rs.3.rs-1597835/v1

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Advancing small-animal perfusion models: Normothermic dual-vessel ex vivo rat liver machine perfusion based on a clinical setup

https://doi.org/10.21203/rs.3.rs-1597835/v1

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Normothermic ex vivo liver machine perfusion (NEVLP) has been developed to address organ shortage in liver transplantation. To establish new therapeutic concepts for the expansion of the donor pool, standardized animal models are needed, that simulate clinical NEVLP. Rat liver grafts were perfused in a dual-vessel NEVLP system using varying doses of the clinically used vasodilator epoprostenol, with and without the addition of the Kupffer cell inhibitor glycine. Cell culture medium supplemented with rat plasma was compared to Steen^TM-based perfusion solution, which is used in clinical NEVLP. Perfusion pressures and bile production were recorded, perfusate transaminases levels were measured and tissue was analyzed for cytokines and 8-Isoprostane. Increasing levels of epoprostenol and the addition of glycine resulted in a stepwise decrease of transaminase secretion and improved bile production. Steen^TM significantly decreased transaminase secretion as well as IL-1β production, resulting in lowest levels of oxidative stress and best-preserved liver integrity. In conclusion, high doses of epoprostenol seem to ameliorate liver function and prevent cellular damage, with glycine acting synergistically. Steen^TM as perfusion basis seems superior. Our rodent NEVLP system may be used to rapidly test new agents for pharmacologic conditioning of livers and to translate these findings from bench-to-bedside.

Liver transplantation

organ shortage

extended criteria donor

normothermic machine perfusion

ischemia reperfusion injury

rat model

The prevalence of end stage liver diseases have been rising for the last decades¹, while the number of liver transplantations – still the only curative treatment option – has recently been declining across Europe². The increased demand for liver allografts is challenged by a limited supply, mainly due to the effects of an aging population² as well as the prevalence of fatty liver disease and fibrosis on the quality of liver grafts³. Substantial advances have been made in recent years in the clinical use of normothermic ex vivo liver perfusion (NEVLP) for improved preservation and quality assessment of organs that were considered suboptimal or even unfit for transplantation^4–7. Using conventional static cold storage, the decision to accept or decline an organ mostly depends on donor specific parameters, duration of ischemia, histopathological assessment – if available – and ultimately the transplant surgeon’s subjective assessment of macroscopic liver appearance. In contrast, NEVLP provides several objective viability parameters, e.g. bile production, lactate clearance, physiological perfusate pH and glucose, that have proven to be relevant for the outcome after transplantation^8–10. Moreover, through the promise of increasing organ preservation time¹¹ and potential expansion of the donor pool through pharmaceutical conditioning and resuscitation of grafts from extended criteria donors¹², the interest of the scientific community to conduct research in the field of NEVLP has surged.

For the sake of investigating future applications of NEVLP, translatable animal models are necessary that simulate the conditions in human trials as closely as possible and allow rapid transfer from bench-to-bedside. In a reverse, “bedside-to-bench” approach, the aim of this study was to develop a reproducible small animal NEVLP model by scaling down a clinically used NEVLP system. The presented rodent NEVLP system combines the dual-vessel setup previously described by our group¹³ with the use of perfusate composition and vasodilators as described by Selzner et al.¹⁴ in a human NEVLP trial using the OrganOx metra (OrganOx Ltd., Oxford, United Kingdom). Our prior set-up was primarily based on culture medium and plasma as well as metamizole for pressure control¹³, however due to the drug not being approved for use in humans in wide parts of the world, it did not fulfil our requirements of creating a translatable model. Although perfusing the liver through portal vein as well as hepatic artery constitutes the standard in clinical NEVLP, the dual-vessel setup is currently not commonly used in small animal models^15− 17. Vasodilation is necessary in general, because the liver artery is prone to spasms after intraoperative manipulation, cold storage, and upon reperfusion.^18,19 Moreover, physiological flow conditions represent a major viability criterion for liver grafts. Prostacyclin analogues such as epoprostenol are the most widely used vasodilators and have also been shown to reduce hepatic ischemia reperfusion injury (IRI) through inhibition of inflammatory signalling²⁰. After already having proven to positively influence perfusion outcome in a single-vessel system¹⁵, the supplementation of glycine was systematically evaluated at the same concentration in a dual-vessel setup for its interactive potential. While not a clinical standard, glycine is known to decrease the expression of proinflammatory cytokines through temporary inhibition of Kupffer cell activation²¹. In a final step, the culture medium was replaced with Steen™ solution. While culture medium is commonly used in experimental small animal NEVLP systems^13,15,16,22, Steen™ solution constitutes one of the most frequently used perfusates in clinical NEVLP.

Animals and study design

Male Sprague-Dawley rats (Janvier, Le Genest-Saint-Isle, France) were kept under species appropriate conditions in the Charité’s Research Institute for Experimental Medicine (Forschungseinrichtung für Experimentelle Medizin). Livers were procured at a weight of 255–355 g and perfused for 6 h in a normothermic dual-vessel NEVLP system with different protocols. In a first set of experiments, n = 4 livers per group were treated with varying doses of epoprostenol (E) (group E0|G- 0 ng/h, group E250|G- 250 ng/h, group E1000|G- 1000 ng/h, and group E2000|G- 2000 ng/h). The same concentrations were evaluated in 4 groups where glycine (G) was additionally supplemented to the circuit at a concentration of 12 mM (E0|G+, E250|G+, E1000|G+, E2000|G+). In a second set of experiments, the protocol of the best-performing group regarding liver damage parameters (E2000|G+) was repeated with Steen™ solution as a basis for perfusion medium instead of the previously used combination of standard cell culture medium (Dulbecco’s Modified Eagles Medium, DMEM) supplemented with rat plasma. Animals were randomized to treatment groups and all experiments were conducted in accordance with the ARRIVE guidelines and the EU Directive 2010/63/EU on the protection of animals used for scientific purposes and were approved by the local animal welfare authority (Landesamt für Gesundheit und Soziales Berlin, project numbers G0012/18 and T0301/17).

Surgical procedures

Surgical procedures were performed as described by Claussen et al.¹³. In short: The animals were anesthetized, the abdominal cavity was opened, and the liver was mobilized. The hepatic artery was dissected all the way to the abdominal aorta with all branches except for the proper hepatic artery ligated. The bile duct was then cannulated. 500 IE Heparin were injected into the vena cava and the abdominal aorta cannulated with a 20-gauge venous catheter (B. Braun, Melsungen, Germany) to allow for blood withdrawal, whereby the blood was saved to be used as part of the perfusion medium. An incision of the superior vena cava and clamping of the supradiaphragmatic aorta were conducted and followed by flushing of the liver with 20 mL of 4°C histidine-tryptophan-ketoglutarate (HTK)-solution (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany), with or without the addition of 12 mM glycine respectively. The celiac trunk was cannulated via an aortic patch using a shortened 24-gauge venous catheter. The portal vein was cannulated using a shortened 16-gauge venous catheter and the liver was again flushed with 20 mL of HTK or sterile HTK/glycine-solution respectively. The inferior vena cava was cannulated using a customized luer connector. The liver was then removed and stored in 4°C HTK ± glycine.

Perfusate composition and perfusion setup

The basis of the perfusion medium consisted of DMEM containing 1 g/L D-glucose and pyruvate (Gibco, Thermo Fischer, Waltham, USA) or Steen Solution™ (XVIVO Perfusion, Sweden). When DMEM was employed, 35 mL were mixed with 5 mL of frozen rat plasma and 10mL of isolated rat erythrocytes. In case of the use of Steen™ solution, 40 mL were mixed with 10 mL of rat erythrocytes. Further additives in both groups included 1.3 mL Aminoplasmal 10% (B. Braun, Melsungen, Germany), 25 mg cefazoline (MIP Pharma, Blieskastel, Germany), 12.5 mg metronidazole (B. Braun, Melsungen, Germany), 0.5 mmol Sodium-Bicarbonate (B. Braun, Melsungen, Germany), and 250 IE heparin (Ratiopharm, Ulm, Germany). Insulin (Humalog, Lilly, Indianapolis, USA) was added at 1.5 IE/h, taurocholic acid (Acros Organics, Fair Lawn, USA) at 3.5 mg/h via syringe driver. In groups where the effect of glycine (AppliChem, Darmstadt, Germany) was evaluated, it was added at a concentration of 12 mM. Epoprostenol (Panpharma, Trittau, Germany) was injected in varying concentrations in respective groups via the hepatic artery canula.

The dual-vessel perfusion system was sustained for 6 hours at 37°C. The organ was weighed, flushed with 20 mL of NaCl 0.9% and placed on a silicone mesh inside a custom-made bioreactor (Glas Gaßner, Oberhaching, Germany), that also served as perfusate reservoir (Fig. 1 A-B). The medium was pumped into a glass oxygenator (Radnoti LTD Dublin, Irland) with a 90% oxygen concentration at a flow of 15 l/min. A glass bubble trap prevented air embolisms into the organ. The final flow was 1.2 mL/min/g liver weight, out of which 1 mL/min/g¹⁵ were directly channeled into the portal vein and 0.2 mL/min/g²³ were subtracted by an additional roller pump and channeled into the hepatic artery. Pressure sensors (Hugo Sachs Electronics, March-Hugstetten, Deutschland) were attached to both vessels, flow and pressure was continuously recorded using the BDAS 2.0 Software (Harvard Apparatus, Holliston, MA, USA).

Pressure recordings were cut off at 198.2 mmHg due to technical limitations. Supraphysiological pressure was defined at above 114 mmHg for the hepatic artery and 12 mmHg for the portal vein²⁴. Organ outflow through the inferior vena cava was guided back into the reservoir. A multichannel pump (Ismatec, Oak Harbour, WA, United States) provided a flow of 10 mL/min into a dialysis cartridge (Spectrum Labs MidiKros 30 kDa, Gardena, USA) on the perfusate as well as the dialysate side, where 500 mL of low potassium MultiBic (multiBic®2 mmol/L potassium, Fresenius Medical Care, Bad Homburg, Germany) were recirculated in countercurrent principle. In groups where the effect of glycine was to be evaluated, a concentration of 12 mM was created in the dialysate. Bile was collected through the cannula in the bile duct.

Sample procurement

Bile was collected, weighed and snap-frozen hourly. Perfusate samples for laboratory measurements as well as blood-gas analyses (BGA) measured after reperfusion, after 3 h and after 6 h from the outflow of the organ as well as after oxygenation. Dialysate samples were additionally measured after reperfusion and after 6 h. BGA were conducted with a ABL800 Flex blood gas analyzer (Radiometer GmbH, Berlin, Deutschland). Photometric measurements of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), urea and total bilirubin were performed in the perfusate by Labor Berlin – Charité Vivantes GmbH. At the end of the perfusion, the livers were flushed with 4°C NaCl 0.9% and weighed. Tissue samples were either snap-frozen or fixated in formalin. Bile ducts were dissected in the liver hilum and snap frozen.

Calculation of hepatic oxygen uptake rate

Hepatic oxygen uptake ratio (HOUR) was determined in accordance with Tolboom et al. ²⁵.

$$HOUR=\frac{({\left[{\text{O}}_{2}\right]}_{in}- {\left[{\text{O}}_{2}\right]}_{out} )\times \text{f}\text{l}\text{o}\text{w} (\text{m}\text{l}/\text{m}\text{i}\text{n})}{100 \times \text{l}\text{i}\text{v}\text{e}\text{r}\text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \left(\text{g}\right)}$$

Total oxygen content was calculated as follows

$${O}_{2} = 1.39 \times Hb \times F{O}_{2}Hb + 0.00314 \times p{O}_{2}$$

Histological analyses

Mayers’ haematoxylin eosin (HE) staining (AppliChem, Darmstadt, Germany) was performed on all sectioned liver lobes as well as bile ducts according to standard protocol. TdT-mediated dUTP-biotin nick end labelling (TUNEL) staining (In situ cell death detection kit, Roche, Basel, Switzerland) and 4’,6-diamidine-2-phenylindol (DAPI) counter staining (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) was performed for left and right-median liver lobes as well as bile ducts according to published protocols. Images were taken with a Zeiss Axio Observer.Z1 microscope using an AxioCam 1Cc5 for HE and AxioCam 506 mono for immunofluorescence stains and analyzed with the Zen 2.3 Pro software (Carl Zeiss AG, Oberkochen, Germany).

Quantification of tissue edema

Livers from the E2000G + and Steen™ groups were weighed before and after perfusion and the difference was recorded. Additionally, tissue from the three largest lobes of the same groups, as well as four non-perfused livers were lyophilized, and the tissue water content was estimated through the difference in dry to wet weight.

Analysis of cytokines in tissue samples

Tissue cytokine analysis was performed for IL-1β, IL-6, IL-10, and TNF-α with a multiplex magnetic bead-based assay (Milliplex Rat Cytokine/Chemokine Magnetic Bead Panel, Merck, Darmstadt, Deutschland). 50 mg of tissue from groups with the highest and lowest epoprostenol concentrations as well as the Steen™ group were homogenized in 750 µl of lysis buffer (Milliplex MAP Lysis Buffer, Merck, Darmstadt, Germany) and 112.5 µl of protease inhibitor (complete Mini protease Inhibitor, Roche, Basel, Switzerland) for 1 min in a vibratory mill (Retsch MM 400, Haan, Deutschland). After centrifugation, the assay was performed with supernantant in 1:25 dilution, the plate was read in a Luminex MagPix (Austin, Texas, USA). Cytokine concentrations were normalized to total protein concentration, measured using a bicinchoninic acid assay (BCA, Thermo Fisher, Waltham, Massachusetts, USA).

Analysis of 8-Isoprostane in tissue samples

8-Isoprostane (8-Iso-PGF-2α) was measured via ELISA (Cayman Chemical, Ann Arbor, Michigan, USA) from liver tissue lysates. 100 mg of tissue was added to 1 mL of 0.1 M phosphate buffer (pH 7.4) substituted with 1 mM Ethylenediaminetetraacetic acid and 0,005% butylated hydroxytoluene. After homogenization and centrifugation, the supernatant was frozen at -80°C. For dissolving esterified 8-Isoprostane, hydrolysis was performed in 15% following manufactures instructions. After purification through C-18 SPE cartridges (Cayman Chemical, Ann Arbor, Michigan, USA), 8-Isoprostane was eluted in methanol. The eluate was dried under a constant stream of nitrogen, samples were resuspended in ELISA buffer and subsequently measured in a 1:10 or 1:20 dilution. Normalization was performed for total protein concentration as mentioned above.

Statistical analysis

Inter-group variables were analyzed for equivalence with one-way ANOVA test. Groups of the first work package (evaluation of glycine and epoprostenol) were analyzed using Kruskal-Wallis test, multiple comparisons were performed with Tukey’s post-hoc test. Groups in the second set of experiments (evaluation of the perfusion medium) were compared using multiple t-tests for each timepoint. Unless stated otherwise, data is presented as mean ± standard error of mean (SEM), a p-value ≤ 0.05 was considered significant. Calculations and graphs were generated using GraphPad Prism Version 8.3.1 (GraphPad Software, La Jolla, CA, USA).

Intergroup variance

There was no significant variance between groups concerning animal weight (302.8 ± 4.7 g, p = 0.623), liver weight (12.9 ± 0.2 g, p = 0.383), or cold ischemia time (70.5 ± 1.3 min, p = 0.109). Electrolytes were kept in close to physiological range for all experiments over the whole course of perfusion (Suppl. Table 1).

Epoprostenol, glyine and Steen™ improved organ quality

The release of transaminases and LDH was considerably less pronounced in groups where glycine was added to the perfusate, with significant differences between the E0|G- vs. E0|G + as well as the E1000|G- vs. E1000|G + groups (Fig. 2 A-B, Suppl. Figure 1 A). Epoprostenol showed a dose-dependent effect in the groups not containing glycine, with significant differences for all enzymes between E0|G- and E2000|G- groups after 6 h (AST: p < 0.0001; ALT: p < 0.0001; LDH: p < 0.0001).

Interestingly, at the highest dose of epoprostenol, no additional effect of glycine was seen. Steen™ solution led to a significant further decrease in all markers of cellular damage when compared to its counterpart using DMEM (E2000|G+; AST: p = 0.019, ALT: p = 0.009; LDH: p = 0.011).

Vascular resistance was decreased through epoprostenol, but increased with application of glycine

Livers perfused without or with 250 ng/h of epoprostenol frequently surpassed the upper limits of physiological hepatic arterial pressure (Fig. 2 C). Using 250 ng/h of epoprostenol kept pressures in a physiological range for the first half of the perfusion duration but not until the end. Doses of 1000 ng/h achieved physiological hepatic arterial pressures over the whole 6-hour perfusion period if glycine was not added (E1000|G-) but failed to control hepatic artery pressure in the E1000|G + group (pressure at 6 h 46.8 ± 6.3 mmHg vs. 139.8 ± 20.1 mmHg; p

= 0.032). 2000 ng/h of epoprostenol was sufficient in groups with and without additional glycine. The basic medium used for perfusion had no significant effect on the development of hepatic arterial pressure after 6 h of perfusion. Portal venous pressure remained below 12 mmHg in all but one experiment (E2000|G+), where it continuously evened out around 15 mmHg (Suppl. Figure 2 A).

Livers remained metabolically active and maintained physiological homeostasis

The perfusate pH was at near-physiological range above 7.2 in all perfusions at all timepoints. Baseline pH surpassed the physiological limit of 7.45 in some cases, usually trended downwards after 3 h to rise again towards the end of the perfusion period (Suppl. Figure 2 B). This development was concomitant with constant CO₂ production of the livers. HOUR was highest at the start of perfusion in most cases and significantly declined over the course of the perfusion (p < 0.0001). Initial oxygen uptake, as well as oxygen uptake after 6 h tended to be higher in groups where glycine was added and with higher doses of epoprostenol. Significant differences were observed between the E1000|G- and E2000|G- (p = 0.018) as well as E2000|G + groups (p = 0.049, Suppl. Table 1).

Bile production peaked in the second hour of perfusion in all experiments (Fig. 2 D). In groups without glycine, there was a dose dependant increase of bile production with rising epoprostenol concentrations. This effect was most pronounced in the final hour of perfusion, with a significant difference between E2000|G- and E0|G- groups (p = 0.0002). Livers with additional glycine tended to keep producing more bile than their counterparts in the latter half of perfusion duration, with a significant difference between E0|G- and E0|G + groups after 5 h (p = 0.012) and 6 h (p = 0.014). The use of Steen™ Solution led to slightly reduced bile production in the second hour of perfusion (p = 0.020).

Glucose levels exceeded physiological limits at the beginning of perfusion in almost all experiments and consistently declined throughout the perfusion period in a highly significant manner (p < 0.0001), reaching the physiological range in most cases (Suppl. Figure 2 C). Lactate clearance was inconsistent throughout groups, and we found no clear corelation to a specific treatment. Individual livers in all groups showed clearance below the threshold of 2.5 mmol/l (based on Mergental et al.⁴) over the course of perfusion. However, a significant variance was seen between subjects within groups. In most cases, lactate concentration increased from 0 to 3 h post-reperfusion and declined towards the end of perfusion (Fig. 2 E). Interestingly, when accounting for plasma expansion through the dialysis circuit by calculation of absolute values of lactate produced, lowest values at the end of perfusion were seen in the E1000|G + and E2000|G + groups (587.13 ± 73.94 mg and 607.75 ± 127.23 mg, respectively (Suppl. Figure 2 D).

Urea concentration consistently increased over the course of perfusion (Suppl. Figure 2E). Livers perfused at all epoprostenol dosages with glycine showed significantly increased urea production compared to their counterparts without glycine (epoprostenol dosage of 0ng/h p = 0.0004; 250 ng/h p 0.0024; 1000 ng/h p = 0.015; 2000 ng/h p < 0.0001) at the end of the perfusion. Total bilirubin remained below measurability in almost all livers, with no significant differences between groups.

Optimized perfusion conditions preserve the liver architecture

Sinusoidal congestion and large areas of necrosis were seen in groups without sufficient arterial pressure control. In contrast, in livers perfused with the highest epoprostenol concentrations and Steen™ only small areas of pressure necrosis was found in areas of the liver where it rested on the mesh (Fig. 3 A-E). Only a few TUNEL positive cells located near to the liver capsule and vascular lining were seen in groups with the highest epoprostenol concentrations and the Steen™ group (Fig. 3 F-J), while larger areas of TUNEL-positive cells were found in the liver parenchyma as well as endothelial cells in groups with higher transaminase secretion during perfusion.

Bile ducts showed generally well-preserved epithelium with little to no apoptotic cells, while the submucosa and muscularis layers exhibited varying amounts of apoptotic cells and some structural defects, potentially owing in part to sample preparation (Fig. 3 K-T).

Epoprostenol and Steen™ influence the cytokine profile

No clear correlation was seen between the addition of glycine or higher doses of epoprostenol alone and levels of either pro-inflammatory or anti-inflammatory cytokines in the liver grafts Fig. 4 A-D). However, the combination of high doses of epoprostenol as well as glycine (E2000|G+) lead to higher levels of IL-1β in liver tissue compared to the E0|G- group (p = 0.0194). Using Steen™ solution significantly reduced IL-1β levels compared to DMEM and plasma (0.933 ± 0.044 pg/µg protein vs. 1.435 ± 0.142 pg/µg protein; p = 0.0148). Other pro-inflammatory cytokines trended lower as well, while IL-10 trended higher, but differences did not reach significance.

The optimized protocol leads to lowest amounts of oxidative stress

8-Isoprostane as marker for reactive oxygen species (ROS) production did not show a clear correlation with a specific treatment, however, all groups using the maximum dosage of epoprostenol consistently displayed low values (Fig. 4 E). The tissue content of 8-Isoprostane varied significantly between groups, with the highest values in the E250|G + group (0.3472 ± 0.0235 pg/µg protein), while the lowest values were seen in the Steen™ group (0.123 ± 0.013 pg/µg protein, p < 0.0001). While not statistically significant, the Steen™ group also trended lower than its counterpart with DMEM and plasma (0.152 ± 0.022 pg/µg protein).

No relevant edema formation was seen with either protocol

Weight change post perfusion was evaluated for groups which used Steen™ solution versus DMEM as basic perfusion medium with the same additives. On average, livers in both groups gained weight after 6 h of perfusion (p = 0.059), but no differences could be seen between the two perfusion solutions (Suppl. Data Fig. 1 B). Tissue water content was also estimated by comparing the ratio of dry weight after lyophilisation to wet weight. Upon comparison of both perfused groups to a set of native snap-frozen rat liver tissue (n = 4), no statistically significant differences were observed (Suppl. Figure 1 C).

Within the last years, NEVLP has quickly evolved from an emerging technology to an established clinical practice. Basic research in the field focuses on using this technology to prolong the preservation period and improve donor pool utilization through assessment of graft quality and pharmacological intervention. Future experiments to in this research field cannot be performed with human livers in adequate numbers due to obvious ethical and logistical challenges. Thus, extensive experimental work in animal NEVLP models will be needed. However, currently available small animal models lack standardization, often using protocols that differ heavily from clinical application and almost exclusively perfusing livers solely through the portal vein^{15–17,26−28}. Based on these prerequisites, our goal was to create an easily translatable, yet simple rat NEVLP model that closely resembles currently used clinical NEVLP systems and should therefore allow for high throughput testing of protocols and interventions in the future.

We opted for the use of a dual-vessel system, as our previous work showed dual-vessel perfusion to be superior to single-vessel perfusion¹³ and the later omits an essential component of clinical NEVLP. We systematically investigated the dose-response relationship to find the ideal concentration of the clinically used vasodilator for the rat model. We conclude that the highest dose of epoprostenol tested (2000 ng/h) most consistently keeps hepatic arterial pressures in a physiological range. This represents a multiple of the dose commonly used in the clinical setting. The cause for this increased need in vasodilation may be a physiological particularity in the rodent liver, however supraphysiological pO₂ in the portal vein has recently been hypothesised to increase the need for vasodilation, and may also be a contributing factor¹¹. High perfusion pressures were associated with increased levels of transaminase secretion to the perfusate and compromised histological liver integrity. This could likely be explained by endothelial cell damage, indicated by TUNEL staining, as well as through macroscopically visible parenchymal haemorrhages in groups without sufficient pressure control. Although these detrimental effects would be less pronounced in a pressure-controlled model, suboptimal vasodilation would lead to insufficient flow conditions, and is therefore no less relevant. As another interesting insight, higher doses of epoprostenol showed a positive effect on markers of hepatocellular cellular damage, independent of the vasodilatory quality. Although this cytoprotective effect has been known for a long time²⁹, a dose-dependency in a liver perfusion setup has not been examined before. No adverse effects were observed with higher doses. Although other vasodilators with similar anti-inflammatory effects recently displayed potential advantages over epoprostenol in a comparative study by Echeverri et al., our results suggest that using higher doses of epoprostenol may achieve equivalent results¹⁹.

While not a clinical standard, glycine has proven to ameliorate organ perfusion conditions in a previous study by our team in a mono-vessel setup¹⁵. In the dual-vessel set-up, glycine indeed resulted in a decrease in markers for cellular damage, but adversely affected hepatic arterial pressure and thus increased the need for vasodilation. This effect has not been described yet and may be traced back to an NMDA-co-agonism of glycine on smooth cells of the arterial wall, which triggers arterial spams³⁰. Due to its overall beneficial effects and its cost-efficiency and availability, glycine could be incorporated as a standard additive to liver perfusions, however potential increases in arterial pressure must be accounted for. As interactions with the arterial vascular system may also occur with other drugs, we strongly advocate the use of a dual-vessel model for further pharmacological research.

Steen™ solution improved transaminase secretion compared to DMEM and plasma, possibly through reduction of inflammatory signalling and ROS production. As the exact composition of Steen™ solution is not communicated, we can only speculate as to the underlying mechanism. Simply substituting a major component of Steen™ solution – humane serum albumin (HSA) – to phosphate-buffered saline did not achieve the same reduction of pro-inflammatory cytokines or ROS-markers in cultured endothelial cells in a study by Pagano et al. They postulated that some components of Steen Solution ^TM reduce the activity of NADPH oxidases, especially the subtype NOX2³¹, that is also expressed by Kupffer cells and hepatocytes³², possibly helping to explain the minimized ROS production seen in our optimized protocol. The Toronto group recently examined different perfusion media in a porcine NEVLP model and showed Steen™ solution to best protect endothelial cells, measurable via reduced hyaluronic acid production. This effect, possibly attributable to the dextrane-content of Steen™ ³³, also corelated with lower levels of necrosis, lower levels of post-transplant lactate and increased overall survival ³⁴.

Common rat models of NEVLP use plasma as colloid expander^13,15,16,22, this was not considered necessary when using HSA-containing Steen™ solution. Constituents of humoral immunity in plasma may explain the difference in performance of basic media. In a recent clinical trial, Liu et al. reported generally favourable outcomes in the use of fresh frozen plasma as an additive, yet without another perfused control group, which makes the influence of plasma-addition hard to quantify³⁵. The supposed primary benefit of Steen™ – protection against edema formation – does not seem to be a factor in the small animal model, as no relevant edema formation was seen after 6 h with either basic perfusion solution. Therefore, Steen™ Solution should be considered as a standard for optimal organ perfusion, however DMEM and plasma may be a less cost-intensive alternative for basic research. The high bicarbonate content of MultiBic® provided excellent buffer capacity, maintaining perfusate pH closer to the physiological range than dialysates previously used by our group^13,15. TNF-α levels in liver tissue were halved compared previously published data from a single vessel protocol, even though ischemic time was longer due to more complicated graft preparation¹⁵. Lactate clearance is regarded one of the most relevant parameters for assessment of liver graft quality, was not consistently seen throughout all groups. This phenomenon is, however, very common in small animal models^{17,36− 38}. The greater perfusate-volume to liver weight ratio compared to human or porcine liver perfusion may be a contributing factor, as erythrocytes are a large source of lactate production. The threshold, usually considered to be around 1.7–2.5 mmol/l after 2–4 hours of perfusion³⁹, is therefore not necessarily relevant for our model.

Our study has several limitations considering the goal of simulating clinical NEVLP devices. Portal vein flow in our system was lower compared to in vivo measurements. However, sufficient perfusion was previously achieved using the same settings by our team¹⁵, and an additional increase in flow also yields little functional improvement in the porcine liver according to Hardison et al.⁴⁰. Even though longer perfusion periods have been described in human trials, we perfused rat livers for 6 h only, as this timeframe seems to be the viability limit for transplantation in the small animal model^16,28,41. We also opted for the use of a dialysis membrane, which is currently not seeing widespread use in human or porcine NEVLP. Once more, this is most likely necessary because of greater ratio of perfusate volume to liver weight in this model, which leads to significant accumulation of potassium and urea over the course of perfusion if no dialysis is employed ^15,16. The use of a dialysis membrane might influence the predictive value of the levels of lactate and glucose as well as the pH in our system. However, in the trial by Eshmuminov et al., the effect of a dialysis membrane on the evaluability of lactate clearance seemed to be negligible, as lactic acid only passed the membrane slowly⁴². While further steps are on our scope, above all proving the viability of the perfused organs in a transplantation model, we were able to show for the first time the feasibility of translating a clinically used protocol of NEVLP to a small animal model. Our current setup is easy enough to handle to allow for perfusion of two livers simultaneously. Besides its applicability in transplant research, it may also be used as a whole-organ-perfusion-model for pharmacological research with the premise of reducing animal experiments in accordance with the 3R concept.

We report on a stepwise improvement of our rodent NEVLP system, designed to enhance clinical transferability back from bench-to-bedside. The optimized perfusion protocol closely mimics clinical NEVLP protocols and shows the least amount of transaminase release, cytokines, and markers of oxidative stress. Our protocol allows for rapid testing of new therapeutic options for re-conditioning of marginal liver grafts and may aid in the struggle against donor organ shortage.

ALT Alanine transaminase

AST Aspartate transaminase

BGA Blood gas analyses

DMEM Dulbecco's Modified Eagle Medium

HE Haematoxylin & Eosin

HOUR Hepatic oxygen uptake ratio

HSA Human serum albumin

HTK Histidine-tryptophan-ketoglutarate

IL-1β Interleukin 1 beta

IL-6 Interleukin 6

IL-10 Interleukin 10

IRI Ischemia reperfusion injury

LDH Lactate dehydrogenase

NEVLP Normothermic ex vivo liver perfusion

ROS Reactive oxygen species

TNF-α Tumor necrosis factor alpha

TUNEL TdT-mediated dUTP-biotin nick end labelling

Grants and financial support

This work was funded by institutional financial support of the Charité – Universitätsmedizin Berlin and by the German Research Foundation (grant number: RA 3044/3-1). Simon Moosburner, Nathanael Raschzok, and Joseph Gassner are participants of the BIH Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health.

Data Availability Statement

Raw data supporting the findings in this study are either incorporated in the main text or can be found in supplementary data files. Any additional data will be provided upon request.

Authors contributions

Writing of the original manuscript was done by FS. Conceptualization was performed by JMGVG, NR and FS. Methodology and supervision were provided by JMGVG and NR. FS, JM, JMGVG, VM, KK and MZ were involved in conducting the investigations (surgery, perfusion experiments, laboratory analyses). Formal analysis and visualization were performed by FS and SM. Resources and funding were provided by NR, IMS, JP. All authors were involved in reviewing and editing of the manuscript.

Acknowledgments

The authors would like to thank Dr. rer. med. Anja Reutzel-Selke and Dr. rer. nat. Jörg Mengwasser for their methodological and general scientific support.

Competing Interests Statement

All authors have read the journal’s authorship agreement and policy on potential conflicts of interest and concluded that there were no relevant competing interest to declare.

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No competing interests reported.

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Advancing small-animal perfusion models: Normothermic dual-vessel ex vivo rat liver machine perfusion based on a clinical setup

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Abstract

Figures

Introduction

Methods

Animals and study design

Surgical procedures

Perfusate composition and perfusion setup

Sample procurement

Calculation of hepatic oxygen uptake rate

Histological analyses

Quantification of tissue edema

Analysis of cytokines in tissue samples

Analysis of 8-Isoprostane in tissue samples

Statistical analysis

Results

Intergroup variance

Epoprostenol, glyine and Steen™ improved organ quality

Vascular resistance was decreased through epoprostenol, but increased with application of glycine

Livers remained metabolically active and maintained physiological homeostasis

Optimized perfusion conditions preserve the liver architecture

Epoprostenol and Steen™ influence the cytokine profile

The optimized protocol leads to lowest amounts of oxidative stress

No relevant edema formation was seen with either protocol

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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