Improved rat liver preservation by hypothermic continuous machine perfusion using polysol, a new, enriched preservation solution

Abstract

For experimental machine perfusion (MP) of the liver, the modified University of Wisconsin solution (UW-G) is most often used. In our search for an enriched MP preservation solution, Polysol was developed. Polysol is enriched with various amino acids, vitamins, and other nutrients for the liver metabolism. The aim of this study was to compare Polysol with UW-G for MP preservation of the liver. Rat livers were preserved during 24 hours with hypothermic MP using UW-G (n = 5) or Polysol (n = 5). Hepatocellular damage (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], alpha-glutathione-S-transferase [alpha-GST]) and bile production were measured during 60 minutes of reperfusion (37°C) with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours of cold storage in UW (n = 5). MP using UW-G or Polysol showed less liver damage when compared with controls. Livers machine perfused with Polysol showed less enzyme release when compared to UW-G. Bile production was higher after MP using either UW-G or Polysol compared with controls. In conclusion, machine perfusion using Polysol results in better quality liver preservation than cold storage with UW and machine perfusion using UW-G. (Liver Transpl 2005;11:539–546.)

Liver transplantation is the treatment of choice in patients with end-stage liver disease.1, 2 The quality of the liver graft depends, among other factors, on the preservation method and the length of the preservation period (i.e., the cold ischemic time). The current gold standard in liver preservation3 is wash-out of the liver using an appropriate preservation solution, followed by cold storage (CS), enabling human liver allografts to be safely preserved for a period of up to 16 hours.4 In this setting, the liver is implanted in the recipient after the preservation phase without any objective knowledge on graft viability. Reliable methods for prior assessment of hepatocellular damage and liver function are lacking in the statically cold stored organ. Donor history, macroscopic evaluation, and liver biopsy analysis can merely give an indication of the viability of the cold stored liver graft.3

The limits of CS in the preservation of most abdominal organs have been reached. As an alternative, machine perfusion (MP) preservation of the liver has come into focus again in experimental studies. MP was already applied in the early sixties.3, 5, 6 After wash-out to clear blood remnants, the liver is connected to a recirculating MP system in which it is perfused with a hypothermic preservation solution for the duration of transport. Several advantages of MP have been postulated over CS: (1) continuous supply of oxygen and nutrients, (2) continuous removal of end-products of metabolism, (3) assessment of liver viability during preservation,7 and (4) potential resuscitation of ischemically damaged organs such as non-heart-beating donor organs8

Experimental studies have shown superior results in posttransplantation function of liver grafts after MP compared with CS.9–11 These results can be explained by the fact that although the organ is cooled to 4°C, 7% to 35% of the intrinsic metabolism is maintained.12 This metabolism, although reduced, could benefit from energy substrates and oxygen, which can only be provided by continuous oxygenated MP. The modified University of Wisconsin solution (UW-Gluconate [UW-G]), most often used in experimental MP, lacks substrates for energy, carbohydrate, and fat metabolism of the liver.10, 11, 13, 15 Although literature on the role of nutrients in solutions for hypothermic organ preservation is scarce,16–18 we hypothesize that a perfusion solution enriched with nutrients results in better quality liver preservation. This led to the development of a new preservation solution, Polysol, which contains the required nutrients for liver metabolism along with potent buffers and free radical scavengers. The components which, among others, make the difference between Polysol and other MP preservation solutions are amino acids, such as glutamine, histidine, tryptophan and arginine, and vitamins, such as ascorbic acid and alpha-tocopherol (Table 1).

Table 1. Important Components in UW, UW-G, and Polysol

Components	UW	UW-G	Polysol
Colloid	HES (5%)	HES (5%)	HES (5%)
Na/K ratio	30/120 mmol/L	125/25 mmol/L	120/20 mmol/L
Buffer	KH2PO4	HEPES	HEPES
		KH2PO4	KH2PO4
			Histidine
Antioxidants	Allopurinol	Allopurinol	Glutathion
	Glutathion	Glutathion	Alpha-tocopherol
			Ascorbic acid
Energy substrates		Glucose	Glucose
Impermeants	Lactobionate	Na-Gluconate	Na-Gluconate
		K-Gluconate	K-Gluconate
		Mg-Gluconate	Raffinose
	Raffinose	Raffinose	Trehalose
Amino acids	−	−	+
Vitamins	−	−	+
pH-indicator	−	−	Phenol-red

The aim of this study was to assess MP of rat livers using Polysol and to compare results with MP using UW-G, both in relation to the gold standard CS method using UW. To this end, both preservation methods and MP solutions were assessed in the isolated perfused rat liver model.

CS, cold storage; MP, machine perfusion; UW(-G), University of Wisconsin(-Gluconate); RP, reperfusion; KHB, Krebs-Henseleit buffer; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; alpha-GST, alpha-glutathione-S-transferase.

Materials and Methods

Animals and Surgery

Male Wistar rats (Harlan, Horst, The Netherlands) weighing 350 g (±50 g) were used as liver donors. The animals were housed under standardized conditions with a 12/12 hour dark/light cycle and ad libitum access to water and a standard pellet chow (Hope Farms, Woerden, The Netherlands), until directly prior to the experiment. All animals were handled in accordance to Dutch regulations and principles of animal care, under approval of the Animal Ethical Committee of the University of Amsterdam.

Rats were anesthesized with O₂/air/isoflurane (1 L/min: 1 L/min: 3%) and an intraperitoneal injection of 0.1 mL/100 g body weight FFM (Hypnorm/Dormicum/aquadest, 1:1:2). During surgery, anesthesia was maintained with inhalation of O₂/air/isoflurane through a mask.

After median laparotomy followed by bilateral subcostal incisions, the liver was mobilized and the bile duct canulated with a 0.9-mm catheter (B-Braun, Melsungen, Germany). Before canulation of the portal vein, the animal was heparinized via the caval vein with 0.1 mL heparin (5000 IU/mL, Leo Pharma, Malmö, Denmark). The liver was washed out with 50 mL of Ringer Lactate (37°C, 10 cm H₂O, Baxter, Utrecht, The Netherlands) via the portal vein cannula (0.8 Fr, enteral feeding tube, Vygon, Valkenswaard, The Netherlands). During washout the animal was bled via incision of the abdominal caval vein. The suprahepatic caval vein was cannulated with a 0.6-Fr cannula (Vygon), the infrahepatic caval vein was ligated, and after trimming of surrounding tissue the liver was excised and weighed.

Machine Perfusion System

A dual machine perfusion system was developed by the Medical Technical Development Department of the Academic Medical Center (AMC, Amsterdam, The Netherlands) enabling both MP and reperfusion (RP) phase in a single setup (Fig. 1). Prior to connection of the excised liver, the circuit was rinsed with 200 mL of sterile aquadest and subsequently with 50 mL of preservation solution. The pressure-controlled perfusion system consists of a reservoir containing 350 mL of sterile MP solution. After connecting the liver to the system, the first 100 mL of perfusion solution was collected. The remaining 250 mL of solution was recirculated by a rollerpump (Ismatec, Glattbrugg, Switzerland). The perfusion solution was oxygenated with carbogen (95% O₂/5% CO₂, 1 L/min, Hoekloos Medical, The Netherlands) by a glass oxygenator, resulting in a prehepatic oxygen tension of approximately 700 mmHg. Air emboli were removed from the system by a bubble trap, after which the solution was cooled using a heat exchanger (HMT-200, Heto, Breda, The Netherlands). The perfusion solution passed through an inline flow meter (HT-207, Transonic Systems Inc., Maastricht, The Netherlands), entered the liver through the portal vein cannula, and ran freely via the suprahepatic caval vein cannula into the reservoir.

Figure 1. The double perfusion system for both 24 hours MP and 60 minutes reperfusion. The system consists of a reservoir from which the perfusion solution is pumped by a roller pump through the glass oxygenator. After oxygenation and removal of air emboli, the solution is either cooled or heated in the heat exchanger. After passing a flow probe, the solution perfuses the liver via the portal vein canula and runs off freely via the caval vein into the perfusate reservoir. Before entering the reservoir, samples can be taken for assessment of liver damage and function.

RP was performed along the same circuit as described above, with a second reservoir now containing 400 mL of Krebs-Henseleit buffer (KHB) solution at 37°C. Before reconnecting the liver, the system was rinsed with 200 mL sterile aquadest and 50 mL KHB. After reconnection of the liver, the first 100 mL was drained to prevent it from re-entering the circuit. The remaining 250 mL of perfusate was oxygenated with carbogen. Samples were obtained from the tubing directly pre- or posthepatically. Temperature was recorded by a probe (Laméris, Nieuwegein, The Netherlands) placed under the liver. After each procedure, the circuit was rinsed and steam-sterilized (134°C for 16 minutes).

Experimental Groups and Preservation Conditions

This study comprised 3 experimental groups: (1) CS-UW (N = 5); (2) MP-UW-G (N = 5), and (3) MP-Polysol (N = 5). The isolated livers were preserved by either CS or MP for 24 hours and thereafter reperfused.

After wash-out with RL (4°C), the liver was flushed in situ with the preservation solution. CS livers were flushed with 50 mL UW (4°C), placed in a sterile cup containing 100 mL UW and stored on melting ice in a cold chamber (4°C) for 24 hours. MP livers were connected to the perfusion system via the portal vein directly after washout and harvesting, flushed with 100 mL of either UW-G or Polysol, and continuously perfused with this solution at 4°C for 24 hours. After the preservation period, all livers were reperfused for 60 minutes at 37°C with oxygenated KHB.

Preservation Solutions

For cold storage, the University of Wisconsin preservation solution (Viaspan, Bristol-Myers Squibb, New York, NY) was used. The UW-G solution for MP was prepared according to Belzer's prescription (pH 7.4 at 4°C, 330 mOsm/kg).11 The MP preservation solution Polysol (pH 7.4 at 4°C, 330 mOsm/kg) was developed at the Surgical Laboratory of the AMC. For RP, KHB without bovine serum albumin (pH 7.4 at 37°C, 320 mOsm/kg) was used.

UW-G, Polysol and KHB were all prepared in our laboratory using analytical reagent grade (or better) chemicals from Sigma-Aldrich (Zwijndrecht, The Netherlands), Merck (Haarlem, The Netherlands), Cambrex (Verviers, Belgium), Centrafarm (Etten-Leur, The Netherlands), and Novo Nordisk (Alphen aan den Rijn, The Netherlands). The Hydroxyethylstarch (HES) was obtained from Fresenius (Taunusstein, Germany). Prior to use, the solutions were sterilized by filtration through a 0.45-μm ampul filter (DowCorning, Allesley, United Kingdom) and a 0.22-μm filter (Millipack 60, Millipore, Amsterdam, The Netherlands).

Assessment of Hepatocellular Damage and Liver Function

Samples for hepatocellular damage assessment were taken every 10 minutes during 60 minutes of RP.

Liver damage was assessed by direct analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) in posthepatic perfusate samples (Laboratory of Clinical Chemistry, AMC, The Netherlands). Alpha-GST (alpha-glutathione-S-transferase) levels were determined using a rat alpha-GST ELISA kit (Biotrin, Dublin, Ireland).

Liver function was assessed by monitoring bile production during 60 minutes of RP. Furthermore, lactate production (Laboratory of Clinical Chemistry, AMC, The Netherlands) and perfusate pH (ABL, Radiometer, Zoetermeer, The Netherlands) were measured during RP.

Histology and Dry/Wet Weight Ratio

At the end of the RP phase biopsies were taken from the caudate and right lateral lobes. Biopsies were stored in formaldehyde (10%) and embedded in paraffin. Paraffin sections (4 μm) were stained with hematoxylin and eosin and evaluated with light microscopy. A 9-point scale was used for morphological classification of hepatic injury graded on a scale of 1 (excellent) to 9 (poor)19, 20: 1. normal rectangular structure; 2. rounded hepatocytes with an increase of sinusoidal spaces; 3. vacuolization in zone 3; 4. vacuolization in zone 2; 5. vacuolization in zone 1; 6. vacuolization and nuclear pyknosis in zone 3; 7. vacuolization and nuclear pyknosis in zone 2; 8. vacuolization and nuclear pyknosis in zone 1; and 9. necrosis.

For dry/wet weight ratios liver biopsies were weighted immediately after reperfusion and then stored in a 60°C stove. Biopsies were weighed again every 7 days, until reduction of liver weight had stopped. To demonstrate the amount of liver edema, the following calculation was used: 1 − (dry weight / wet weight) × 100%.

Statistical Analysis

The Kruskal-Wallis test was used for overall comparison of the three groups. If significant differences were shown, differences between individual groups were evaluated by the non-parametric Mann-Whitney test. Results in text and graphs are shown as mean ± SEM. Statistical significance was defined as P < 0.05.

Results

Perfusion Parameters

Liver weights did not differ significantly between experimental groups (16.5 ± 0.5 g). During both hypothermic MP and normothermic RP the perfusion pressure was constantly kept at 20 cm H₂O (gravity controlled). The perfusion flow during hypothermic MP reached 1 mL/min/gram liver maximally. During normothermic RP a maximum flow of 3 mL/min/gram liver was recorded. Oxygenation during hypothermic MP resulted in a perfusate pO₂ of approximately 700 mmHg and during normothermic RP, due to the higher temperature, in a pO₂ of approximately 500 mmHg. The temperature recorded during normothermic RP was 37.1 ± 0.4°C.

Hepatocellular Damage

ALT release after 24 hours cold ischemic time was significantly higher after CS with UW compared with MP using UW-G at t = 0 (4.6 ± 2.4 vs. 0.4 ± 0.2) and t= 10 minutes (5.4 ± 1.7 vs. 1.4 ± 0.2 U/L) (Fig. 2A). However, when CS-UW is compared to MP-Polysol, ALT levels are significantly lower after MP-Polysol at all time points except t = 0 and t = 50 minutes. LDH levels appear higher after 24 hours CS-UW, without reaching significancy. LDH is significantly higher after CS-UW at t = 10 minutes (Fig. 2B) compared with MP using either UW-G or Polysol. Perfusate flow, pH, and lactate production were not significantly different (data not shown).

Figure 2. (A) Perfusate ALT levels during 60 minutes of normothermic reperfusion with KHB. Reduction in ALT is shown at all time points for MP vs. CS. A reduction in ALT release in MP livers using Polysol vs. CS in UW was found at t = 10–20–30–40–60 minutes of RP and in MP livers using UW-G vs. CS in UW at t = 0–10 minutes of RP. (B) Perfusate LDH levels during 60 minutes of normothermic reperfusion with KHB. Decreased LDH levels are shown for MP vs. CS. Values for MP using Polysol vs. CS in UW are lower at t = 10 minutes of RP. (C) Perfusate AST levels during 60 minutes of normothermic reperfusion with KHB. Decreased release of AST after 24 hours MP vs. CS in UW. Significant reduction of AST release after MP using UW-G vs. CS in UW at t = 10. Significantly decreased levels of AST are shown after MP using Polysol vs. MP using UW-G at t = 40–50–60 minutes of RP. Values (N = 5) are expressed as mean ± SEM.

When comparing the two MP solutions, less damage after 24 hours of MP-Polysol was seen, as shown by the lower AST levels (Fig. 2C). Although there was a trend in favor of Polysol at all time points, there were no significant differences in ALT (Fig. 2A), LDH (Fig. 2B), perfusate flow, pH, and lactate production (data not shown).

Release of alpha-GST (Fig. 3) at t = 40 was lower after MP-Polysol compared with CS-UW (125.5 ± 10.5 vs. 46.4 ± 9.1, respectively, P < 0.02) and MP-UW-G (101.6 ± 12.0 vs. 46.4 ± 9.1, respectively, P < 0.02).

Figure 3. Perfusate alpha-GST levels during 60 minutes of normothermic RP with KHB. A significant reduction in alpha-GST after 24 hours MP using Polysol vs. MP using UW-G is demonstrated. Values (N = 5) are expressed as mean ± SEM.

Hepatocellular Function

Bile production appeared higher after MP-Polysol than after MP-UW-G or CS-UW (355 ± 82.3 vs. 256 ± 26.2 vs. 180 ± 61.99 μL, respectively). However, this did not reach significance (Fig. 4).

Figure 4. Bile production during 60 minutes of normothermic reperfusion with KHB. Bile production is increased after MP using Polysol compared with CS in UW and MP using UW-G. Values (N = 5) are expressed as mean ± SEM.

Histology

After histopathological scoring of the liver sections, a better median score was assigned to the MP groups using UW-G and Polysol (2.0 ± 0.6 and 1.6 ± 0.4 points, respectively) compared with the CS-UW livers (4.5 ± 0.9 points) (P = 0.06 for UW-G and P = 0.03 for Polysol). There were no significant differences between the MP groups (Fig. 5).

Figure 5. Histopathological appearance of livers following 60 minutes of normothermic reperfusion with KHB: (A) After 24 hours CS: Widened sinusoids (arrows), vacuolization in zones 1–3 (circles), pycnosis and areas of necrosis. (B) After 24 hours MP using UW-G: decreased sinusoidal spaces (arrows), vacuolization in zone 3 (circles), no necrosis. (C) After 24 hours MP using Polysol: normal sinusoidal structure and hepatocytes, no vacuolization or necrosis.

The dry/wet weight ratio of liver sections was highest in the MP groups, accounting for the lowest percentage of edema (Fig. 6). Percentages were 76 ± 1.0 vs. 72 ± 0.5 vs. 72 ± 0.7, respectively.

Figure 6. Dry/wet weight ratio of liver biopsies, after reperfusion (N = 5). Dry/wet weight ratio (%) is highest in the CS group compared with both MP-UW-G and MP-Polysol. Values expressed as mean ± SEM.

Discussion

MP of donor organs has been applied for many years5, 6 and has been the method of choice before CS was introduced by Collins in 1969.21 For the preservation of ischemically damaged human kidneys, MP was introduced clinically in 1972.22 In clinical liver transplantation, graft preservation is up to now limited to CS.23, 24 Several restrictions related to MP have hampered widespread application of MP. Compared with the simple method of CS, MP is more complex, expensive, and labor-intensive, and it requires highly skilled personnel. Not withstanding these disadvantages, recent developments aimed at enlarging the donor pool have shown that organs of non-heart-beating donors can be improved by MP. These organs have sustained warm ischemic damage before harvesting and require a higher quality of preservation in order to maintain post-transplantation viability.25–31 In this respect, CS has severe limitations in that supply of oxygen and nutrients is not possible. Preservation of donor grafts by MP, however, does provide these requirements. Recent experimental studies on preservation of the liver have shown superior results for MP compared with CS. Compagnon et al. studied rat liver preservation by 24 and 48 hours MP using Celsior with hydroxyethylstarch compared to cold storage using Celsior. Reperfusion results were improved after MP.9 Schön et al. and Friend et al. studied MP of the non–heart-beating donor pig liver under normothermic instead of hypothermic conditions, with superior results for MP compared with CS.8, 32–34 Hypothermic MP of the NHBD liver also resulted in superior preservation compared with CS,11, 35–38 but no comparisons have been made between normothermic and hypothermic MP of the donor liver.

For clinical MP of the kidney, the modified University of Wisconsin solution (UW-Gluconate) is used. This solution has been further modified for application of MP in the liver, by substituting mannitol with raffinose. The resulting solution has been extensively used in experimental liver preservation10, 11, 14 but is not commercially available. We have developed a new preservation solution for MP of liver and kidney, Polysol, containing the nutrients that are in our view necessary to support the suppressed metabolism at 4°C. Although our ultimate goal is the preservation of organs of marginal donors, we first sought to test Polysol in a well-established heart-beating donor model in order to obtain baseline values.

In this study we have shown the benefits of MP over CS in a heart-beating donor rat liver model. Hepatocellular damage was significantly lower in the MP-preserved livers. This has also been shown by other groups9, 10, 39 and can be explained by the continuous oxygenation of the perfusion system and the continuous supply of nutrients during MP. Furthermore, liver function as expressed by bile production is improved after 24 hours MP. Although sinusoidal endothelial cell damage has been postulated to be more prominent after MP,40 we have shown no differences in perfusate flow (a surrogate marker for sinusoidal endothelial cell and microvascular damage) during reperfusion.

MP using Polysol resulted in lower hepatocellular damage values and improved post-preservation function in terms of bile production. We have enhanced the buffering capacity of the new solution, optimized oxygen-free radical scavenger content, and added specific nutrients for amino acid, energy and fat metabolism. Furthermore, this solution is prepared at pH 7.4, but after connection to the oxygenated liver the pH decreases to pH 7.2. It has been postulated that mild acidosis of the perfusate enhances cytoprotection during hypothermia, which can be considered an additional advantage.41

Although we have shown promising results for the use of Polysol as an MP preservation solution, these results will have to be validated in a large animal liver allo-transplantation model. In such a model, clinically relevant endpoints such as through interaction with recipient blood and survival need to be analyzed.

In conclusion, preservation of the heart-beating donor rat liver by MP results in a better quality liver preservation compared with CS. MP using the new, enriched preservation solution, Polysol, results in equal to better quality liver preservation when compared with UW-G.