Successful Nonfreezing, Subzero Preservation of Rat Liver with 2,3-Butanediol and Type I Antifreeze Protein

Journal of Surgical Research 96, 30 –34 (2001) doi:10.1006/jsre.2000.6053, available online at http://www.idealibrary.com on

Successful Nonfreezing, Subzero Preservation of Rat Liver with 2,3-Butanediol and Type I Antifreeze Protein Kyle A. Soltys, M.D., Arun K. Batta, Ph.D.,* and Baburao Koneru, M.D. Department of Surgery, Transplant Research Laboratory, and *Department of Medicine, University of Medicine and Dentistry–New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103 Submitted for publication February 23, 2000; published online December 21, 2000

INTRODUCTION Background. Organ cryopreservation is hindered by ice inflicted damage. Nonfreezing preservation of livers at subzero temperatures might offer advantages over current preservation. Methods. Sprague–Dawley rats were divided into three groups. UW livers (n ⴝ 6) were stored in University of Wisconsin (UW) solution at ⴙ4°C. UWB livers (n ⴝ 6) were perfused ex vivo with UW ⴙ 10% 2,3butanediol at <7°C and stored at ⴚ4°C. AFP livers (n ⴝ 4) were preserved identical to UWB livers, except for addition of 1 mg/ml of type I antifreeze protein. After 24 h livers were perfused with Krebs–Henseleit buffer (37°C) for 60 min. Bile production, O 2 consumption (O 2C), taurocholate extraction, and lactate dehydrogenase (LDH) release during perfusion and liver adenine nucleotide content and energy charge at the end of perfusion were measured. Cell membrane integrity was determined by trypan blue infusion. Results. Ice formation was prevented in all livers stored at ⴚ4°C. Bile production, O 2C, and taurocholate extraction were similar among three groups. Livers stored at ⴚ4°C contained significantly more adenine nucleotides than livers stored at ⴙ4°C but the energy charge was similar. LDH release was significantly greater (P < 0.05) in the AFP group vs UWB and UW (63 vs 28 and 21 mU/min/g liver, respectively). Hepatocyte and sinusoidal cell trypan blue uptake was similar in all three groups. Conclusions. Butanediol with or without AFP was effective in preventing ice formation up to 24 h in rat livers stored at ⴚ4°C. Although as effective as current ⴙ4°C protocols, subzero preservation for longer periods needs to be achieved prior to clinical application. © 2001 Academic Press Key Words: cryopreservation; subzero preservation; organ preservation; cryoprotectants; antifreeze proteins.

0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Advances in organ preservation techniques and more efficacious immunosuppressive drugs have vastly improved the results of liver transplantation. In current liver preservation protocols, the donor liver is flushed with hypothermic (⫹4°C) University of Wisconsin (UW) 1 solution and is stored in the same solution until the time of implantation. While this protocol allows safe transplantation up to 12 h after removal from the donor, primary nonfunction still occurs at an unacceptable rate of 5–10% [1] and remains an important cause of death and emergent retransplantation. Severe preservation injury is also associated with an increased incidence of rejection, nonanastomotic bile duct strictures and decreased long-term graft survival [1–3]. Clearly, the development of improved preservation techniques is important to the further development of clinical transplantation. Further decreasing the metabolic rate of donor organs should in theory increase the time that organs can be stored for. This would allow the recipient operation to become a more elective procedure leading to better allocation of health care resources and also make the interregional sharing of organs more practical. Subzero preservation of livers has the potential of making these goals feasible. Based on mathematical models, the metabolic rate of an organ is depressed approximately 10-fold by current ⫹4°C preservation protocols [2]. Further decreasing the storage temperature to ⫺4°C would theoretically decrease the metabolic rate of an organ 17-fold. However, the decreased rate of metabolism would be asso1 Abbreviations used: AFP, antifreeze protein; 2,3-BD, 2,3-butanediol; CP, cryoprotectant/s; DMSO, dimethyl sulfoxide; HPLC, highperformance liquid chromatography; KHB, krebs–Henseleit buffer; UW, University of Wisconsin; UWB, University of Wisconsin solution with butanediol.

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SOLTYS, BATTA, AND KONERU: NONFREEZING PRESERVATION OF RAT LIVER

ciated with potential deleterious effects caused by the process of supercooling and freezing. The mechanisms of damage caused by both freezing and supercooling have been extensively researched and are naturally overcome by several species of amphibians, fish, and insects [4, 5]. The principle adaptation that allows several species of frogs to survive low temperatures is the rapid synthesis and accumulation of lowmolecular-weight cryoprotectants (CP) such as glucose and glycerol in their blood and tissues [6, 7]. Several other small molecular weight solutes including ethylene glycol, dimethylsuloxide (DMSO), 1,3-propanediol, and glycerol have been employed as CP in experimental models [8, 9]. The alcohol 2,3-butanediol (2,3-BD) has shown a great deal of promise as a CP, although it has been found to be toxic at high concentrations (⬎10% v/v) [10]. Several species of fish and insects produce unique CP peptides known as antifreeze proteins (AFPs) [11, 12]. AFPs have been found to modify the propagation of ice crystal formation and efficiently inhibit recrystallization of ice during the thawing process [13]. Type I AFP, from the arctic flounder, is the prototype of these molecules and has been utilized in several different experiments involving cryopreservation, with varied success [14, 15]. While subzero preservation of isolated mammalian cells has seen some success, freezing preservation of solid organs has not been successful. Ice formation and osmotic stress cause severe disruption of microvasculature and cell membranes. These factors cause poor organ function that has been almost universally witnessed on reperfusion of these organs. Nonfreezing preservation at subzero temperatures is an alternative to cryopreservation. Very scant information is available in the literature regarding nonfreezing subzero preservation of whole livers and prompted us to conduct this study [16]. Our aims in the following experiments were to examine the feasibility of subzero preservation of whole rat livers to ⫺4°C and to avoid gross ice formation with 2,3-butanediol and type I AFPs as cryoprotectants. MATERIALS AND METHODS Experimental design. In the first set of experiments the function of rat livers stored at ⫺4°C in a 10% 2,3-butanediol in UW solution was compared to livers preserved in UW at ⫹4°C (n ⫽ 6 each). Pilot studies in our laboratory showed that 10% 2,3-BD in University of Wisconsin solution (UWB) consistently inhibited gross ice crystallization, even with seeding, at temperatures as low as ⫺5.3°C. In the second set of experiments the effect of addition of type I antifreeze proteins (a kind gift of A/F Protein Inc., Waltham, MA) to UWB on the function of livers stored at ⫺4°C was examined (n ⫽ 4). The concentration of type I AFP chosen in our study (1 mg/mL) has been shown to inhibit the crystallization of ice at subzero temperatures [15]. Following 24 h of preservation, livers were evaluated during 60 min of perfusion in an ex vivo recirculating system. Bile production, O 2 consumption, LDH release, and taurocholate extraction during

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perfusion, as well as liver content of adenine nucleotides at the end of perfusion, were measured. Hepatocyte and sinusoidal lining cell membrane integrity was determined by light microscopic analysis of trypan blue uptake at the end of the perfusion. Animals. Specific pathogen-free male Sprague–Dawley rats (Taconic Farms, Germantown, NY) were housed in standard rodent cages at 25°C with light/dark cycles of 12 h. Rats weighed 300 – 400 g and had free access to water and lab chow (Harlan Teklad, Madison, WI). All rats were maintained and treated in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and all experimental procedures were approved by the New Jersey Medical School’s Animal Care Committee. Liver procurement. Nonfasted rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). After celiotomy was performed, the bile duct was canulated with PE-10 tubing and 500 U of heparin was injected into the inferior vena cava. The aorta was canulated with a 20-gauge catheter and flushed with 10 cc of cold (4°C) UW solution with immediate blanching of the liver parenchyma. The suprahepatic cava was divided, the portal vein was quickly canulated with a 16-gauge catheter, and the liver was perfused with an additional 30 cc of cold UW solution. A 14-gauge catheter was then inserted into the infrahepatic cava and the portal catheter was capped. The liver was rapidly released from the abdomen and transported to the perfusion chamber in cold flush solution. Livers in the control group were placed in a storage bag containing 100 cc of UW solution and stored on ice. Subzero preservation. A standard recirculating liver perfusion chamber [17] was utilized to gradually increase the concentration of the cryoprotectants to their final concentrations over a period of 30 min. The basal solution for all hypothermic perfusion and storage was nonoxygenated UW solution at ⱕ⫹7°C. Flow rates through the liver were adjusted to maintain a portal pressure of ⱕ10 cm H 2O, which was continuously measured with a manometer. The CPs (2,3BD ⫾ AFP) were added to the perfusate reservoir at a constant rate over 30 min, until full-strength concentrations (10% 2,3-BD ⫾ 1 mg/ml AFP) were reached. After an additional 15 min of perfusion, the liver was removed from the system and weighed. A 40-g type T thermocouple (Physitemp, Clifton, NJ) was inserted through the wall of the suprahepatic inferior vena cava into the parenchyma of the median lobe. The liver was then sealed into an impermeable bag containing 100 cc of a group-specific storage solution (UW ⫹ 10% 2,3-BD ⫾ 1 mg/ml AFP), suspended in a precooled beaker of alcohol, and rapidly cooled (⬵10°C/min) from ⫹4 to ⫺4°C. The liver temperature was constantly monitored by the parenchymal thermocouple and the temperature maintained at ⫺4°C for 24 h by a cooling plate (Thermoelectronics, Wilmington, DE). Rewarming and perfusion. Following 24 h of preservation, livers were rapidly warmed to 37°C by submersion into a 40°C bath and were weighed and perfused with 37°C oxygenated (PO 2 ⱖ 500 mm Hg) Krebs–Henseliet Buffer (KHB), which initially contained fullstrength concentrations of the group-specific CP solution. The concentration of the CP was decreased over 30 min by continuous dilution of the perfusate in the reservoir with pure KHB. When the level of the CP approximated zero, the livers were allowed to equilibrate with unsupplemented KHB for 15 min prior to the 60 min of experimental period. The flow rate of the perfusate was approximately 3 mL/g/min, again keeping the portal pressure ⱕ10 cm H 2O. During the experimental period, the pH of the perfusate was maintained at approximately 7.4 by addition of aliquots of a bicarbonate solution to the perfusate as indicated. Bile was collected into a graduated syringe and quantified as microliters per gram of liver per minute. Portal vein inflow and cavil outflow perfuse PO 2 were measured every 30 min and hepatic oxygen consumption was expressed in milliliters of O 2 per gram per minute. LDH release into the perfuse was quantified at 30 and 60 min using a calorimetric assay (Sigma– Aldrich, St. Louis, MO) and expressed in milliunits per gram per minute. Sodium taurocholate (TC), 200 ␮M, was added to the KHB buffer just prior to the experimental period. Inflow perfusate was

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TABLE 1 Weight Change during Preservation, Bile Output, and O 2 Consumption during 60 min of Perfusion Group

N

Wt Change (% initial)

Bile output (␮L/g liver/min)

O 2 Consumption (mL O 2/g liver/min)

UW UWB AFP

6 6 5

⫺7.7 ⫾ 1.6 ⫺11.5 ⫾ 5.5 ⫺13.0 ⫾ 4

84 ⫾ 12 91 ⫾ 9 83 ⫾ 6

2.1 ⫾ 0.13 1.8 ⫾ 0.17 1.9 ⫾ 0.21

Note. Rat livers were preserved either at ⫹4°C in University of Wisconsin solution (UW) or at ⫺4°C in UW ⫹ 10% 2,3-butanediol without (UWB) or with type I antifreeze protein (AFP). Following 24 h of storage, livers were perfused ex vivo with recirculating acellular buffer. Data are expressed as means ⫾ SD. The differences between various groups are not significant. collected after 30 and 60 min of perfusion and taurocholate levels were determined by gas chromatography [18]. ATP determination. Tissue concentrations of adenine nucleotides in snap-frozen liver samples were measured using HPLC as described by Wynants and van Belle [19]. Briefly, the frozen tissue was ground with a liquid-nitrogen-cooled mortar and pestle, weighed, and extracted with perchloric acid. The extract was filtered with a 0.22-nm filter and an aliquot was injected onto a C-18 column (Supelcosil 3␮ LC ⫺ 15 cm ⫻ 4.6 mm; Supelco, Bellafonte, PA). The mobile phase consisted of 0.1 M KH 2PO 4 (pH 6.0) at 1 ml/min. Absorbance at 254 nm was recorded with a UV detector (Model 490, Waters Inc., Milford, MA), and results were analyzed with a software package. Distinct, reproducible peaks were identified for adenine tri-, di-, and monophosphates (ATP, ADP, AMP) and were quantified using nucleotide standards (Sigma–Aldrich). The protein concentration of liver was determined by the bicinchoninic acid method (Pierce, Rockford, IL). Adenine nucleotide content was expressed as picograms per milligram of protein and as hepatic energy charge. Trypan blue uptake. After sampling for ATP analysis, the liver was perfused for 8 min with trypan blue (2.5% v/v) in KHB and fixed with glutaraldehyde (2.5%) in Tris buffer. Representative samples of liver were stored in formalin and embedded in paraffin and 4-␮mthin sections were stained with eosin [20]. Trypan blue uptake by both hepatocytes and sinusoidal lining cells was determined by examination of 160 high-powered (100⫻) fields in a blinded fashion. Statistical analysis of the data was performed using analysis of variance and multiple comparisons were made with Tukey–Kramer test.

FIG. 1. Lactate dehydrogenase (LDH) release (left) and taurocholate uptake (right) by rat livers preserved either at ⫹4°C in University of Wisconsin (UW) solution or at ⫺4°C in UW ⫹ 10% 2,3-butanediol without (UWB) or with (AFP) type I antifreeze protein. Following 24 h of storage livers were perfused ex vivo with a recirculating acellular buffer. *P ⬍ 0.05 vs UWB and UW groups.

ered to indicate true physiologic clearance rates because the concentration of taurocholate in the perfusate was not kept constant. The content of ATP, ADP, and AMP was significantly lower in livers stored for 24 h at ⫹4°C when compared to those stored at ⫺4°C. However, the overall energy charge was found to be similar between the groups (Table 2). On light microscopic analysis of tissue sections from the three groups, both sinusoidal lining cells and hepatocytes demonstrated similar degrees of trypan blue uptake from the perfusate (Fig. 2). However, AFP livers preserved at ⫺4°C released significantly more LDH (63 ⫾ 15 mU/g/min) than livers stored at ⫺4°C in UWB (38 ⫾ 8.2) and at ⫹4°C in UW (21 ⫾ 5.6, Fig. 1, left). DISCUSSION

In our study, we were uniformly successful in avoiding gross ice formation in rat livers stored at ⫺4°C with 10% TABLE 2

RESULTS

In the group of livers stored for 24 h at ⫺4°C, gross ice crystallization was invariably prevented by 10% BD with or without the addition of type I antifreeze proteins. These livers did not differ significantly in the relative weight change during preservation when compared to those stored at ⫹4°C in UW solution (Table 1). Livers stored at ⫺4°C in UW solution supplemented with either 10% 2,3-BD or 10% 2,3-BD and 1 mg/ml type I AFP produced similar quantities of bile when compared to livers stored at ⫹4°C in UW (Table 1). The O 2 consumption was also similar among the three groups (Table 1). The ability of the livers to remove taurocholate from the perfusate was similar in all groups (Fig. 1, right). However, this cannot be consid-

Adenine Nucleotides (pg/mg protein) Content and Energy Charge (EC) in Livers after 60 min of Reperfusion Group N UW UWB AFP

6 6 5

ATP

ADP

AMP

EC

162 ⫾ 35* 265 ⫾ 97 343 ⫾ 95

552 ⫾ 209† 1796 ⫾ 480 1264 ⫾ 420

310 ⫾ 27* 574 ⫾ 201 750 ⫾ 280

0.42 ⫾ 0.01 0.41 ⫾ 0.06 0.44 ⫾ 0.02

Note. Rat livers were preserved either at ⫹4°C in University of Wisconsin solution (UW) or at ⫺4°C in UW ⫹ 10% 2,3-butanediol without (UWB) or with type I antifreeze protein (AFP). Following 24 h of storage, livers were perfused ex vivo with recirculating acellular buffer. Energy charge calculated as ATP ⫹ 1/2 ADP ⫼ ATP ⫹ ADP ⫹ AMP. Data are expressed as means ⫾ SD. * P ⬍ 0.05 vs AFP group. † P ⬍ 0.05 vs UWB.

SOLTYS, BATTA, AND KONERU: NONFREEZING PRESERVATION OF RAT LIVER

FIG. 2. Trypan blue uptake by hepatocytes and sinusoidal cells in rat livers preserved either at ⫹4°C in University of Wisconsin (UW) solution or at ⫺4°C in UW ⫹ 10% 2,3-butanediol without (UWB) or with (AFP) type I antifreeze protein (UWB). After 24 h of storage, livers were perfused ex vivo with recirculating acellular buffer for 60 min. At the end of perfusion, trypan blue was infused. Fixed liver sections were counterstained with eosin, and cells with trypan blue uptake were counted in 160 high-power fields (⫻100). The differences between groups were not significant.

2,3-BD with or without type 1 AFP. Prior to our study there has been only a single brief report of successful nonfreezing subzero preservation of whole livers. One of the first attempts at subzero preservation of whole liver was by Brown et al., with exposure of canine livers to 2 M dimethyl sulfoxide or glycerol with subsequent 5-day storage at ⫺6°C. After orthotopic transplantation, no recipient animals survived greater than 12 h [21]. Subsequent attempts employing varying concentrations of glycerol, DMSO, and ethylene glycol have been met with equally disappointing results [22]. In the present state of cryobiology, we feel avoidance of gross ice formation is critical to successful subzero whole organ preservation. During slow freezing, ice forms first in the intravascular space and, to maintain osmotic equilibrium, water leaves the cells to freeze in the extracellular space and vasculature. This results in cell shrinkage and mechanical disruption of the vasculature, preventing adequate reperfusion upon thawing [4, 5]. Although gross ice formation was prevented in our study, one cannot rule out the possibility of microscopic intra- and extracellular ice crystallization. The cryoprotective action of small molecular weight solutes in nature, such as glucose and glycerol, is based on their colligative properties. The cryoprotectant properties of propylene glycol, DMSO, and 2,3butanediol are believed to be similar to those of glycerol. These molecules are more rapidly equilibrated than glycerol across cell membranes and thus cause fewer osmotic shifts. In addition there may be beneficial direct interactions among 2,3-BD, DMSO, and the phospholipids of cell membranes [23, 24]. In our study, livers perfused and stored in 10% 2,3-BD demonstrated a similar pattern of weight loss compared with those stored in UW alone. This suggests that the net osmotic shifts were similar in the two groups and that there was an adequate penetration of 2,3-BD into the cells.

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There was minimal toxicity of 10% 2,3-BD, based on LDH release and trypan blue uptake data. Eschewedge et al. reported ⫺4°C preservation of whole rat liver for 72 h using 8% 2,3-BD in UW solution [16]. However, the actual functional properties of these livers were not detailed in their report [16], and we were not able to reproduce their findings. When we attempted to increase subzero storage times beyond 24 h, gross ice formation was noted in nearly half of the livers (data not shown). Another cryoprotective system found in nature is illustrated by the winter flounder, Platystethus americanus. On exposure to decreasing temperatures, the liver of P. americanus begins to produce large quantities of a type I AFP [11]. Type I AFP binds to ice crystals, modifies the crystalline structure of ice into a less damaging configuration, and inhibits the recrystallization of ice [13]. Antifreeze proteins also have the capacity to protect cells and membranes from damage caused by simple hypothermia, possibly by their ability to suppress K⫹ and Ca 2⫹ currents at low temperature [25]. In our study, the addition of 1 mg/mL of type I AFP to a 10% solution of 2,3-BD in UW did not result in further improvement in liver function after 24-h storage at ⫺4°C. Interestingly, the hepatic concentration of adenine nucleotides was found to be highest in this group. The mechanisms and significance of increased LDH release seen in the AFP group are not clear. Experiments in our laboratory demonstrated that 1 mg/ml type I AFP alone, when added to UW solution, was unable to preserve any hepatic function once gross ice formation was noted (data not shown). This experience is similar to that of Rubinsky et al. with preservation of rat whole livers frozen at ⫺3°C for 6 h with antifreeze glycoproteins and glycerol [15]. The degree of cryoprotection in that study was poor, with only a 31% return of function after freezing. Isolated ex vivo liver perfusion is well established as a method for evaluating ischemia/reperfusion injury to the liver and the effects of various preservation solutions [26 –28]. When compared to liver transplants in rats, it is much less expensive and less time consuming and provides an opportunity to control more variables than is possible in liver transplantation. Rat liver transplantation is more suitably used in evaluating preservation methods demonstrated to be superior in an isolated perfusion model. When subzero preserved livers were tested in an ex vivo perfused liver system in our study, bile production, taurocholate removal, and membrane permeability to trypan blue were comparable to the livers preserved in a conventional manner. These data suggest that if microscopic ice crystallization did occur, it did not lead to significant cell injury. The tenet of cryopreservation is that decreases in storage temperatures below those used in current protocols would result in a further decrease in the metabolic rate

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of an organ and thus preserve energy stores and decrease accumulation of potentially toxic metabolites [2]. A strong correlation exists among donor liver ATP concentration, the ability to regenerate ATP after transplantation, and graft viability [29]. Studies have shown improved preservation of high-energy phosphates after storage at subzero temperatures with and without butanediol [30, 31]. Although our data showed improved preservation of tissue adenine nucleotides in the subzero preserved livers over those of conventionally preserved livers, this, however, did not translate into improved function. We do not have an adequate explanation for this disparity. In conclusion, our study shows that subzero nonfreezing preservation of rat whole livers with adequate return of function is feasible for up to 24 h. However, despite further lowering of core organ temperatures by about 8 –10°C, subzero preservation did not result in improvement over conventional preservation. Successful preservation for periods of 48 h and longer with improved cryoprotective agents and techniques may offer advantages over the current liver preservation protocols.

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