The isolated perfused rat liver (IPRL) has proven to be a useful model for the study of physiology and pathology of the liver. For research in nonparenchymal cell (NPC) function that includes measurement of cytokine production (eg, TNF), it is necessary to have a sterilized perfusion system. We have modified the IPRL apparatus so as to be able to perform sterile perfusions of two livers simultaneously. The perfusion apparatus is a recirculating closed system in which the oxygenator is a plastic container separated into two chambers by a fenestrated plastic wall. A disposable macropore filter functions as both a bubble trap and perfusate filter. The sterilization process is done by immersing the various components in Benz-All solution. The tubing is disinfected by irrigation with 10% Clorox followed by 0.9% sodium chloride solution. The perfusate used is filter-sterilized Krebs buffer solution containing 0.5 g Mandol/250 mL perfusate. Not only can two organs be conveniently perfused simultaneously, but the entire system can be reliably sterilized for up to 20 consecutive perfusions. Bile production is higher and more stable with less leakage of intracellular enzymes. Many of the components are disposable and can be altered to suit the needs of a particular experiment. © 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

Flock E V

Gores G J

Hamilton R L

Hans Mischinger

Miller L L

Ontell S J

Prakash N. Rao

Premalatha Chelvakumar

Randy Rubin

Tao Liu

Thomas E. Starzl

Thomas R. Walsh

Wolkoff A M

PubMed

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A Modified Apparatus for Dual, Sterilized, Isolated Perfusion of the Rat Liver

Liu, T

Walsh, TR

Mischinger, H

Rao, PN

Chelvakumar, P

Rubin, R

Starzl, TE

Name not available

A modified apparatus for dual, sterilized isolated perfusion of the rat liver

D-Scholarship@Pitt

Journal of Investigative Surgery, Volume 3, pp. 365-372 Printed in the UK. All rights reserved. A Modified Apparatus for Dual, Sterilized, Isolated Perfusion of the Rat Liver TAO LIU, MD2 THOMAS R. WALSH, MDI HANS MISCHINGER, MD3 PRAKASH N. RAO, PhD i PREMALATHA CHELVAKUMAR, DVM 1 RANDY RUBIN, BS1 THOMAS E. ST ARZL, MDI 1 Department of Surgery University of Pittsburgh and the Veterans Administration Medical Center Pittsburgh, PA 15261 2 Department of Surgery First Teaching Hospital Bethune University of Medical Sciences Changchun, Jilin Peoples Republic of China 3 Department of Surgery Medical School Karl-Franzens University Graz, Austria Abstract The isolated perfused rat liver (IPRL) has proven to be a useful model for the study qf physiology and pathology of the liver, For research in nonparenchy-mal cell (NPC) function that includes measurement of cytokine production (eg, TNF), it is necessary to have a sterilized perfusion system, We have modified the IPRL apparatus so as to be able to perform sterile perfusions of two livers simulta-neously. The perfusion apparatus is a recirculating closed system in which the oxygenator is a plastic container separated into two chambers by a fenestrated plastic wall. A disposable macropore filter functions as both a bubble trap and perti/sate filter. The sterilization process is done by immersing the various compo-nents in Benz-All solution. The tubing is disinfected by irrigation with 10% Clorox followed by 0.9% sodium chloride solution. The perfusate used isfilter-sterilized Krebs buffer solution containing 0.5 g Mandolj25 0 mL perfusate, Not only can two organs be conveniently perfused simultaneously, but the entire system can be reli-ably sterilized for up to 20 consecutive perfusions. Bile production is higher and more stable with less leakage of intracellular enzymes. Many of the components are disposable and can be altered to suit the needs of a particular experiment. Keywords: Isolated perfused rat liver (IPRL), dual, sterilized. Supported by research grants from the Veterans Administration and Project Grant DK 29961 from the National Institutes of Health, Bethesda, Maryland. Dr. Walsh is a National Research Service Award Fellow. Address correspondence to Thomas R. Walsh, MD, Department of Surgery, University of Pittsburgh School of Medicine, 497 Scaife Hall, Pittsburgh, PA 15261. 365 366 T. Liu et al. The technique and apparatus used for ex vivo perfusion of the isolated rat liver (IPRL) have been greatly improved since they were first used about 100 years ago. IPRL has proven to be a useful model for the study of normal and abnormal physiol-ogy ofliver. 1- 3 In recent years, research in hepatology has expanded to studies of the NPC populations of the liver. Whole bacteria as well as bacterial particles can alter NPC function. At certain concentrations, bacteria and bacterial particles activate NPC. It has therefore become necessary to develop a sterilized IPRL apparatus and perfusate for studies of the intact liver. For example, measurement of the effects of cold and warm ischemia on the Kupffer cells, endothelial cells, and hepatocytes, or the measurement of cytokine production (eg, tumor necrosis factor (TNF), which is produced by the Kupffer cells), requires a sterile perfusion system. Sterilization of the IPRL apparatus and perfusate is time-consuming. However, we have further modi-fied the IPRL apparatus so as to be able to perfuse two livers simultaneously. There-fore, two intact livers can be perfused simultaneously for 2-!- to 3 h, each in less time than it would take to do them separately. Liver perfusions can be done with reproduc-ible bile production, leakage of hepatocellular enzymes, and production of tumor necrosis factor. Material and Methods Male Lewis rats (250-350 g) were used as liver donors. All animals are induced and maintained with inhalational methoxyflurane. Prior to harvesting the liver, 300 units of heparin are injected via the penile vein. The surgical procedure is similar to that previously reported4- 6 except for some minor modifications. A transverse incision is used to expose the upper abdominal cavity, which allows access to all the supporting ligaments of the liver and to the suprahepatic inferior vena cava (IYC). The portal vein is cannulated with a 16-gauge angiocatheter, while the infrahepatic IYC is can-nulated with a 14-gauge catheter. The larger angiocatheter in the IYC allows a de-creased perfusion pressure (10-14 em H20) at high flow rates (2.5-3.5 mL/min g liver-I). This improves the delivery of oxygen without barotrauma to the hepatic vasculature as is evidenced by decreased swelling and weight gain of the livers during a perfusion. The suprahepatic IYC is ligated at the level of the diaphragm immedi-ately after cannulation of the infrahepatic IYC. Harvest of the two livers is staggered from 30 to 45 min to allow for adequate spacing of sample collection during the perfusion. The second liver is harvested during the beginning of the first perfusion. Each liver lies on a separate petri dish, which is covered by a second, inverted dish (Fig 2). We routinely allow a 15-min period of equilibration (I = 15 to t = 0) after the liver is placed on the perfusion apparatus for the bile production and temperature of the liver to stabilize. The perfusion apparatus is a recirculating system and has three major parts that are housed within a thermostatically controlled cabinet: a perfusion flow controller, a pump, and an oxygenator.2,7,8 The following modifications have been made in the apparatus (Fig 1) and the technique of perfusion. The Oxygenator A covered plastic container that is used for the outer housing of the oxygenator has been separated into two chambers by a piece of plastic that has been fenestrated. This allows free flow of oxygen and carbon dioxide between the two chambers. The oxy-GAS FLOW 95% 02 + 5% CO2 -Isolated Perfused Rat Liver 367 9 o go Figure 1. Schematic diagram of dual perfusion apparatus: 1, pulsatile pump; 2, oxygenator; 3, filter and debubbler and reservoir; 4, perfusate sample port; 5, three-way stopcock; 6, manome-ter: 7, liver platform and collecting basin; 8, bile collector; 9, constant temperature circulator. genator tubing is thin-walled silicon (T 5715-6, American Scientist, Warrendale, PA), which yields a partial pressure of oxygen of 500-600 mm Hg in each perfusion circuit when provided at 5 L/min of95% O2/5% CO2 , The Perfusate Flow Regulator A Master Flex pump controller (Model 7016-20) with silicon tubing (No. 6411-16 or 6411-14, Cole Parmer Instrument Co., Chicago, IL) is used in each of the indepen-dent perfusion systems. The flow rate of perfusate is kept at 2.5-3.5 mL/g Ii ver min -1. The perfusate used is blood-free Krebs buffer with 2% bovine serum albumin. This allows measurements of oxygen consumption and release of various intracellular enzymes to be performed without interference from red blood cells. Higher rates of flow rapidly increase the pressure within the circuit and cause the liver to swell presumably secondary to vascular barotrauma. The percentage of prehepatic oxygen that is extracted is stable with minimal acidosis throughout at least 21 h of perfusion. Perfusion Pressure Monitor The pressure within each perfusate circuit is continuously monitored through a three-way stopcock placed immediately before the portal vein cannula (Fig 1 V This is connected to a pressure manometer (No. 4607, Abbott Laboratories, North Chi-368 T. Liu et al. Figure 2. Close-up diagram of liver platform and collection basin: 1, perfusate inflow; 2, three-way stopcock connected with manometer and portal vein cannula; 3, lye cannula; 4, perfusate bypass; 5, perfusate outflows. The platform is made from petri dishes. Another dish placed upside down covers the platform. cago, IL), which is mounted on the side of the cabinet. Normal perfusion pressure for a control perfusion is 10-14 cm H20. After a period of ischemia (cold or warm), it is important to slowly increase the rate of perfusate flow while keeping the pressure below 15 cm H20. Too rapid resumption of the normal rate of flow will lead to greatly increased perfusion pressures, swelling of the liver, and release ofhepatocellu-lar enzymes. Bubble Trap and Perfusion Filter We use a macropore filter (Blood set 64, Abbott) as both a bubble trap and perfusate filter. A filter is placed in-line within each inflow circuit and is disposed of after each perfusion. Removal of this disposable filter permits a more thorough irrigation ofthe tubing after each perfusion and simplifies sterilization. Collection Basin for IVC Effluent A 50-mL disposable sterile centrifuge tube (Corning Glass Works, Corning, NY) is attached to a petri dish (No. 5-757-13, Fisher Scientific Co.) in which an appropri-ately sized opening has been cut (Fig 2). Silicone Sealant (Bostik 9732, Middletown, MA) is used. This collection basin should also be covered with another inverted petri dish to assure a closed perfusion system and to avoid contamination during the perfusion. If the height of the centrifuge tube is adjusted properly, it can lie within the water bath, which helps to maintain the perfusate at the desired temperature. Isolated Perfused Rat Liver 369 200 -E 150 --(J) -C :::J 100 ---u. z 50 t--0- TNF STERILE O 0 25 50 75 100 125 LENGTH OF PERFUSION (min) Figure 3. TNF production during sterile perfusion. TNF rises gradually during 120 min of perfusion due to recirculation and mild Kupffer cell activation. Sterilization of the Perfusion System During all surgical procedures and the assembly of the perfusion apparatus. a surgical mask and gloves are worn. A sterile pack that contains towels, 4 x 4 gauze pads, and cotton applicators is available for each liver harvest. Between perfusions, the basin for collection of Ive effluent and its cover are immersed in 1:750 Benz-All aqueous solution (XTTRIUM Lab, Chicago, IL) for at least 15 min and then rinsed with sterile water. The sterile blood set filter is opened immediately prior to the perfusion, 150 -0- SGOT STERILE • SGOT NONSTERILE -- 100 E t---a (J) (!J -en c :::J 50 o +----,r---.---.--......------,r----,----1 5 1 5 45 75 105 135 LENGTH OF PERFUSION (min) Figure 4. SGOT production during sterile and nonsterile perfusions. SGOT gradually rises in a linear fashion due to a recirculation in each system. After 90 min of perfusion, there is a large leakage of SGOT in the nonsterile perfusions. (N = 6 sterile and 5 nonsterile perfusions.) 370 T. Liu et al. 100 80 --0-- SGPT STERILE -• SGPT NONSTERILE I- ~ 60 a. en CJ -en c: 40 ::::J -20 o+---~K---~----~--~K---~---15 15 45 75 105 135 LENGTH OF PERFUSION (min) Figure 5. SGPT production during sterile and nonsterile perfusions. SGPT rises minimally during 120 min of sterile perfusion. During the nonsterile perfusions, there is a great rise in SGPT leakage after 90 min. (N = 6 sterile perfusions.) and the ends of the tubing and connectors (three-way stopcocks) are wiped with gauze saturated with 70% alcohol during assembly. The ports ofthe stopcocks should also be wiped with alcohol when a sample of perfusate is collected. The lumen of the apparatus tubing is disinfected by perfusion first with 10% Clorox (Oakland, CA) for 10 min. Then, 1 L of 0.9% sodium chloride is used to irrigate each perfusion circuit. When the tubing is empty, the collection basin is filled with sterilized Krebs buffer 2.0 -c: 1.5 E -- BILE STERILE E ro 1.0 BILE NONSTERILE "- P < .001 O"l --:J 0.5 -lKl~-----D----~K-----K------r-----D----15 15 45 75 105 135 LENGTH OF PERFUSION (min) Figure 6. Bile production during sterile and nonsterile perfusions. Bile production is much higher in the modified IPRL system and stays stable up to 180 min. (N = 6 sterile and 5 nonsterile perfusions.) Isolated Perfused Rat Liver 371 solution containing O.S g of cefamandol/2S0 mL of perfusate (Eli Lilly, Indianapo-lis, IN). Samples for culture are taken after 10 min by injection of 1 mL of perfusate into trypticase soy agar with S% sheep blood (Becton Dickinson, Cockeysville, MD). We have found that, if we follow each of the above steps, an average of IS, and up to 20, consecutive perfusions with the same tubing will be sterile. The production of TNF, SGOT, and SGPT (Figs 3-S) and bile production (Fig 6) are reproducible. There is a gradual rise over small ranges in levels ofTNF, SOOT, and SGPT due to recircula-tion. The leakage of enzymes is much less than before modification of the apparatus (Figs 3-6), especially after the first 90 min of reperfusion. Hepatocytes and nonparen-chymal cells are intact histologically throughout 120 min of perfusion. Advantages of the Modified Perfusion System 1. The perfusion system is further simplified and made convenient to use and maintain because of a disposable inflow filter and a bubble trap that is inex-pensive and commercially available. 2. The 9S% 02/S% CO2 mixture is more efficiently used, because two simulta-neous perfusions in parallel can be completed in less time than it takes to do two separate sequential perfusions. 3. The length of tubing used for each perfusion is kept at a minimum, because the collection basin efficiently warms the perfusate. This simplifies the process of sterilization and lessens the cost of the apparatus. 4. The effluent collection basin and its cover are easily disinfected, which makes this a closed perfusion system. This avoids contamination during the perfusion. In summary, we have improved the isolated rat liver perfusion system such that not only can two organs be perfused simultaneously, but the tubing and perfusates can be reliably sterilized for up to 20 consecutive perfusions. A gradual rise in trans-aminase levels occurs over a small range due to recirculation. Bile production is greatly increased over perfusions done before the present modifications and is stable throughout a perfusion. References I. Miller LL. History of isolated liver perfusion and some still unsolved problems. In: Bartosek I, Guaitani A, Miller AA, eds. Isolated Liver Perfusion and Its Application. New York: Raven; 1972:1-9. 2. Wolkoff AM, Johansen KL, Goeser T. The isolated perfused rat liver: preparation and application. Anal Biochem. 1987;167:1-14. 3. Gores GJ, Kost LJ, Larusso NF. The isolated perfused rat liver: conceptual and practical considerations. Hepato!ogy. 1986;6(3):511-517. 4. Miller LL, Bly CG, Watson ML, et a1. The dominant role of the liver in plasma protein synthesis. J Exp Med. 1951;94:431-453. 5. Flock EV, Owen CA Jr. Metabolism of thyroid hormones and some derivatives in isolated perfused rat liver. Am J Physioi. 1965;209: 1039-1045. 372 T. Liu et at. 6. Ontell SJ, Colella MS, Horowitz T, et al. Applications of isolated perfused rat liver in transplantation research. J Invest Surg. 1988; 1 :25-27. 7. Miller LL, Bly CG, Watson ML, et al. The dominant role of the liver in plasma protein synthesis. J ExpMed. 1951;94:431-453. 8. Hamilton RL, Berry MN, William Me. A simple and inexpensive membrane lung for small organ perfusion. J Lipid Res. 1974; 15: 182-186. 

A modified apparatus for dual, sterilized, isolated perfusion of the rat liver

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A modified apparatus for dual, sterilized, isolated perfusion of the rat liver

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