- Email: [email protected]
A Comparison of a Sucrose-Based Solution with Other Preservation Media for Cold Storage of Isolated Hepatocytes
Cryobiology 41, 315–318 (2000) doi:10.1006/cryo.2000.2286, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION A Comparison of a Sucrose-Based Solution with Other Preservation Media for Cold Storage of Isolated Hepatocytes I. V. Shanina,* L. P. Kravchenko,* B. J. Fuller,† and V. I. Grischenko* *Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 23 Pereyaslavskaya St., Kharkov 310015, Ukraine; and †University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, United Kingdom A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall’s citrate solution, with Dulbecco’s medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco’s medium and Marshall’s citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane “blebs” and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 ⫾ 7%; 72 h, 65 ⫾ 6%) and UW solution (48 h, 72 ⫾ 5%; 72 h, 63 ⫾ 4%) than in Marshall’s citrate (48 h, 31 ⫾ 5%; 72 h, 10 ⫾ 1%) and Dulbecco’s medium (48 h, 8 ⫾ 2%; 72 h, 5 ⫾ 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes. © 2000 Academic Press
Key Words: hepatocytes; hypothermic storage; morphological parameters.
previous studies suggested that a sucrose-based solution (SBS) might be helpful for cold preservation of isolated hepatcoytes (6), and this approach has been supported by some work on whole-organ preservation (1) in which sucrose was found to be a good impermeant to protect viability. In the current work, we have compared the effects of storage at hypothermia on rat hepatocytes for up to 72 h in four solutions (Dulbecco’s solution as a control, and then in Marshall’s citrate solution, UW solution, and the sucrose-based solution) and assessed the membrane integrity of the cells by dye exclusion and morphology by light microscopy. Hepatocytes were isolated from liver of rats (200 –300 g body weight) under anesthesia according to our method of perfusion with a sucrose medium containing 4 mM EDTA (6). Studies were performed on five cell isolates. Analysis of the suspensions of 1–5 ⫻ 10 6 cells/ml was made in duplicate using 0.6% trypan blue solution, prepared in physiological solution (NaCl, 0.15 M; pH 7.4). This was
Use of isolated hepatocytes as a model system in cryobiological research allows a better understanding of the mechanisms of cell damage under hypothermic temperatures, which is important for the development of reliable methods of long-term organ preservation (8). It also is important for a better understanding of hepatocyte storage for cell transplantation, which is currently undergoing reevaluation in the clinical setting (2). Until now, the solution developed by the University of Wisconsin (UW) for organ preservation has been recognized as an effective medium for preservation of hepatocytes following cold hypoxia (7). However, this solution contains several ingredients which either are expensive or cannot easily be obtained in some countries, such as the Ukraine. To facilitate studies on isolated hepatocytes, a less complex but efficacious storage solution is required. Our Received March 6, 2000; accepted October 31, 2000. Parts of the collaborative study were made possible by support of the Wellcome Trust Grant N 056648/Z/99/Z. 315
0011-2240/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
316
BRIEF COMMUNICATION TABLE 1 Composition of the Solutions Used for Hepatocyte Preservation
Potassium phosphate (monobasic) Sodium chloride Sodium phosphate (dibasic) Calcium chloride Magnesium sulfate Mannitol Sucrose Raffinose Potassium citrate Sodium citrate Magnesium chloride Potassium chloride Sodium phosphate (monobasic) Potassium hydroxide Sodium hydroxide Lactobionic acid Glutathione (reduced) Allopurinol BSA pH at 4°C
Dulbecco’s solution
Marshall’s solution
Sucrose-based solution
Mod UW solution
0.2 8 1 0.1 — — — — — — 0.2 0.2 1.5 — — — — — — 7.3
— — — — 10 33.8 — — 8.6 8.6 — — — — — — — — — 7.1
0.06 0.372 0.067 0.17 0.12 — 85 — — — — — — — — — — — 10 7.4
3.55 — — — 2.62 — — 17.8 — — — — — 5.85 1.54 38.61 0.96 0.14 — 7.4
Note. Values are in g/liter.
mixed in equal volumes with cell suspensions, and the exposure time was not less than 3 min at room temperature. The calculation was performed after 200 cells were counted. Initial viability of the cells was 91 ⫾ 5%, as judged by trypan blue exclusion. Hypothermic storage of the isolated hepatocytes was achieved by suspension of the cells in the corresponding solution at a volume of 3 ml with a cell concentration of 3–5 ⫻ 10 6/ml at 0 –2°C in ice. Four solutions were investigated and their compositions are shown in Table 1. Dye exclusion was performed at 30 min and at 24, 48, and 72 h of hypothermic storage. The cell morphologies were recorded using a light microscope equipped with a video camera, connected with an IBM PC. Statistical significance for dye exclusion was assessed using a one-way ANOVA t test. Significance levels of P ⬍ 0.01 only were reported to allow for multiple comparisons (Bonferoni correction). In all groups, dye exclusion was similar after 30-min cold exposure and in the range of 80 –
90%. The cells at this stage exhibited normal spherical morphology, with a distinct cell membrane. Over the next 48 –72 h, however, there was a clear difference in cell stability in the different solutions. In both Dulbecco’s solution and Marshall’s citrate solution, there was a continued and significant fall in dye excluding ability, such that by 72 h dye exclusion had fallen to 5 ⫾ 0.5 and 10 ⫾ 0.9%, respectively (Fig. 1). In the other two solutions (mod UW and SBS), there was a different long-term outcome, with dye exclusion maintained at higher values in both. After 48 and 72 h, dye exclusion was 72 ⫾ 5 and 63 ⫾ 4% in the mod UW solution. At the same time, the viability of liver cells stored in the SBS remained quite high (75 ⫾ 7.1 and 65 ⫾ 5.9%) at both these stages of cold storage (48 and 72 h, respectively). At 72 h, both mod UW- and SBS-stored cells had higher dye excluding abilities than cells in Dulbecco’s solution (P ⬍ 0.01), and similarly both were higher than cells stored in Marshall’s citrate solution (P ⬍ 0.01).
BRIEF COMMUNICATION
317
and integrity of the plasma membrane of rat liver cells to 72 h at hypothermia. The role of effective nonpermeating agents in organ preservation has been a key area of investigation in organ preservation research over the past three decades (3), and it is becoming apparent that these agents may exert several subtle beneficial effects by achieving better control of cell vol-
FIG. 1. Trypan blue exclusion of isolated hepatocytes at the various stages of hypothermic storage in the different preservation solutions. Data are means ⫾ SE; n ⫽ 5. Initial values are for cells exposed for 30 min to each solution. Cells were exposed to UW (Œ), SBS (■), Marshall’s solution (䊐), and Dulbecco’s solution (}). SBS- and UW-stored cells showed higher dye exclusion (P ⬍ 0.01) than cells in Dulbecco’s solution or Marshall’s solution after 48 and 72 h.
In parallel to the differences in dye exclusion, there was a divergence in appearance of the cells stored in Dulbecco’s or Marshall’s citrate and the cells stored in the other two solutions, which became more pronounced with time. An example of each type of morphology is shown in Fig. 2. In Dulbecco’s and Marshall’s solutions, a high percentage of cells exhibited large blebs on the plasma membrane by 72 h cold storage (Fig. 2A). This did not influence dye exclusion directly, as many of the cells that exhibited blebs also excluded trypan blue. However, it remains questionable whether such grossly abnormal cells could recover cohesive metabolic function on rewarming, even though they were excluding trypan blue at this stage. In the two other solutions, the cells (both dyeexcluding and dye-permeable cells) retained a spherical morphology, and where blebs could be recognised, these were seen as much smaller extrusions of the plasma membrane (Fig. 2B). Our results confirm previous studies which demonstrated that UW solution, with its unique mixture of nonpermeating agents, is a good preservation medium for storage of isolated hepatocytes (7, 10). We have additionally demonstrated that a simpler, sucrose-based solution is equally effective at maintaining morphology
FIG. 2. (A) Hepatocytes at the 72-h stage of hypothermic storage in Marshall’s hypertonic citrate solution. Note the huge blebs on the plasma membrane of the hepatocytes (magnification ⫻400). (B) Hepatocytes after 72-h storage in SBS solution. The cells have retained their spherical morphology and only small blebs are seen on the plasma membrane of some cells (arrows; magnification ⫻400).
318
BRIEF COMMUNICATION
ume at hypothermia. These include avoiding activation of proteolytic enzymes within cells and the development of pro-apoptotic stimuli (4). Sucrose-based solutions have been shown to be effective in preserving function in whole organs at hypothermia (1), and the use of the SBS solution for storage of isolated hepatocytes may facilitate hepatocyte research in situations in which the more complex UW solution (or its important ingredients) is not easily available, as in some countries outside the West. We felt that it was important to establish the morphology and membrane integrity of liver cells in SBS at hypothermia because no data were available for these parameters. The ability of cells to extrude trypan blue dye is only one parameter with which to assess structural integrity and does not necessarily provide information about more subtle postrecovery functions such as protein synthesis (5). However, breaching of the hepatocyte plasma membrane permeability to such an extent that trypan blue dye can enter the cell compartments during hypothermic storage probably represents a catastrophic destruction of the cell membrane, with rewarming to body temperatures likely to cause further damage. It is also becoming established that cold injury itself is complex in relation to time of exposure, with early and late components of damage (10), which may be reflected in our studies by the development of the gross membrane blebbing. More detailed investigations will now be required to assess the metabolic and molecular characteristics of hepatocytes after rewarming from the SBS solution and whether these are comparable to those seen with cells stored in UW solution. These studies are currently underway.
ACKNOWLEDGMENT The authors thank Dr. Alexander Sukach for technical assistance with the light microscopy and image processing. REFERENCES 1. Ahmed, N., Kashi, H., Helmy, H., Hoddingham, J., Potts, D., and Lodge, P. Renal preservation with phosphate-buffered sucrose: A comparison with hyperosmolar citrate in a prospective trial. Transplant. Proc. 29, 355–356 (1997). 2. Fox, I., Roy Chowdhury, J., Kaufman, S., Goetzen, T., Roy Chowdhury, N., Warketin, P., Dorko, K., Sauter, B., and Strom, C. Treatment of Criggler–Najjar Syndrome Type 1 with hepatocyte transplantation. N. Engl. J. Med. 338, 1422–1426 (1998). 3. Fuller, B. J. Effects of cooling on mammalian cells. In “Clinical Applications of Cryobiology” (B. J. Fuller and B. W. W. Grout, Eds.), pp. 1–21. CRC Press, Boca Ration, FL, 1991. 4. Gao, W., Bentley, R., Madden, J., and Clavien, P. Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation. Hepatology 27, 1652–1660 (1998). 5. Innes, G., Fuller, B., and Hobbs, K. Functional testing of hepatocytes following recovery from cryopreservation. Cryobiology 25, 23–30 (1988). 6. Kravchenko, L., Andrienko, A., Belous, A., and Shanina, I. Long-term storage of isolated liver cells at hypothermia. Cryo-Letters 15, 135–145 (1994). 7. Mamprin, M., Guibert, E., and Rodriguez, J. Glutathione content during rinsing and rewarming of rat hepatocytes preserved in University of Wisconsin solution. Cryobiology 40, 270 –276 (2000). 8. Marsh, D., Vreugdenhil, P., Mack, V., Belzer, F. O., and Southard, J. Hypothermic preservation of hepatocytes. 1. Role of cell swelling. Cryobiology 26, 524 –534 (1989). 9. Southard, J., Van Gulik, T., and Ametani, M. Importent components of the UW-solution. Transplantation 49, 251–257 (1990). 10. Vreugdenhil, P. K., Ametani, M. S., Haworth, R. A., and Southard, J. H. Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes. Transplantation 67, 1468 –1473 (1999).
BRIEF COMMUNICATION A Comparison of a Sucrose-Based Solution with Other Preservation Media for Cold Storage of Isolated Hepatocytes I. V. Shanina,* L. P. Kravchenko,* B. J. Fuller,† and V. I. Grischenko* *Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 23 Pereyaslavskaya St., Kharkov 310015, Ukraine; and †University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, United Kingdom A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall’s citrate solution, with Dulbecco’s medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco’s medium and Marshall’s citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane “blebs” and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 ⫾ 7%; 72 h, 65 ⫾ 6%) and UW solution (48 h, 72 ⫾ 5%; 72 h, 63 ⫾ 4%) than in Marshall’s citrate (48 h, 31 ⫾ 5%; 72 h, 10 ⫾ 1%) and Dulbecco’s medium (48 h, 8 ⫾ 2%; 72 h, 5 ⫾ 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes. © 2000 Academic Press
Key Words: hepatocytes; hypothermic storage; morphological parameters.
previous studies suggested that a sucrose-based solution (SBS) might be helpful for cold preservation of isolated hepatcoytes (6), and this approach has been supported by some work on whole-organ preservation (1) in which sucrose was found to be a good impermeant to protect viability. In the current work, we have compared the effects of storage at hypothermia on rat hepatocytes for up to 72 h in four solutions (Dulbecco’s solution as a control, and then in Marshall’s citrate solution, UW solution, and the sucrose-based solution) and assessed the membrane integrity of the cells by dye exclusion and morphology by light microscopy. Hepatocytes were isolated from liver of rats (200 –300 g body weight) under anesthesia according to our method of perfusion with a sucrose medium containing 4 mM EDTA (6). Studies were performed on five cell isolates. Analysis of the suspensions of 1–5 ⫻ 10 6 cells/ml was made in duplicate using 0.6% trypan blue solution, prepared in physiological solution (NaCl, 0.15 M; pH 7.4). This was
Use of isolated hepatocytes as a model system in cryobiological research allows a better understanding of the mechanisms of cell damage under hypothermic temperatures, which is important for the development of reliable methods of long-term organ preservation (8). It also is important for a better understanding of hepatocyte storage for cell transplantation, which is currently undergoing reevaluation in the clinical setting (2). Until now, the solution developed by the University of Wisconsin (UW) for organ preservation has been recognized as an effective medium for preservation of hepatocytes following cold hypoxia (7). However, this solution contains several ingredients which either are expensive or cannot easily be obtained in some countries, such as the Ukraine. To facilitate studies on isolated hepatocytes, a less complex but efficacious storage solution is required. Our Received March 6, 2000; accepted October 31, 2000. Parts of the collaborative study were made possible by support of the Wellcome Trust Grant N 056648/Z/99/Z. 315
0011-2240/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
316
BRIEF COMMUNICATION TABLE 1 Composition of the Solutions Used for Hepatocyte Preservation
Potassium phosphate (monobasic) Sodium chloride Sodium phosphate (dibasic) Calcium chloride Magnesium sulfate Mannitol Sucrose Raffinose Potassium citrate Sodium citrate Magnesium chloride Potassium chloride Sodium phosphate (monobasic) Potassium hydroxide Sodium hydroxide Lactobionic acid Glutathione (reduced) Allopurinol BSA pH at 4°C
Dulbecco’s solution
Marshall’s solution
Sucrose-based solution
Mod UW solution
0.2 8 1 0.1 — — — — — — 0.2 0.2 1.5 — — — — — — 7.3
— — — — 10 33.8 — — 8.6 8.6 — — — — — — — — — 7.1
0.06 0.372 0.067 0.17 0.12 — 85 — — — — — — — — — — — 10 7.4
3.55 — — — 2.62 — — 17.8 — — — — — 5.85 1.54 38.61 0.96 0.14 — 7.4
Note. Values are in g/liter.
mixed in equal volumes with cell suspensions, and the exposure time was not less than 3 min at room temperature. The calculation was performed after 200 cells were counted. Initial viability of the cells was 91 ⫾ 5%, as judged by trypan blue exclusion. Hypothermic storage of the isolated hepatocytes was achieved by suspension of the cells in the corresponding solution at a volume of 3 ml with a cell concentration of 3–5 ⫻ 10 6/ml at 0 –2°C in ice. Four solutions were investigated and their compositions are shown in Table 1. Dye exclusion was performed at 30 min and at 24, 48, and 72 h of hypothermic storage. The cell morphologies were recorded using a light microscope equipped with a video camera, connected with an IBM PC. Statistical significance for dye exclusion was assessed using a one-way ANOVA t test. Significance levels of P ⬍ 0.01 only were reported to allow for multiple comparisons (Bonferoni correction). In all groups, dye exclusion was similar after 30-min cold exposure and in the range of 80 –
90%. The cells at this stage exhibited normal spherical morphology, with a distinct cell membrane. Over the next 48 –72 h, however, there was a clear difference in cell stability in the different solutions. In both Dulbecco’s solution and Marshall’s citrate solution, there was a continued and significant fall in dye excluding ability, such that by 72 h dye exclusion had fallen to 5 ⫾ 0.5 and 10 ⫾ 0.9%, respectively (Fig. 1). In the other two solutions (mod UW and SBS), there was a different long-term outcome, with dye exclusion maintained at higher values in both. After 48 and 72 h, dye exclusion was 72 ⫾ 5 and 63 ⫾ 4% in the mod UW solution. At the same time, the viability of liver cells stored in the SBS remained quite high (75 ⫾ 7.1 and 65 ⫾ 5.9%) at both these stages of cold storage (48 and 72 h, respectively). At 72 h, both mod UW- and SBS-stored cells had higher dye excluding abilities than cells in Dulbecco’s solution (P ⬍ 0.01), and similarly both were higher than cells stored in Marshall’s citrate solution (P ⬍ 0.01).
BRIEF COMMUNICATION
317
and integrity of the plasma membrane of rat liver cells to 72 h at hypothermia. The role of effective nonpermeating agents in organ preservation has been a key area of investigation in organ preservation research over the past three decades (3), and it is becoming apparent that these agents may exert several subtle beneficial effects by achieving better control of cell vol-
FIG. 1. Trypan blue exclusion of isolated hepatocytes at the various stages of hypothermic storage in the different preservation solutions. Data are means ⫾ SE; n ⫽ 5. Initial values are for cells exposed for 30 min to each solution. Cells were exposed to UW (Œ), SBS (■), Marshall’s solution (䊐), and Dulbecco’s solution (}). SBS- and UW-stored cells showed higher dye exclusion (P ⬍ 0.01) than cells in Dulbecco’s solution or Marshall’s solution after 48 and 72 h.
In parallel to the differences in dye exclusion, there was a divergence in appearance of the cells stored in Dulbecco’s or Marshall’s citrate and the cells stored in the other two solutions, which became more pronounced with time. An example of each type of morphology is shown in Fig. 2. In Dulbecco’s and Marshall’s solutions, a high percentage of cells exhibited large blebs on the plasma membrane by 72 h cold storage (Fig. 2A). This did not influence dye exclusion directly, as many of the cells that exhibited blebs also excluded trypan blue. However, it remains questionable whether such grossly abnormal cells could recover cohesive metabolic function on rewarming, even though they were excluding trypan blue at this stage. In the two other solutions, the cells (both dyeexcluding and dye-permeable cells) retained a spherical morphology, and where blebs could be recognised, these were seen as much smaller extrusions of the plasma membrane (Fig. 2B). Our results confirm previous studies which demonstrated that UW solution, with its unique mixture of nonpermeating agents, is a good preservation medium for storage of isolated hepatocytes (7, 10). We have additionally demonstrated that a simpler, sucrose-based solution is equally effective at maintaining morphology
FIG. 2. (A) Hepatocytes at the 72-h stage of hypothermic storage in Marshall’s hypertonic citrate solution. Note the huge blebs on the plasma membrane of the hepatocytes (magnification ⫻400). (B) Hepatocytes after 72-h storage in SBS solution. The cells have retained their spherical morphology and only small blebs are seen on the plasma membrane of some cells (arrows; magnification ⫻400).
318
BRIEF COMMUNICATION
ume at hypothermia. These include avoiding activation of proteolytic enzymes within cells and the development of pro-apoptotic stimuli (4). Sucrose-based solutions have been shown to be effective in preserving function in whole organs at hypothermia (1), and the use of the SBS solution for storage of isolated hepatocytes may facilitate hepatocyte research in situations in which the more complex UW solution (or its important ingredients) is not easily available, as in some countries outside the West. We felt that it was important to establish the morphology and membrane integrity of liver cells in SBS at hypothermia because no data were available for these parameters. The ability of cells to extrude trypan blue dye is only one parameter with which to assess structural integrity and does not necessarily provide information about more subtle postrecovery functions such as protein synthesis (5). However, breaching of the hepatocyte plasma membrane permeability to such an extent that trypan blue dye can enter the cell compartments during hypothermic storage probably represents a catastrophic destruction of the cell membrane, with rewarming to body temperatures likely to cause further damage. It is also becoming established that cold injury itself is complex in relation to time of exposure, with early and late components of damage (10), which may be reflected in our studies by the development of the gross membrane blebbing. More detailed investigations will now be required to assess the metabolic and molecular characteristics of hepatocytes after rewarming from the SBS solution and whether these are comparable to those seen with cells stored in UW solution. These studies are currently underway.
ACKNOWLEDGMENT The authors thank Dr. Alexander Sukach for technical assistance with the light microscopy and image processing. REFERENCES 1. Ahmed, N., Kashi, H., Helmy, H., Hoddingham, J., Potts, D., and Lodge, P. Renal preservation with phosphate-buffered sucrose: A comparison with hyperosmolar citrate in a prospective trial. Transplant. Proc. 29, 355–356 (1997). 2. Fox, I., Roy Chowdhury, J., Kaufman, S., Goetzen, T., Roy Chowdhury, N., Warketin, P., Dorko, K., Sauter, B., and Strom, C. Treatment of Criggler–Najjar Syndrome Type 1 with hepatocyte transplantation. N. Engl. J. Med. 338, 1422–1426 (1998). 3. Fuller, B. J. Effects of cooling on mammalian cells. In “Clinical Applications of Cryobiology” (B. J. Fuller and B. W. W. Grout, Eds.), pp. 1–21. CRC Press, Boca Ration, FL, 1991. 4. Gao, W., Bentley, R., Madden, J., and Clavien, P. Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation. Hepatology 27, 1652–1660 (1998). 5. Innes, G., Fuller, B., and Hobbs, K. Functional testing of hepatocytes following recovery from cryopreservation. Cryobiology 25, 23–30 (1988). 6. Kravchenko, L., Andrienko, A., Belous, A., and Shanina, I. Long-term storage of isolated liver cells at hypothermia. Cryo-Letters 15, 135–145 (1994). 7. Mamprin, M., Guibert, E., and Rodriguez, J. Glutathione content during rinsing and rewarming of rat hepatocytes preserved in University of Wisconsin solution. Cryobiology 40, 270 –276 (2000). 8. Marsh, D., Vreugdenhil, P., Mack, V., Belzer, F. O., and Southard, J. Hypothermic preservation of hepatocytes. 1. Role of cell swelling. Cryobiology 26, 524 –534 (1989). 9. Southard, J., Van Gulik, T., and Ametani, M. Importent components of the UW-solution. Transplantation 49, 251–257 (1990). 10. Vreugdenhil, P. K., Ametani, M. S., Haworth, R. A., and Southard, J. H. Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes. Transplantation 67, 1468 –1473 (1999).