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Respiratory Activity of Isolated Rat Hepatocytes Following Cold Storage and Subsequent Rewarming: A Comparison of Sucrose-Based and University of Wisconsin Solutions
Cryobiology 42, 218–221 (2001) doi:10.1006/cryo.2001.2317, available online at http://www.academicpress.com on
BRIEF COMMUNICATION Respiratory Activity of Isolated Rat Hepatocytes Following Cold Storage and Subsequent Rewarming: A Comparison of Sucrose-Based and University of Wisconsin Solutions L. P. Kravchenko,*† A. Yu. Petrenko,* A. Yu. Somov,* V. I. Grischenko,* and B. J. Fuller‡,1 *Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of the Ukraine, 23 Pereyaslavskaya Str., Kharkov 61015, Ukraine, and †University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, United Kingdom Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 ⫾ 3 and 23 ⫾ 3 nmol O2/min/106 cells, respectively) compared to control cell values (31 ⫾ 3 and 33 ⫾ 5 nmol O2/min/106 cells; P ⬍ 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 ⫾ 6 and 40 ⫾ 5 nmol O2/min/106 cells; for control cells: 43 ⫾ 5 and 46 ⫾ 6 nmol O2/min/106 cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended. Key Words: isolated hepatocytes; hypothermic storage; respiratory function; sucrose-based solution; University of Wisconsin solution.
The search for effective and simple preservation solutions for both whole liver and isolated hepatocytes has been an active area of research over the past decade. The University of Wisconsin (UW) solution has significantly improved the practice of liver preservation for transplantation since its first development (10) and is based on a mixture of impermeants such as lactobionate and raffinose, which effectively control cell volume during hypothermia. We (4, 9) and others (1) have been interested in the use of sucrose as an alternative osmotic agent for cell and organ storage, for a variety of reasons, including
Received January 2, 2001; accepted April 27, 2001; published online July 18, 2001. † Deceased 1 To whom correspondence should be addressed. This work was a collaborative study made possible by the support of the Wellcome Trust Grant 056648/Z/99/Z. 0011-2240/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
easier availability in some countries where organ preservation procedures are only recently being developed. An additional reason in the case of isolated hepatocytes would be that in situations in which the cells might be used for transplantation, periods of both cold storage (when the liver tissues are initially harvested for cell isolation) and cryopreservation (when sufficient cell banks are accumulated for clinical use) are necessary (2). A sucrose-based medium has been found to be effective for both hypothermic storage as judged by morphological criteria (9) and freeze preservation (7) of isolated hepatocytes in our experience, and the use of a single solution for both steps may simplify the protocols for establishment of an hepatocyte bank. However, cell morphology and membrane integrity are only basic indices of functional survival after hypothermia, and the current studies were undertaken to evaluate hepatocyte respira-
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tion as a more stringent metabolic test (3), both immediately after cold preservation and after a period of rewarming to allow expression of any latent damage during reperfusion. Isolation of rat hepatocytes was achieved by the nonenzymatic method described in our previous papers. Briefly, rat livers were isolated under anesthesia induced by injection (I.P. administration of sodium pentabarbitone) and were perfused with a sucrose–EDTA medium (4). The minced tissue was then subjected to controlled mechanical disaggregation by vibration (6). Cells were recovered by centrifugation, washed, counted in a hemocytometer, and resuspended in the relevant storage medium. Hypothermic storage of the samples was carried out in glass vials (at a cell concentration of 3–5 ⫻ 106/ml and a volume of 5 ml) at a temperature of 4°C for 45 min (the period of initial isolation, washing, and resuspension as control) and after 24 and 48 h. In this study we compared the sucrose-based solution (SBS) (4, 9), a solution based on sucrose and containing a low ionic content, with the more widely used UW solution (10). For assessment of the stability of fresh and stored hepatocytes, incubations were conducted in Krebs–Ringer bicarbonate (pH 7.4). Cell suspensions were studied either immediately after different stages of cold storage or after subsequent periods of 60 min normothermic incubation (designated as rewarming) with shaking to mimic reperfusion injury. Cell membrane integrity was estimated by the exclusion of vital dye (trypan blue at a final concentration of 0.3%) as described previously (4, 9). Endogenous respiration was measured by polarographic means in a 1-ml cuvette at 36°C, using a Clark’s platinum oxygen electrode (in Krebs–Ringer– phosphate solution, pH 7.4, as described (6)). Values were calculated per 106 cells in the sample. A further metabolic evaluation of cell integrity was made by stimulation of respiration by succinate (2 mM). This substrate slowly crosses the plasma membrane of normal hepatocytes and causes little stimulation of the endogenous respiration rate. However, in cells with compromised membrane integrity, succinate may more easily pass into the cells (5) and pro-
219
duce a marked stimulation of respiration greater than twofold, as shown in chemically permeabilized cells (3). The total measurement time, including the succinate test, was 7 min. The data were processed with ORIGIN software, statistical significance was determined by the Student–Fisher method, and a value of P ⬍ 0.01 was accepted as significant to allow for multiple comparisons between groups (Bonferroni correction). The initial suspension of freshly isolated hepatocytes contained not less than 87–95% of cells excluding vital dye (mean value of 93 ⫾ 4%, see Table 1). Freshly isolated hepatocytes possessed a high level of endogenous respiration after exposure to SBS (Table 1), which statistically increased following the period of 60 min normothermic incubation. Similar levels of endogenous respiration for freshly isolated cells were demonstrated in previous studies (4, 5). Respiration indices in the presence of succinate immediately after cell isolation demonstrated a slight increase (expressed as a ratio of the rates Vsucc./Vend.), were recorded as mean values of 1.12 for fresh cells and 1.13 for fresh cells after rewarming, and again are in line with the literature data (5). After the first 24 h of storage of isolated hepatocytes in SBS, dye exclusion remained high at a mean value of 85%, whereas endogenous respiratory activity statistically decreased (P ⬍ 0.01) by 25% (Table 1). However, after incubation at 37°C, respiration recovered to values similar to those in fresh cells. Exposure to succinate caused a significant increase in respiration after cold storage as measured by the ratios Vsucc./Vend. of 1.60 (P ⬍ 0.01), whereas it remained high (at mean values of 1.50 after incubation) but not significantly different from than seen with fresh cells. After 48 h storage in SBS, cell dye exclusion was decreased by a significant but relatively small degree to mean values of 78 ⫾ 4% after storage and 75 ⫾ 4% after rewarming (Table 1). Nevertheless, respiratory activity of the rewarmed cells was again reduced after cold storage compared to that of the controls, and, more notably, no recovery of respiration followed the period of normothermic incuba-
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tion. In this situation after 48 h storage, exposure to succinate caused a further sustained rise in respiration (to mean values of 1.88 and 1.71 of the endogenous rate after incubation—Table 1; P ⬍ 0.01 for each compared to respective fresh cell values). For cells exposed to UW solution, a very similar outcome was obtained (see Table 1). Rewarming for 60 min in freshly isolated cells allowed an increase in endogenous respiration (from mean values of 33 ⫾ 5 to 46 ⫾ 6 nmol O2/min/106 cells), whereas exposure to succinate again caused a small increase for fresh cells immediately tested (Vsucc./Vend. of 1.14) and after rewarming (Vsucc./Vend. of 1.12). Endogenous respiration after 24 h cold storage was reduced compared to that in fresh cells, but recovered to levels similar to that in controls after 60 min rewarming. Whereas dye excluding ability was little changed after 24 h storage in UW solution, exposure to succinate again caused a more notable increase in respiration (Vsucc./Vend. to mean values of 1.59; P ⬍ 0.01 compared to fresh cells) in cells tested immediately after storage and the slightly lower mean value of 1.38 after rewarming. As seen for the SBS-
stored cells, 48 h of hypothermic storage caused a significant reduction in respiration which did not recover after normothermic rewarming. A small but significant decrease in dye-excluding cells followed 48 h storage in UW solution, whereas there was a marked stimulation of respiration in the presence of succinate both after cold exposure alone and after 60 min rewarming (Vsucc./Vend. mean values of 1.86 and 1.81; both P ⬍ 0.01 compared to respective values for fresh cells—Table 1). The results reported here demonstrate several important points about preservation of isolated hepatocytes. Endogenous respiration in intact hepatocytes is a good index of metabolic integrity and is depressed in pathological states such as septic shock (3). Our results demonstrated that the procedure of isolation and washing resulted in cells with a slightly depressed respiratory activity, and this returned to values typical for isolated hepatocytes after rewarming at 37°C. This may reflect loss of soluble substrates and cofactors during isolation, which were replenished during preincubation. When the cells were stored in either SBS or UW solution for 24 h, respiration was further
TABLE 1 Dye Exclusion and Indices of the Respiration Activity (nmol O2/min/106 cells) in Freshly Isolated Hepatocytes, Hepatocytes after Cold Storage Alone in Either SBS or UW Solutions, and after Storage with Subsequent Normothermic Incubation for 60 min Media
SBS
UW
Indices
Endogenous respiration Respiration, stimulated by succinate Vsucc./Vend. Viability, % Endogenous respiration Respiration, stimulated by succinate Vsucc./Vend. Viability, %
Freshly isolated hepatocytes, 45 min 4°C
Incubated 37°C, 60 min
31 ⫾ 3
43 ⫾ 5*
35 ⫾ 4
49 ⫾ 6*
1.12 ⫾ 0.07 95 ⫾ 4 33 ⫾ 5
1.13 ⫾ 0.05 93 ⫾ 3 46 ⫾ 6*
38 ⫾ 4
50 ⫾ 5*
1.14 ⫾ 0.06 95 ⫾ 4
1.12 ⫾ 0.06 96 ⫾ 6
Cold storage, 24 h
4°C
Cold storage, 48 h
Incubated 37°C, 60 min
4°C
21 ⫾ 3⫹
41 ⫾ 6*
22 ⫾ 3⫹
17 ⫾ 3**
34 ⫾ 5
60 ⫾ 8*
41 ⫾ 5
29 ⫾ 5
1.60 ⫾ 0.07⫹ 88 ⫾ 4 23 ⫾ 3⫹ 36 ⫾ 6 1.60 ⫾ 0.09⫹ 83 ⫾ 5
1.50 ⫾ 0.13 88 ⫾ 5 40 ⫾ 5* 55 ⫾ 6* 1.40 ⫾ 0.08 86 ⫾ 4
1.90 ⫾ 0.09⫹ 78 ⫾ 4⫹ 23 ⫾ 3⫹ 43 ⫾ 5 1.90 ⫾ 0.07⫹ 76 ⫾ 4⫹
Incubated 37°C, 60 min
1.70 ⫾ 0.12⫹ 75 ⫾ 4⫹ 21 ⫾ 4** 38 ⫾ 5 1.80 ⫾ 0.13⫹ 74 ⫾ 4+
Note. Also given are the ratios of the rates of respiration in the presence or absence of succinate (Vsucc./Vend.). Values are given as means ⫾ SE (n ⫽ 6–12). Denotes groups that are statistically significant in comparison with * the respective groups exposed only to cold storage; ⫹ the respective values for freshly isolated cells; and ** the respective values for groups exposed to cold storage and subsequent normothermic incubation. For purposes of the Ukraine, the methods for isolating cells and producing the sucrose medium have been patent registered (N 14080:1997 & N 16946:1997).
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BRIEF COMMUNICATION
reduced, but again recovered to within the normal range after rewarming. However, by 48 h cold storage, respiration did not recover after preincubation, suggesting severe disruption to oxidative metabolism. This is likely to be a multifactorial damage resulting from several factors including both depletion of cell solutes and disruption of mitochondrial architecture (8). The depression of respiration after prolonged storage was much greater (⬍50% in cells after 48 h storage and preincubation compared with control values) than might be expected by the small changes at the same time in dye exclusion and serves as another reminder that morphology and metabolism are not always linked in cells in the acute poststorage period after hypothermic preservation. The succinate exposure test was more predictive of the progressive damage experienced by the hepatocytes as storage times lengthened and already showed a marked deviation from that of fresh cells in the hepatocytes studied after cold storage alone even by 24 h storage, when endogenous respiration after rewarming and dye exclusion were both similar to those values for control cells. By 48 h, in either preservation solution, the succinate stimulation of respiration had reached levels approaching those in permeabilized cells which have lost metabolic control. It was also of note that, under these conditions of study, the damage expressed during longer periods of cold storage related more to the preservation time than to reperfusion damage (i.e., the increase in succinate-stimulated respiration was similar in the hepatocytes both at the end of the cold period and after rewarming at 37°C). The presented data also confirm that the SBS solution was, to all intents and purposes, an equally effective solution for hepatocyte cold storage compared with the UW solution. In situations in which liver tissues are being harvested and processed for hepatocyte banking for trans-
plantation, the SBS solution could provide a single solution for both organ cold storage and subsequent cryopreservation, simplifying the required protocols. Further studies will be needed to investigate the interaction of different cryoprotective agents with cells in SBS for hepatocyte banking. 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. Kantrow, S., Taylor, D., Carraway, M., and Piantadosi, C. Oxidative metabolism in rat hepatocytes and mitochondria during sepsis. Arch. Biochem. Biophys. 345, 278–288 (1997). 4. Kravchenko, L., Andrienko, A., Shanina, I., and Belous, A. Long-term storage of isolated liver cells at hypothermia. CryoLetters 15, 133–144 (1994). 5. Mapes, J., and Harris, R. On the oxidation of succinate by parenchymal cells isolated from rat liver. FEBS Lett. 51, 80–83 (1975). 6. Petrenko, A., and Sukach, A. Isolation of intact mitochondria and hepatocytes using vibration. Anal. Biochem. 194, 326–329 (1991). 7. Petrenko, A., Grischuk, B., Roslyakov, A., Mazur, P., and Belous, A. Survival, metabolic activity and transport of potassium ions of rat hepatocytes after rapid freeze–thaw under protection of dimethyl sulphoxide and separation in Percoll density gradient. CryoLetters 13, 87–98 (1992). 8. Sammut, I., Thornilley, M., Simpkin, S., Fuller, B., Bates, T., and Green, C. Impairment of hepatic mitochondrial respiratory function following storage and orthotopic transplantation of rat livers. Cryobiology 36, 49–60 (1998). 9. Shanina, I., Kravchenko, L., Fuller, B., and Grischenko, V. A comparison of a sucrose-based solution with other preservation media for cold storage of hepatocytes. Cryobiology 41, 315–318 (2000). 10. Southard, J., Van Gulik, T., and Ametani, M. Important components of the UW-solution. Transplantation 49, 251–257 (1990).
BRIEF COMMUNICATION Respiratory Activity of Isolated Rat Hepatocytes Following Cold Storage and Subsequent Rewarming: A Comparison of Sucrose-Based and University of Wisconsin Solutions L. P. Kravchenko,*† A. Yu. Petrenko,* A. Yu. Somov,* V. I. Grischenko,* and B. J. Fuller‡,1 *Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of the Ukraine, 23 Pereyaslavskaya Str., Kharkov 61015, Ukraine, and †University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, United Kingdom Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 ⫾ 3 and 23 ⫾ 3 nmol O2/min/106 cells, respectively) compared to control cell values (31 ⫾ 3 and 33 ⫾ 5 nmol O2/min/106 cells; P ⬍ 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 ⫾ 6 and 40 ⫾ 5 nmol O2/min/106 cells; for control cells: 43 ⫾ 5 and 46 ⫾ 6 nmol O2/min/106 cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended. Key Words: isolated hepatocytes; hypothermic storage; respiratory function; sucrose-based solution; University of Wisconsin solution.
The search for effective and simple preservation solutions for both whole liver and isolated hepatocytes has been an active area of research over the past decade. The University of Wisconsin (UW) solution has significantly improved the practice of liver preservation for transplantation since its first development (10) and is based on a mixture of impermeants such as lactobionate and raffinose, which effectively control cell volume during hypothermia. We (4, 9) and others (1) have been interested in the use of sucrose as an alternative osmotic agent for cell and organ storage, for a variety of reasons, including
Received January 2, 2001; accepted April 27, 2001; published online July 18, 2001. † Deceased 1 To whom correspondence should be addressed. This work was a collaborative study made possible by the support of the Wellcome Trust Grant 056648/Z/99/Z. 0011-2240/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
easier availability in some countries where organ preservation procedures are only recently being developed. An additional reason in the case of isolated hepatocytes would be that in situations in which the cells might be used for transplantation, periods of both cold storage (when the liver tissues are initially harvested for cell isolation) and cryopreservation (when sufficient cell banks are accumulated for clinical use) are necessary (2). A sucrose-based medium has been found to be effective for both hypothermic storage as judged by morphological criteria (9) and freeze preservation (7) of isolated hepatocytes in our experience, and the use of a single solution for both steps may simplify the protocols for establishment of an hepatocyte bank. However, cell morphology and membrane integrity are only basic indices of functional survival after hypothermia, and the current studies were undertaken to evaluate hepatocyte respira-
218
BRIEF COMMUNICATION
tion as a more stringent metabolic test (3), both immediately after cold preservation and after a period of rewarming to allow expression of any latent damage during reperfusion. Isolation of rat hepatocytes was achieved by the nonenzymatic method described in our previous papers. Briefly, rat livers were isolated under anesthesia induced by injection (I.P. administration of sodium pentabarbitone) and were perfused with a sucrose–EDTA medium (4). The minced tissue was then subjected to controlled mechanical disaggregation by vibration (6). Cells were recovered by centrifugation, washed, counted in a hemocytometer, and resuspended in the relevant storage medium. Hypothermic storage of the samples was carried out in glass vials (at a cell concentration of 3–5 ⫻ 106/ml and a volume of 5 ml) at a temperature of 4°C for 45 min (the period of initial isolation, washing, and resuspension as control) and after 24 and 48 h. In this study we compared the sucrose-based solution (SBS) (4, 9), a solution based on sucrose and containing a low ionic content, with the more widely used UW solution (10). For assessment of the stability of fresh and stored hepatocytes, incubations were conducted in Krebs–Ringer bicarbonate (pH 7.4). Cell suspensions were studied either immediately after different stages of cold storage or after subsequent periods of 60 min normothermic incubation (designated as rewarming) with shaking to mimic reperfusion injury. Cell membrane integrity was estimated by the exclusion of vital dye (trypan blue at a final concentration of 0.3%) as described previously (4, 9). Endogenous respiration was measured by polarographic means in a 1-ml cuvette at 36°C, using a Clark’s platinum oxygen electrode (in Krebs–Ringer– phosphate solution, pH 7.4, as described (6)). Values were calculated per 106 cells in the sample. A further metabolic evaluation of cell integrity was made by stimulation of respiration by succinate (2 mM). This substrate slowly crosses the plasma membrane of normal hepatocytes and causes little stimulation of the endogenous respiration rate. However, in cells with compromised membrane integrity, succinate may more easily pass into the cells (5) and pro-
219
duce a marked stimulation of respiration greater than twofold, as shown in chemically permeabilized cells (3). The total measurement time, including the succinate test, was 7 min. The data were processed with ORIGIN software, statistical significance was determined by the Student–Fisher method, and a value of P ⬍ 0.01 was accepted as significant to allow for multiple comparisons between groups (Bonferroni correction). The initial suspension of freshly isolated hepatocytes contained not less than 87–95% of cells excluding vital dye (mean value of 93 ⫾ 4%, see Table 1). Freshly isolated hepatocytes possessed a high level of endogenous respiration after exposure to SBS (Table 1), which statistically increased following the period of 60 min normothermic incubation. Similar levels of endogenous respiration for freshly isolated cells were demonstrated in previous studies (4, 5). Respiration indices in the presence of succinate immediately after cell isolation demonstrated a slight increase (expressed as a ratio of the rates Vsucc./Vend.), were recorded as mean values of 1.12 for fresh cells and 1.13 for fresh cells after rewarming, and again are in line with the literature data (5). After the first 24 h of storage of isolated hepatocytes in SBS, dye exclusion remained high at a mean value of 85%, whereas endogenous respiratory activity statistically decreased (P ⬍ 0.01) by 25% (Table 1). However, after incubation at 37°C, respiration recovered to values similar to those in fresh cells. Exposure to succinate caused a significant increase in respiration after cold storage as measured by the ratios Vsucc./Vend. of 1.60 (P ⬍ 0.01), whereas it remained high (at mean values of 1.50 after incubation) but not significantly different from than seen with fresh cells. After 48 h storage in SBS, cell dye exclusion was decreased by a significant but relatively small degree to mean values of 78 ⫾ 4% after storage and 75 ⫾ 4% after rewarming (Table 1). Nevertheless, respiratory activity of the rewarmed cells was again reduced after cold storage compared to that of the controls, and, more notably, no recovery of respiration followed the period of normothermic incuba-
220
BRIEF COMMUNICATION
tion. In this situation after 48 h storage, exposure to succinate caused a further sustained rise in respiration (to mean values of 1.88 and 1.71 of the endogenous rate after incubation—Table 1; P ⬍ 0.01 for each compared to respective fresh cell values). For cells exposed to UW solution, a very similar outcome was obtained (see Table 1). Rewarming for 60 min in freshly isolated cells allowed an increase in endogenous respiration (from mean values of 33 ⫾ 5 to 46 ⫾ 6 nmol O2/min/106 cells), whereas exposure to succinate again caused a small increase for fresh cells immediately tested (Vsucc./Vend. of 1.14) and after rewarming (Vsucc./Vend. of 1.12). Endogenous respiration after 24 h cold storage was reduced compared to that in fresh cells, but recovered to levels similar to that in controls after 60 min rewarming. Whereas dye excluding ability was little changed after 24 h storage in UW solution, exposure to succinate again caused a more notable increase in respiration (Vsucc./Vend. to mean values of 1.59; P ⬍ 0.01 compared to fresh cells) in cells tested immediately after storage and the slightly lower mean value of 1.38 after rewarming. As seen for the SBS-
stored cells, 48 h of hypothermic storage caused a significant reduction in respiration which did not recover after normothermic rewarming. A small but significant decrease in dye-excluding cells followed 48 h storage in UW solution, whereas there was a marked stimulation of respiration in the presence of succinate both after cold exposure alone and after 60 min rewarming (Vsucc./Vend. mean values of 1.86 and 1.81; both P ⬍ 0.01 compared to respective values for fresh cells—Table 1). The results reported here demonstrate several important points about preservation of isolated hepatocytes. Endogenous respiration in intact hepatocytes is a good index of metabolic integrity and is depressed in pathological states such as septic shock (3). Our results demonstrated that the procedure of isolation and washing resulted in cells with a slightly depressed respiratory activity, and this returned to values typical for isolated hepatocytes after rewarming at 37°C. This may reflect loss of soluble substrates and cofactors during isolation, which were replenished during preincubation. When the cells were stored in either SBS or UW solution for 24 h, respiration was further
TABLE 1 Dye Exclusion and Indices of the Respiration Activity (nmol O2/min/106 cells) in Freshly Isolated Hepatocytes, Hepatocytes after Cold Storage Alone in Either SBS or UW Solutions, and after Storage with Subsequent Normothermic Incubation for 60 min Media
SBS
UW
Indices
Endogenous respiration Respiration, stimulated by succinate Vsucc./Vend. Viability, % Endogenous respiration Respiration, stimulated by succinate Vsucc./Vend. Viability, %
Freshly isolated hepatocytes, 45 min 4°C
Incubated 37°C, 60 min
31 ⫾ 3
43 ⫾ 5*
35 ⫾ 4
49 ⫾ 6*
1.12 ⫾ 0.07 95 ⫾ 4 33 ⫾ 5
1.13 ⫾ 0.05 93 ⫾ 3 46 ⫾ 6*
38 ⫾ 4
50 ⫾ 5*
1.14 ⫾ 0.06 95 ⫾ 4
1.12 ⫾ 0.06 96 ⫾ 6
Cold storage, 24 h
4°C
Cold storage, 48 h
Incubated 37°C, 60 min
4°C
21 ⫾ 3⫹
41 ⫾ 6*
22 ⫾ 3⫹
17 ⫾ 3**
34 ⫾ 5
60 ⫾ 8*
41 ⫾ 5
29 ⫾ 5
1.60 ⫾ 0.07⫹ 88 ⫾ 4 23 ⫾ 3⫹ 36 ⫾ 6 1.60 ⫾ 0.09⫹ 83 ⫾ 5
1.50 ⫾ 0.13 88 ⫾ 5 40 ⫾ 5* 55 ⫾ 6* 1.40 ⫾ 0.08 86 ⫾ 4
1.90 ⫾ 0.09⫹ 78 ⫾ 4⫹ 23 ⫾ 3⫹ 43 ⫾ 5 1.90 ⫾ 0.07⫹ 76 ⫾ 4⫹
Incubated 37°C, 60 min
1.70 ⫾ 0.12⫹ 75 ⫾ 4⫹ 21 ⫾ 4** 38 ⫾ 5 1.80 ⫾ 0.13⫹ 74 ⫾ 4+
Note. Also given are the ratios of the rates of respiration in the presence or absence of succinate (Vsucc./Vend.). Values are given as means ⫾ SE (n ⫽ 6–12). Denotes groups that are statistically significant in comparison with * the respective groups exposed only to cold storage; ⫹ the respective values for freshly isolated cells; and ** the respective values for groups exposed to cold storage and subsequent normothermic incubation. For purposes of the Ukraine, the methods for isolating cells and producing the sucrose medium have been patent registered (N 14080:1997 & N 16946:1997).
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BRIEF COMMUNICATION
reduced, but again recovered to within the normal range after rewarming. However, by 48 h cold storage, respiration did not recover after preincubation, suggesting severe disruption to oxidative metabolism. This is likely to be a multifactorial damage resulting from several factors including both depletion of cell solutes and disruption of mitochondrial architecture (8). The depression of respiration after prolonged storage was much greater (⬍50% in cells after 48 h storage and preincubation compared with control values) than might be expected by the small changes at the same time in dye exclusion and serves as another reminder that morphology and metabolism are not always linked in cells in the acute poststorage period after hypothermic preservation. The succinate exposure test was more predictive of the progressive damage experienced by the hepatocytes as storage times lengthened and already showed a marked deviation from that of fresh cells in the hepatocytes studied after cold storage alone even by 24 h storage, when endogenous respiration after rewarming and dye exclusion were both similar to those values for control cells. By 48 h, in either preservation solution, the succinate stimulation of respiration had reached levels approaching those in permeabilized cells which have lost metabolic control. It was also of note that, under these conditions of study, the damage expressed during longer periods of cold storage related more to the preservation time than to reperfusion damage (i.e., the increase in succinate-stimulated respiration was similar in the hepatocytes both at the end of the cold period and after rewarming at 37°C). The presented data also confirm that the SBS solution was, to all intents and purposes, an equally effective solution for hepatocyte cold storage compared with the UW solution. In situations in which liver tissues are being harvested and processed for hepatocyte banking for trans-
plantation, the SBS solution could provide a single solution for both organ cold storage and subsequent cryopreservation, simplifying the required protocols. Further studies will be needed to investigate the interaction of different cryoprotective agents with cells in SBS for hepatocyte banking. 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. Kantrow, S., Taylor, D., Carraway, M., and Piantadosi, C. Oxidative metabolism in rat hepatocytes and mitochondria during sepsis. Arch. Biochem. Biophys. 345, 278–288 (1997). 4. Kravchenko, L., Andrienko, A., Shanina, I., and Belous, A. Long-term storage of isolated liver cells at hypothermia. CryoLetters 15, 133–144 (1994). 5. Mapes, J., and Harris, R. On the oxidation of succinate by parenchymal cells isolated from rat liver. FEBS Lett. 51, 80–83 (1975). 6. Petrenko, A., and Sukach, A. Isolation of intact mitochondria and hepatocytes using vibration. Anal. Biochem. 194, 326–329 (1991). 7. Petrenko, A., Grischuk, B., Roslyakov, A., Mazur, P., and Belous, A. Survival, metabolic activity and transport of potassium ions of rat hepatocytes after rapid freeze–thaw under protection of dimethyl sulphoxide and separation in Percoll density gradient. CryoLetters 13, 87–98 (1992). 8. Sammut, I., Thornilley, M., Simpkin, S., Fuller, B., Bates, T., and Green, C. Impairment of hepatic mitochondrial respiratory function following storage and orthotopic transplantation of rat livers. Cryobiology 36, 49–60 (1998). 9. Shanina, I., Kravchenko, L., Fuller, B., and Grischenko, V. A comparison of a sucrose-based solution with other preservation media for cold storage of hepatocytes. Cryobiology 41, 315–318 (2000). 10. Southard, J., Van Gulik, T., and Ametani, M. Important components of the UW-solution. Transplantation 49, 251–257 (1990).