CRYOBIOLOGY
27, 143-152 (1990)
The Effects of Cryopreservation Transport, and Protein
on Membrane Integrity, Membrane Synthesis in Rat Hepatocytes
ROBERT DE LOECKER, BARRY J. FULLER,* JACQUES GRUWEZ, AND WILLIAM DE LOECKER Afddingen Biochemie and Klinische en Experimentele Heelkunde, Katholieke Universiteit te Leuven, 3oW Leuven, Belgium; and *Academic Department of Surgery, Royal Free Hospital and School of Medicine, London NW3 2QG, United Kingdom
The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.g M of Me,90 during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me,SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+ ,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-“Clisoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of a-amino[l-W]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180min, subsequent addition of labeled a-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at sign& cantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined. 0 1990Academic Press,Inc.
ever, the freeze-thaw cycle inflicts considerable damage to the isolated hepatocytes. Nonspecific hydrolysis due to lysosomal hydrolases released upon progressive destruction of the hepatocytes during incubation may be responsible for the constantly low degree of metabolic recovery (12). As documented by the ultrastructural studies, the observed metabolic activities appear to be carried out only by a relatively few remaining intact cells, the number of which rapidly decreases during incubation (11). Received December 14, 1988; accepted July 19, Indeed current methods of cryopreserva1989.
The cryopreservation of isolated hepatocytes is of particular interest in view of the possible transplantation of these cells to treat inborn disorders of metabolism. As freshly prepared isolated hepatocytes have been successfully applied to correct bilirubin uridyl diphosphate glucuronyltransferase in rats, the preservation of hepatocytes for longer periods of time becomes an important issue (5, 14,21,26,38,40). HOW-
143 001l-2240/90 $3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in my form reserved.
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LOECKER
tion, apart from reducing cell viability, appear to yield a reduced proportion of cells which are metabolically identical to freshly prepared hepatocytes (11, 16, 34). In contrast cryopreserved hepatocytes transplanted through the portal vein or into the spleen of animals after experimental induction of acute liver failure considerably reduce mortality. This beneficial effect however proves to be due to the presence of a still undefined cytosolic heat-stable factor present even in nonviable hepatocytes and capable of stimulating liver regeneration (l-3, 20, 23-25, 28, 35, 39). Long-term survival of hepatocytes after cryopreservation, as would be required in transplantation studies, will inevitably demand that the recovered cells be capable of synthesizing new macromolecules for turnover of cell components and integrated function of enzyme systems. For this reason we have chosen to study protein synthesis in hepatocytes after freezing. The complex protein synthetic process involves supply of metabolic energy and transmembrane import of amino acids and their incorporation into the growing polypeptide chain. In the present study we have examined both membrane transport of amino acids, using nonmetabolized analogues, and incorporation of radiolabeled amino acids into the protein units. In addition, we have studied the role of the suspending medium in retention of functional cells after cryopreservation, since previous studies from this laboratory have indicated the benefits of an intracellular-type solution (31). MATERIALS
Preparation
AND
METHODS
of Isolated Hepatocytes
Male Wistar R rats weighing 200 g were anesthetized with 0.4 ml of Nembutal (Abbott Laboratories, Chicago, IL), intraperitoneally administered. Through an abdominal midline incision, the portal vein was cannulated. The liver was removed, attached to a closed-circuit perfusion system,
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and perfused at 37°C with 150 ml of calcium free Krebs-Ringer bicarbonate buffer (KR), pH 7.4, containing 20 mg of ethylene glycolbis(2-aminoethyl)tetraacetic acid (EGTA-Fluka AG, CH-9470, Buchs) for 15 min to remove the blood. The KR was constantly gassed with 95% OT5% COZ. Subsequently perfusion was continued with fresh KR with calcium (1 mM) containing 0.05% (w/v) of collagenase (BoehringerMannheim, Germany) for 20 to 30 min. The digestion of the liver was judged to be sufficient when the capsule separated from the parenchyma after applying gentle pressure with forceps. The hepatocytes were gently shaken in KR and filtered through 100~km nylon mesh to remove cell clusters and undigested particles (33). After centrifugation for 30 set at 5Og, the cells were resuspended at a concentration of 5 x IO6viable cells/ml in buffer medium having either an extracellular (KR) or an intracellular complement of electrolytes (Euro-Collins, Fresenius, Bad Homburg, Germany). Viability was assessed by the trypan blue exclusion test. Hepatocytes were incubated at room temperature for 5 min in a medium containing 0.2% (w/v) trypan blue (Merck, Darmstadt, Germany). The total number of cells and those excluding the dye were microscopically counted in a hemocytometer (12). The cells excluding the dye were considered surviving hepatocytes and were expressed as percentages of the total number of cells counted. In all experiments 100 ml of the different buffers contained 4 ml of an L-amino acid supplement (Vamin, Vitrum, Stockholm 12, Sweden) to avoid nitrogen depletion as well as 100,000units of penicillin-K (Sigma, St. Louis, MO), 100mg of streptomycin sulfate (Sigma), and 20 mg of gentamicin sulfate (Sigma). Cryopreservation
The cryopreservation procedure followed was one which has been used previously by this laboratory with minor mod&
CRYOPRESERVED
cations (9). To freshly prepared hepatocytes at a concentration of 5 x lo6 viable cells/ml, dimethyl sulfoxide (Me,SO, Merck, Darmstadt, Germany) was slowly added to a final concentration of 1,8 M over a period of 30 min under gentle agitation at room temperature. Subsequently cell samples (1.5 ml) were placed in polypropylene tubes (Nunc Cryotubes, Denmark) sized 7 x 1.2 cm and progressively cooled in an alcohol bath (Fryka-Therm-FT800, Copenhagen, Denmark) to - 7°C when nucleation was induced by clamping the tubes with Nz cooled forceps. After dissipation of latent crystallization heat, the samples were further cooled at 1”Clmin down to -38°C placed in liquid nitrogen gas phase for 45 min, and submerged into the liquid phase. After 2 days of cryopreservation, thawing took place in a waterbath at 37°C. To remove the cryoprotectant, hepatocyte suspensions were slowly diluted 10X with the appropriate buffer solutions followed by spontaneous sedimentation, removal of the supernatant, and a further 10X dilution. Finally after low speed centrifugation, the cells were resuspended in the original volume of either high Na+, low Kt (KR) or low Naf , high K+ (Euro-Collins) medium.
HEPATOCYTES
145
of Me,SO and progressive slow removal of the cryoprotectaut (D-KR and D-EC); and (E) after exposure to 1.8 M of Me,SO and 0.8 M of sorbitol followed by the removal of the cryoprotectants (E-KR and E-EC). Cryopreserved hepatocytes were incubated without the removal of 1.8 M of Me,SO (Groups F-KR and F-EC), with the subsequent addition of 0.8 M of sorbitol (Groups G-KR and G-EC), after removal of Me,SO (Groups H-KR and H-EC), or after removal of Me,SO and sorbitol together (Groups I-KR and I-EC). To follow the amino acid incorporation into proteins, aliquots of 1 ml of fresh and cryopreserved hepatocyte suspensions after the removal of 1.8 M of Me,SO were incubated in the presence of 0.1 I&i of L-[U-‘4C]isoleucine (sp radioact, 150 mCi/mmol; Amersham International, Amersham, Bucks, UK). After incubation for up to 180 min, 5 ml of trichloroacetic acid (20%) (TCA, Merck) was added to denature the proteins. After a further three washes with 10 ml of TCA (5%), the precipitated proteins were dissolved in 2 ml of Lumasolve (Lumac Systems AG, Basel, Switzerland) by being heated overnight at 60°C. After the addition of 10 ml of the scintillator Lipoluma (Lumac Systems AG), the incorporated radioactivity was asIncubation Procedures sessed by counting in a liquid scintillation Incubations were carried out in either spectrometer (Rack Beta, LKB, Wallac, KR or EC, and the various groups are la- OY, Turku, Finland). Amino acid transport activity was evalubeled with the postscript -KR or -EC to indicate this. In some other groups a prein- ated by incubating hepatocytes in the prescubation at 37°C in KR preceded the test ence of 0.1 &i of a-amino[l-‘4C]isobutyric period and these are labeled with the post- acid (AIB, sp radioact, 60 mCilmmo1; Amscript -KR ~180. To assess viability in dif- ersham International). After different incuferent circumstances, according to the try- bation periods, hepatocytes, incubated in pan blue exclusion test, fresh hepatocytes the presence of the amino acid analog, were were incubated at 37°C for up to 180 min diluted with 10 ml of fresh incubation meunder gentle agitation under the following dium containing 30 mmol of nonradioactive conditions: (A) in the appropriate fresh AIB (Sigma). After low-speed centrifugabuffer solutions (A-KR and A-EC); (B) in tion three more identical washes were carthe presence of 1.8 M of Me,SO (B-KR and ried out and the cells were finally dissolved B-EC); (C) in the presence of 1.8 M of in Lumasolve and further processed to asMe,SO and 0.8 M of sorbitol (Merck) (C- sess the intracellular radioactivity. In another series of experiments, after an KR and C-EC); (D) after exposure to 1.8 M
146
DE LOECKER ET AL.
initial preincubation period of 180 min, at 37”C, 0.1 $i of L-[U-‘4C]isoleucine or 0.1 $i of ol-amino[l-‘4C]isobutyric acid was added to 1 ml of hepatocyte suspensions, which were then incubated further to 180 min (Groups A-KR ~180 and H-KR ~180). The radioactivity measurements were expressed as disintegrations per min (DPM) per I x lo6 viable cells kSEM. Statistical evaluation of significance was carried out according to Student’s t test. RESULTS
Figure 1 shows the effect of incubation time in KR on the membrane integrity of fresh hepatocytes. After the isolation procedure, 95% of the recovered hepatocytes were considered viable according to the trypan blue exclusion test. Incubation at 37°C in pure KR (Group A-KR) reduced the number of dye-excluding cells by approximately 8% over a period of 60 min. For the cells initially exposed to 1.8 M of Me,SO (Group D-KR) or to 1.8 M of Me,SO together with 0.8 M of sorbitol (Group E-KR) incubation in fresh KR after removal of the cryoprotectants resulted in a fall in dyeexcluding cells amounting to respectively 38 and 18%. However in the presence of one or both cryoprotectants (Groups B-KR and C-KR), the number of dye-excluding cells was reduced after 1 hr of incubation to around 5%. It was observed that, apart from the freshly used hepatocytes, a proportionally faster decline in the dyeexcluding ability occurred over the first 15 min of incubation, followed by further linear deterioration of the cells. Figure 2 shows the effects of incubation time in KR on the membrane integrity of cryopreserved hepatocytes. After thawing, around 75% of the cryopreserved hepatocytes retained the ability to exclude trypan blue. In the presence of 1.8 M of Me,SO (Group F-KR) or in the presence of 1.8 M of Me,SO and 0.8 M of sorbitol together (Group G-KR), the dye-excluding ability of
FIG. 1. Survival of fresh hepatocytes after incubation for different periods of time in Krebs-Ringer bicarbonate buffer (KR). Freshly isolated hepatocytes were suspended in fresh KR (5 x lo6 cells/ml) (A23roup A-KR), in KR containing 1.8 M of Me,SO Q-Group B-KR), or in KR containing 1.8 of Me,SO and 0.8 M of sorbitol (C-Group C-KR) and incubated for up to 60 min. After the exposure of fresh hepatocytes for 45 min to 1.8 of Me+30 (CGroup DXR) or to 1.8 M of Me,SO and 0.8 M of sorbitol (CGroup E-KR) the cryoprotectants were removed by dilution twice 10X, and cells were resuspended in fresh KR for incubation. After each incubation period the cells were treated with trypan blue dye and counted in a hemocytometer. The surviving cells were expressed as a percentage of the original number of hepatocytes. Each value represents the mean of eight experiments ASEM. At the end of 60 min Groups D and E showed a significant (P < 0.01 in both cases) fall in dyeexcluding cells compared to that in controls (Group A-KR). However the number of dye-excluding cells in Groups B and C showed a much greater fall (P < 0.001 in both cases).
the cryopreserved hepatocytes was reduced to 30 and 17%, respectively, after 15 min of incubation, rapidly leveling off to a value below 5% after 60 min of incubation. After removal of Me,SO by dilution (Group H-KR) and resuspension, incubation in fresh KR progressively reduced dye exclusion to 34% over a 60-min period. Dye exclusion by the hepatocytes, after freezing and thawing and subsequent exposure to 0,8 M of sorbitol for 30 min followed by the removal of both cryoprotectants (Group IKR), resuspension, and incubation in fresh KR, followed a similar pattern. These sur-
CRYOPRESERVED HEPATOCYTES
FIG. 2. Survival of cryopreserved hepatocytes after incubation for different periods of time in KR. Cryopreserved hepatocytes (5 x 106cells/ml) after thawing were incubated in the presence of 1.8 M Me+30 in which they were preserved (o--Group F-KR) or after the removal by dilution of the cryoprotectant and resuspension in fresh buffer (Wroup H-KR). Some frozen samples after thawing were further exposed to 0.8 M of sorbitol for 30 min and subsequently incubated in the presence of Me,SO and sorbitol (04roup G-KR) or after the removal of both cryoprotectants (Uroup I-KR). Surviving cells were evaluated according to the trypan blue exclusion test. Each value represents the average percentage of surviving cells of eight experiments *SEM. Again those groups incubated still in the presence of cryoprotectants (groups A and I) showed a much greater fall in viability compared to the relevant group in which the cryoprotectants had been removed (P < 0.001 in both cases at 60 min).
FIG. 3. Survival of cryopreserved hepatocytes after incubation in Euro-Collins and KR. Fresh hepatocytes were incubated in Euro-Collins (CGroup A-EC) and in KR (O-Group A-KR) for different periods of time up to IX0 min. Hepatocytes were cryopreserved in Euro-Collins and in KR in the presence of 1.8 M of Me,SO. After the removal of the cryoprotectant by dilution with the corresponding buffer medium, the cells were incubated in the original volume of either Euro Collins (CGroup H-EC) or KR (O--Group HKR) followed by the viability assessment according to the trypan blue exclusion test. Each percentage value represents the mean of eight experiments kSEM. By 180 min hepatocytes incubated in KR (Group A-KR) showed a dye exclusion significantly lower than that of those incubated in EC (Group A-EC) (P < 0.001). The same was true on recovery after freezing (P < 0.001).
vival indices do not take into account the proportion of cells lost during the cryopreservation procedure. Figure 3 shows a comparison of the survival of cryopreserved hepatocytes after incubation in either KR or EC. Dye exclusion by freshly prepared hepatocytes incubated in Euro-Collins medium decreased almost linearly by 16% over a period of 180 min (Group A-EC), while in the analogous circumstances, incubation in KR reduced the number of dye-excluding cells by 87% (Group A-KR). In the latter case particularly after the first hour of incubation, dye exclusion declined sharply. As for the hepatocytes cryopreserved and incubated in
Euro-Collins buffer, the reduction in dyeexcluding cells over a period of 180 min amounted to 47% (Group H-EC), while cryopreservation and incubation in KR reduced dye-excluding ability by 40% after 30 min and by 52% after 60 min with only 2% viability retained after 180 min of incubation (Group H-KR). Figure 4 illustrates the protein synthesizing activity of fresh and cryopreserved hepatocytes. Although the incorporation of L-[U-‘4C]isoleucine into the proteins of freshly prepared hepatocytes and of cryopreserved hepatocytes after removal of Me,SO and resuspension in KR (Groups AKR and H-KR) increased over the incuba-
incubation
time
in min
148
DE LOECKER ET AL.
102030
60
90
120 180 Incubation time in men
FIG. 4. Incorporation of L-[U-‘4C]isoleucine into the proteins of fresh and cryopreserved hepatocytes. Fresh hepatocytes (Wroup A-KR) or hepatocytes cryopreserved in KR (0) containing 1.8 M of Me,SO after removal of the cryoprotectant (Group H-KR) by dilution were incubated in KR (5 x 106cells/ml) in the presence of 0.1 &i of L-[U-“CJisoleucine at 37°C over 180 min. In another series of experiments fresh hepatocytes (U-Group A-KR ~180) and cryopreserved hepatocytes (o--Group I-I-KR ~180) after the removal of Me+ were preincubated without radioactivity for 180 min in KR at 37°C. Subsequently 0. l PCi of L-[U-‘4C]isoleucine was added and incubation was continued for another 180 min during which the amino acid incorporation was evaluated. Each incorporation value expressed as disintegrations per min @PM) per I x 106of originally viabk hepatocytes is the mean of eight experiments zSEM. Cryopreserved hepatocytes (H-KR) showed a significantly lower incorporation of leucine at 60 min (P < 0.001 compared to fresh A-KR). Preincubation for I80 min considerably reduced activity in both fresh and cryopreserved groups subsequently tested for 180 min.
tion period of 180 min, the rate of incorporation progressively leveled off after the first hour. After 180 min of incubation the total radioactivity incorporated by cryopreserved hepatocytes (Group H-KR) amounted to 56% of the fresh samples (Group A-KR). Supplementary addition of 140 mmol of glucose (Merck) to the KR did not affect the rate of protein synthesizing activity. When fresh (Group A-KR ~180) and cryopreserved cells (Group H-KR p180), preincubated for 180 min, were subsequently subjected to a further incubation for up to 180 min in the presence of L-[U-14C]isoleucine, the observed incorpo-
ration of radioactivity was similar for both cell samples and progressed at a much lower level. In Fig. 5 the membrane transport activities of fresh and cryopreserved hepatocytes are shown. The uptake of the amino acid analog, u-amino[l-‘4C]isobutyric acid (a measure of amino acid membrane transport activity) by freshly prepared hepatocytes (Group A-KR) rapidly increased up to a peak value which was reached after 60 min of incubation. Upon prolonged incubation an equally fast de8000 i
I-* 1020304D
= 60
90
n 120 180 Incubation time ID mm
FIG. 5. The uptake of a-amino[l-‘4C]isobutyric acid by fresh and cryopreserved hepatocytes. Fresh hepatocytes (CGroup A-KR) and hepatocytes cryopreserved (O---Group H-KR) in KR containing 1.8 M of Me,SO after the removal of the cryoprotectant by dilution were incubated in KR (5 X lo6 cells/ml) in the presence of 0.1 &i of a-amino[l-14C]isobutyric acid over I80 min. In a further experiment the freshly prepared hepatocytes (Wroup A-KR ~180) and the cryopreserved hepatocytes (U--Group H-KR ~180) after removal of Me,SO were preincubated for I80 min without radioactivity and after the addition of 0.1 &i of u-amino[l-‘4C]isobutyric acid incubation was continued for another 180 min. Each value expressed as DPM per I x lo6 of originally viable hepatocytes is the mean of eight experiments zkSEM. Maximum uptake was seen between 40 and 90 min of incubation, and 60 min was chosen as a convenient point for comparison. Again cryopreserved hepatocytes (H-KR) showed significantly depressed uptake compared to that of controls (A-KR) (P < 0.001). Also as before, preincubation for 180 min (A-KR ~180 and H-KR ~180) diminished uptake greatly.
CRYOPRESERVED
cline in intracellular radioactivity occurred, which after 120 min progressively leveled off. The cryopreserved hepatocytes frozen in 1.8 M of Me,SO, after the progressive removal of the cryoprotectant by dilution (Group H-KR), also showed a peak uptake value of the amino acid analog which was attained after 30 min of incubation. Further incubation progressively reduced the accumulated radioactivity as was observed with the fresh hepatocytes, although again at a lower level of activity. After an initial preincubation for 180 min at 37°C of both freshly preparedhepatocytes (Group A-KR ~180) and cryopreserved cells after removal of 1.8 M of Me,SO (Group H-KR p180), the subsequent incubation for a further 180 min in the presence of 0.1 @Zi of cy-amino[ l-‘4C]isobutyric acid resulted in identical low values similar to nonspecific background radioactivity.
HEPATOCYTES
149
the recuperated hepatocytes after thawing (on the basis of the dye exclusion reaction) remained high, there was a marked decline in viability upon further incubation of the cryopreserved hepatocytes. The beneficial protective actions of the addition of sorbitol before dilution, which was observed with nonfrozen cells, was not apparent after thawing frozen hepatocytes. Since both fresh and cryopreserved hepatocytes incubated in a buffer medium with intracellular electrolyte composition retain viability to a much greater extent, even over longer periods of time, we surmise that the membrane (Naf ,K+)-ATPase pump mechanism is protected by the intracellular buffer composition (6, 7). Under these circumstances, however, viability values obtained with the dye exclusion method cannot be compared with the direct evaluation of metabolic parameters because membrane transport and protein synthesis are considerably inhibited DISCUSSION by the intracellular electrolyte composition of the incubation buffer. It should be During incubation in the KR buffer, which is characterized by an extracellular pointed out here that other workers have electrolyte composition, a progressive slow commented on the beneficial results obdecline in viability of freshly prepared he- tained when using potassium-rich solutions patocytes occurs, while in the presence of during addition and removal of cryoproMe,SO or of Me,SO together with sorbitol, tectants (4, 19, 30). In addition, we previviability is extremely poor. However, cells ously reported that the contractile reinitially exposed to Me,SO or to Me,SO sponses of smooth muscle after cryopreserand sorbitol, which are washed before be- vation were best preserved when freezing ing incubated in fresh buffer, retain a con- was carried out in a potassium-rich intrasiderable degree of dye-excluding ability. cellular buffer (3 1). Thus the importance of Cells exposed to Me,SO together with sor- these types of buffer in cryopreservation bitol before incubation show a higher de- generally is worthy of more research in the gree of viability upon subsequent incuba- future. Our present results show that the assesstion compared with cells exposed to Me,SO ment of viability may also depend on the alone. The removal of the cryoprotectant different parameters examined. Indeed the Me,SO in the presence of the extracellular sorbitol was beneficial as observed in pre- viability values expressed in terms of the vious studies with erythrocytes (6,7). It has trypan blue exclusion test or the ability to been noted previously that after the freeze- metabolize fluorescein diacetate are usually thaw procedure, followed by dilution of the substantially higher than when metabolic cryoprotectants and resuspension in fresh parameters such as gluconeogenesis, glycomedium, the initial number of hepatocytes gen synthesis, urea synthesis, bilirubin conmay already be reduced by around 30% jugation, ability to accumulate 99mT~ (12). Although the immediate survival of HIDA, and particularly protein synthesis
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DE LOECKER
are evaluated (13, 16, 18, 22, 29). This sug gests a lack of correlation between the dye exclusion tests and the metabolic tests; the latter probably more accurately represent metabolic recovery. Reservation of protein synthesizing activity and membrane transport may not always run parallel. In the present system, however, the viability values obtained by the dye exclusion are additionally illustrated and confirmed by the uptake of ol-amino[ l-L4C]isobutyric acid which is a measure of amino acid transport activity and by the incorporation of L-[U-‘~CIisoleucine into the protein. Indeed the initial increase in uptake of the amino acid analog in fresh as well as in cryopreserved hepatocytes was followed by a sharp decline. This is an indication of cell membrane damage and similarly the protein synthesizing activity leveled off as incubation progressed. According to both parameters, the metabolic activities of the cryopreserved hepatocytes were recorded at a lower level than the fresh cells. Dehydration induced by freezing leads to cell shrinkage and the ensuing changes in membrane mechanical properties may damage membrane structures resulting in vesiculation and -even loss of membrane material (10, 15, 17, 27, 29, 32, 36, 37). Apart from morphological anomalies, permeability changes modifying membrane transport functions are concomitant with an inhibited metabolic activity (9). As both fresh and cryopreserved hepatocytes, after an initial preincubation period of 180 min, are unable, upon subsequent incubation, to concentrate a-aminoisobutyric acid, while amino acid incorporation into the proteins still occurs at a low base level, it appears that even in the absence of adequate membrane transport function some residual aspects of protein synthesizing activity may continue to take place. Although hepatocytes prove particularly
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sensitive to cryopreservation, careful handling allows recovery of a considerable proportion of viable cells capable of demonstrating efficient membrane transport activity and protein synthesizing activity even in an incubation medium not containing hepatotrophic factors (8). ACKNOWLEDGMENTS
The authors are indebted to the FGWO (Belgian National Foundation for Medical Research) for a grant to the laboratories and to Mrs. F. De Wever and Miss N. Volders for their excellent technical assistance. REFERENCES
1. Ashwood-Smith, M. .I. Stability of microsomal enzymes associated with the conversion of carcinogens to bacterial mutagens (Ames’SalmonelIal microsome test) to freezing and thawing. Cryobiology 14, 24&244 (1977). 2. Ashwood-Smith, M. J. Low temperature preservation of cells, tissues and organs. In “Low Temperature Preservation in Medicine and Biology” (M. J. Ashwood-Smith and .I. Farrant, Eds.), pp 1944. Pitman, Tunbridge, Wells, 1980. 3. Becker, W. K., and Lillehei, R. C. Transplantation of cryopreserved hepatocytes for experimental acute liver failure. Cryobiology 17,6I7618 (1980). 4. Clark, P., Fahy, G., and Karow, A. M. Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices. Cryobiology 21, 260-273 (1984). 5. Coundouris, J., Hannah, C., Grant, H., and Hawksworth, G. Cryopreservation of isolated rat hepatocytes. Biochem. Sot. Tmns. 14,692693 (1986). 6. De Loecker, R., Penninckx, F., and Kerremans, R. Osmotic effects of rapid dilution of cryoprotectants. I. Effects on human erythrocyte swelling. Cryo-Letl. 8, 130-139 (1987). 7. De Loecker, F., and Penninckx, F. Osmotic effects of rapid dilution of cryoprotectants. II. Effects on human erythrocyte hemolysis. CryoLat. 8, 140-145 (1987). 8. Drochmans, P., Wanson, J. C., May, C., and Bernaert, D. Ultrastructural and metabolic studies of isolated and cultured hepatocytes. In “Hepatotrophic Factors” (R. Porter and J. Whelan, Eds.), Ciba Foundation Symposium 55, pp. 7-
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Mito, M. Transplantation of cryopreserved isolated hepatocytes into the rat spleen. Trunsplant. Proc. 13, 84&854 (1981). Le Cam, A., Guillouzo, A., and Freychet, P. Ultrastructural and biochemical studies of isolated rat hepatocytes prepared under hypoxic conditions. Exp. Cell Res. 98, 382-395 (1976). Makowka, L., Falk, R. E., Rotstein, L. E., Falk, J. A., Nossal, N., Langer, B., Blendis, L. M., and Phillips, M. J. Cellular transplantation in the treatment of experimental hepatic failure. Science 210, 901-903 (1980). Makowka, L., Rotstein, L., Falk, R., Falk, J., Larger, B., Nossal, N., Blendis, I., and Phillips, M. Reversal of toxic and anoxic induced hepatic failure by synergic, allogeneic and xenogeneic hepatocyte transplantation. Surgery 88, 244-253 (1980). Makowka, L., Rotstein, L., Falk, J., Zuk, R., Langer, B., Blendis, L., and Phillips, M. J. Allogeneic and xenogeneic hepatocyte transplantation. Transplunt. Pm. 13, 85=59 (1981). Matas, A. J., Sutherland, D. E. R., Steffes, M. W., Mauer, S. M., Lowe, A., Simmons, R. L., and Najarian, J. S. Hepatocellular transplantations for metabolic deficiencies: Decrease of plasma bilirubin in Gunn rats. Science 192, 892-894 (1976). McGrath, J. J. Thermodynamic modelling of membrane damage. In “Effects of Low Temperature on Biological Membranes” (G. J. Morris and A. Clarke, Eds.), pp. 335-377. Academic Press, London, 1981. Mito, M., Ebata, H., Kusano, T., Or&hi, T., Hiratsuka, M., and Saito, T. Studies on extopic liver utilizing hepatocyte transplantation into the rat spleen. Transplant. Proc. 11, 585-591 (1979). Nutt, L., Attenburrow, V., and Fuller, B. Investigations into repair of freeze-thaw damage in isolated rat hepatocytes. Cryo-Lett. 1, 513-518 (1980). Pegg, D. E., Jacobsen, I. A., Diaper, M. P., and Foreman, J. Perfusion of rabbit kidneys with solutions containing propane- 1,2-diol. Ctyobiology 24, 42Q-428(1987). Penninckx, F., Vandekerckhove, Ph., De Loecker, W., and Kerremans, R. Preservation of Taenia coli by freezing and storage at - 196°C. Cryobiology 23, 222-229 (1986). Pomeroy, M. K., and Siminovitch, D. Seasonal cytological changes in secundary phloem parenchyma cells in Robinia pseudoaccacia in relation to cold hardiness. Cunad. J. Bar. 49, 787795 (1971).
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33. Seglen, P. 0. Preparation of isolated rat liver cells. Metkods Cell Bid. 13, 2W33 (1976). 34. Sekiguchi, S., Kusano, M., O&hi, T., Ebata, H., and Mite, M. Cryogenic preservation of isolated hepatocytes in rat. Cryobiology 15, 724 725 (1978). 3.5. Sommer, B. G., Sutherland, D. E. R., Simmons,
R. L., and Najarian, J. S. Hepatocellular transplantation for experimental ischemic acute liver failure in dogs. J. Surg. Res. 29,3&325 (1980). 36. Steponkus, P. L., and Weist, S. C. Freeze:thaw induced lesions in the plasma membrane. In “Low Temperature Stress in Crop Plants: The Role of Membranes” (J. H. Lyons, D. Graham, and J. Raison, Eds.), pp 231-254. Academic Press, New York, 1979. 37. Steponkus, P. L., Wolfe, J., and Dowgert, M. F. Stress induced by contraction and expansion during a freeze-thaw cycle: Membrane per-
spective. In “Effects of Low Temperature on Biological Membranes” (G. J. Morris and A. Clarke, Eds.), pp. 307-322. Academic Press, London, 1981. 38. Sutherland, D. E. R., Matas, A. J., Steffes, M. W., Simmons, R. L., and Najarian, J. S. Transplantation of liver cells in an animal model of congenital enzyme deficiency disease: The Gunn rat. Transplant. Proc. 9, 317-319 (1977). 39. Sutherland, D. E. R., Numata, M., Matas, A. J.,
Simmons, R. L., and Najarian, J. S. Hepatocellular transplantation in acute liver faiture. Surgery 82, 124-132 (1977). 40. Vroemen, J. P. A. M., Blanckaert, N., Buurman, W. A., Heirwegh, K. P. M., and Kootstra, G. Treatment of enzyme deficiency by hepatocyte transplantation in rats. J. Surg. Res. 39, 267275 (1985).
27, 143-152 (1990)
The Effects of Cryopreservation Transport, and Protein
on Membrane Integrity, Membrane Synthesis in Rat Hepatocytes
ROBERT DE LOECKER, BARRY J. FULLER,* JACQUES GRUWEZ, AND WILLIAM DE LOECKER Afddingen Biochemie and Klinische en Experimentele Heelkunde, Katholieke Universiteit te Leuven, 3oW Leuven, Belgium; and *Academic Department of Surgery, Royal Free Hospital and School of Medicine, London NW3 2QG, United Kingdom
The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.g M of Me,90 during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me,SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+ ,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-“Clisoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of a-amino[l-W]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180min, subsequent addition of labeled a-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at sign& cantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined. 0 1990Academic Press,Inc.
ever, the freeze-thaw cycle inflicts considerable damage to the isolated hepatocytes. Nonspecific hydrolysis due to lysosomal hydrolases released upon progressive destruction of the hepatocytes during incubation may be responsible for the constantly low degree of metabolic recovery (12). As documented by the ultrastructural studies, the observed metabolic activities appear to be carried out only by a relatively few remaining intact cells, the number of which rapidly decreases during incubation (11). Received December 14, 1988; accepted July 19, Indeed current methods of cryopreserva1989.
The cryopreservation of isolated hepatocytes is of particular interest in view of the possible transplantation of these cells to treat inborn disorders of metabolism. As freshly prepared isolated hepatocytes have been successfully applied to correct bilirubin uridyl diphosphate glucuronyltransferase in rats, the preservation of hepatocytes for longer periods of time becomes an important issue (5, 14,21,26,38,40). HOW-
143 001l-2240/90 $3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in my form reserved.
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tion, apart from reducing cell viability, appear to yield a reduced proportion of cells which are metabolically identical to freshly prepared hepatocytes (11, 16, 34). In contrast cryopreserved hepatocytes transplanted through the portal vein or into the spleen of animals after experimental induction of acute liver failure considerably reduce mortality. This beneficial effect however proves to be due to the presence of a still undefined cytosolic heat-stable factor present even in nonviable hepatocytes and capable of stimulating liver regeneration (l-3, 20, 23-25, 28, 35, 39). Long-term survival of hepatocytes after cryopreservation, as would be required in transplantation studies, will inevitably demand that the recovered cells be capable of synthesizing new macromolecules for turnover of cell components and integrated function of enzyme systems. For this reason we have chosen to study protein synthesis in hepatocytes after freezing. The complex protein synthetic process involves supply of metabolic energy and transmembrane import of amino acids and their incorporation into the growing polypeptide chain. In the present study we have examined both membrane transport of amino acids, using nonmetabolized analogues, and incorporation of radiolabeled amino acids into the protein units. In addition, we have studied the role of the suspending medium in retention of functional cells after cryopreservation, since previous studies from this laboratory have indicated the benefits of an intracellular-type solution (31). MATERIALS
Preparation
AND
METHODS
of Isolated Hepatocytes
Male Wistar R rats weighing 200 g were anesthetized with 0.4 ml of Nembutal (Abbott Laboratories, Chicago, IL), intraperitoneally administered. Through an abdominal midline incision, the portal vein was cannulated. The liver was removed, attached to a closed-circuit perfusion system,
ET AL.
and perfused at 37°C with 150 ml of calcium free Krebs-Ringer bicarbonate buffer (KR), pH 7.4, containing 20 mg of ethylene glycolbis(2-aminoethyl)tetraacetic acid (EGTA-Fluka AG, CH-9470, Buchs) for 15 min to remove the blood. The KR was constantly gassed with 95% OT5% COZ. Subsequently perfusion was continued with fresh KR with calcium (1 mM) containing 0.05% (w/v) of collagenase (BoehringerMannheim, Germany) for 20 to 30 min. The digestion of the liver was judged to be sufficient when the capsule separated from the parenchyma after applying gentle pressure with forceps. The hepatocytes were gently shaken in KR and filtered through 100~km nylon mesh to remove cell clusters and undigested particles (33). After centrifugation for 30 set at 5Og, the cells were resuspended at a concentration of 5 x IO6viable cells/ml in buffer medium having either an extracellular (KR) or an intracellular complement of electrolytes (Euro-Collins, Fresenius, Bad Homburg, Germany). Viability was assessed by the trypan blue exclusion test. Hepatocytes were incubated at room temperature for 5 min in a medium containing 0.2% (w/v) trypan blue (Merck, Darmstadt, Germany). The total number of cells and those excluding the dye were microscopically counted in a hemocytometer (12). The cells excluding the dye were considered surviving hepatocytes and were expressed as percentages of the total number of cells counted. In all experiments 100 ml of the different buffers contained 4 ml of an L-amino acid supplement (Vamin, Vitrum, Stockholm 12, Sweden) to avoid nitrogen depletion as well as 100,000units of penicillin-K (Sigma, St. Louis, MO), 100mg of streptomycin sulfate (Sigma), and 20 mg of gentamicin sulfate (Sigma). Cryopreservation
The cryopreservation procedure followed was one which has been used previously by this laboratory with minor mod&
CRYOPRESERVED
cations (9). To freshly prepared hepatocytes at a concentration of 5 x lo6 viable cells/ml, dimethyl sulfoxide (Me,SO, Merck, Darmstadt, Germany) was slowly added to a final concentration of 1,8 M over a period of 30 min under gentle agitation at room temperature. Subsequently cell samples (1.5 ml) were placed in polypropylene tubes (Nunc Cryotubes, Denmark) sized 7 x 1.2 cm and progressively cooled in an alcohol bath (Fryka-Therm-FT800, Copenhagen, Denmark) to - 7°C when nucleation was induced by clamping the tubes with Nz cooled forceps. After dissipation of latent crystallization heat, the samples were further cooled at 1”Clmin down to -38°C placed in liquid nitrogen gas phase for 45 min, and submerged into the liquid phase. After 2 days of cryopreservation, thawing took place in a waterbath at 37°C. To remove the cryoprotectant, hepatocyte suspensions were slowly diluted 10X with the appropriate buffer solutions followed by spontaneous sedimentation, removal of the supernatant, and a further 10X dilution. Finally after low speed centrifugation, the cells were resuspended in the original volume of either high Na+, low Kt (KR) or low Naf , high K+ (Euro-Collins) medium.
HEPATOCYTES
145
of Me,SO and progressive slow removal of the cryoprotectaut (D-KR and D-EC); and (E) after exposure to 1.8 M of Me,SO and 0.8 M of sorbitol followed by the removal of the cryoprotectants (E-KR and E-EC). Cryopreserved hepatocytes were incubated without the removal of 1.8 M of Me,SO (Groups F-KR and F-EC), with the subsequent addition of 0.8 M of sorbitol (Groups G-KR and G-EC), after removal of Me,SO (Groups H-KR and H-EC), or after removal of Me,SO and sorbitol together (Groups I-KR and I-EC). To follow the amino acid incorporation into proteins, aliquots of 1 ml of fresh and cryopreserved hepatocyte suspensions after the removal of 1.8 M of Me,SO were incubated in the presence of 0.1 I&i of L-[U-‘4C]isoleucine (sp radioact, 150 mCi/mmol; Amersham International, Amersham, Bucks, UK). After incubation for up to 180 min, 5 ml of trichloroacetic acid (20%) (TCA, Merck) was added to denature the proteins. After a further three washes with 10 ml of TCA (5%), the precipitated proteins were dissolved in 2 ml of Lumasolve (Lumac Systems AG, Basel, Switzerland) by being heated overnight at 60°C. After the addition of 10 ml of the scintillator Lipoluma (Lumac Systems AG), the incorporated radioactivity was asIncubation Procedures sessed by counting in a liquid scintillation Incubations were carried out in either spectrometer (Rack Beta, LKB, Wallac, KR or EC, and the various groups are la- OY, Turku, Finland). Amino acid transport activity was evalubeled with the postscript -KR or -EC to indicate this. In some other groups a prein- ated by incubating hepatocytes in the prescubation at 37°C in KR preceded the test ence of 0.1 &i of a-amino[l-‘4C]isobutyric period and these are labeled with the post- acid (AIB, sp radioact, 60 mCilmmo1; Amscript -KR ~180. To assess viability in dif- ersham International). After different incuferent circumstances, according to the try- bation periods, hepatocytes, incubated in pan blue exclusion test, fresh hepatocytes the presence of the amino acid analog, were were incubated at 37°C for up to 180 min diluted with 10 ml of fresh incubation meunder gentle agitation under the following dium containing 30 mmol of nonradioactive conditions: (A) in the appropriate fresh AIB (Sigma). After low-speed centrifugabuffer solutions (A-KR and A-EC); (B) in tion three more identical washes were carthe presence of 1.8 M of Me,SO (B-KR and ried out and the cells were finally dissolved B-EC); (C) in the presence of 1.8 M of in Lumasolve and further processed to asMe,SO and 0.8 M of sorbitol (Merck) (C- sess the intracellular radioactivity. In another series of experiments, after an KR and C-EC); (D) after exposure to 1.8 M
146
DE LOECKER ET AL.
initial preincubation period of 180 min, at 37”C, 0.1 $i of L-[U-‘4C]isoleucine or 0.1 $i of ol-amino[l-‘4C]isobutyric acid was added to 1 ml of hepatocyte suspensions, which were then incubated further to 180 min (Groups A-KR ~180 and H-KR ~180). The radioactivity measurements were expressed as disintegrations per min (DPM) per I x lo6 viable cells kSEM. Statistical evaluation of significance was carried out according to Student’s t test. RESULTS
Figure 1 shows the effect of incubation time in KR on the membrane integrity of fresh hepatocytes. After the isolation procedure, 95% of the recovered hepatocytes were considered viable according to the trypan blue exclusion test. Incubation at 37°C in pure KR (Group A-KR) reduced the number of dye-excluding cells by approximately 8% over a period of 60 min. For the cells initially exposed to 1.8 M of Me,SO (Group D-KR) or to 1.8 M of Me,SO together with 0.8 M of sorbitol (Group E-KR) incubation in fresh KR after removal of the cryoprotectants resulted in a fall in dyeexcluding cells amounting to respectively 38 and 18%. However in the presence of one or both cryoprotectants (Groups B-KR and C-KR), the number of dye-excluding cells was reduced after 1 hr of incubation to around 5%. It was observed that, apart from the freshly used hepatocytes, a proportionally faster decline in the dyeexcluding ability occurred over the first 15 min of incubation, followed by further linear deterioration of the cells. Figure 2 shows the effects of incubation time in KR on the membrane integrity of cryopreserved hepatocytes. After thawing, around 75% of the cryopreserved hepatocytes retained the ability to exclude trypan blue. In the presence of 1.8 M of Me,SO (Group F-KR) or in the presence of 1.8 M of Me,SO and 0.8 M of sorbitol together (Group G-KR), the dye-excluding ability of
FIG. 1. Survival of fresh hepatocytes after incubation for different periods of time in Krebs-Ringer bicarbonate buffer (KR). Freshly isolated hepatocytes were suspended in fresh KR (5 x lo6 cells/ml) (A23roup A-KR), in KR containing 1.8 M of Me,SO Q-Group B-KR), or in KR containing 1.8 of Me,SO and 0.8 M of sorbitol (C-Group C-KR) and incubated for up to 60 min. After the exposure of fresh hepatocytes for 45 min to 1.8 of Me+30 (CGroup DXR) or to 1.8 M of Me,SO and 0.8 M of sorbitol (CGroup E-KR) the cryoprotectants were removed by dilution twice 10X, and cells were resuspended in fresh KR for incubation. After each incubation period the cells were treated with trypan blue dye and counted in a hemocytometer. The surviving cells were expressed as a percentage of the original number of hepatocytes. Each value represents the mean of eight experiments ASEM. At the end of 60 min Groups D and E showed a significant (P < 0.01 in both cases) fall in dyeexcluding cells compared to that in controls (Group A-KR). However the number of dye-excluding cells in Groups B and C showed a much greater fall (P < 0.001 in both cases).
the cryopreserved hepatocytes was reduced to 30 and 17%, respectively, after 15 min of incubation, rapidly leveling off to a value below 5% after 60 min of incubation. After removal of Me,SO by dilution (Group H-KR) and resuspension, incubation in fresh KR progressively reduced dye exclusion to 34% over a 60-min period. Dye exclusion by the hepatocytes, after freezing and thawing and subsequent exposure to 0,8 M of sorbitol for 30 min followed by the removal of both cryoprotectants (Group IKR), resuspension, and incubation in fresh KR, followed a similar pattern. These sur-
CRYOPRESERVED HEPATOCYTES
FIG. 2. Survival of cryopreserved hepatocytes after incubation for different periods of time in KR. Cryopreserved hepatocytes (5 x 106cells/ml) after thawing were incubated in the presence of 1.8 M Me+30 in which they were preserved (o--Group F-KR) or after the removal by dilution of the cryoprotectant and resuspension in fresh buffer (Wroup H-KR). Some frozen samples after thawing were further exposed to 0.8 M of sorbitol for 30 min and subsequently incubated in the presence of Me,SO and sorbitol (04roup G-KR) or after the removal of both cryoprotectants (Uroup I-KR). Surviving cells were evaluated according to the trypan blue exclusion test. Each value represents the average percentage of surviving cells of eight experiments *SEM. Again those groups incubated still in the presence of cryoprotectants (groups A and I) showed a much greater fall in viability compared to the relevant group in which the cryoprotectants had been removed (P < 0.001 in both cases at 60 min).
FIG. 3. Survival of cryopreserved hepatocytes after incubation in Euro-Collins and KR. Fresh hepatocytes were incubated in Euro-Collins (CGroup A-EC) and in KR (O-Group A-KR) for different periods of time up to IX0 min. Hepatocytes were cryopreserved in Euro-Collins and in KR in the presence of 1.8 M of Me,SO. After the removal of the cryoprotectant by dilution with the corresponding buffer medium, the cells were incubated in the original volume of either Euro Collins (CGroup H-EC) or KR (O--Group HKR) followed by the viability assessment according to the trypan blue exclusion test. Each percentage value represents the mean of eight experiments kSEM. By 180 min hepatocytes incubated in KR (Group A-KR) showed a dye exclusion significantly lower than that of those incubated in EC (Group A-EC) (P < 0.001). The same was true on recovery after freezing (P < 0.001).
vival indices do not take into account the proportion of cells lost during the cryopreservation procedure. Figure 3 shows a comparison of the survival of cryopreserved hepatocytes after incubation in either KR or EC. Dye exclusion by freshly prepared hepatocytes incubated in Euro-Collins medium decreased almost linearly by 16% over a period of 180 min (Group A-EC), while in the analogous circumstances, incubation in KR reduced the number of dye-excluding cells by 87% (Group A-KR). In the latter case particularly after the first hour of incubation, dye exclusion declined sharply. As for the hepatocytes cryopreserved and incubated in
Euro-Collins buffer, the reduction in dyeexcluding cells over a period of 180 min amounted to 47% (Group H-EC), while cryopreservation and incubation in KR reduced dye-excluding ability by 40% after 30 min and by 52% after 60 min with only 2% viability retained after 180 min of incubation (Group H-KR). Figure 4 illustrates the protein synthesizing activity of fresh and cryopreserved hepatocytes. Although the incorporation of L-[U-‘4C]isoleucine into the proteins of freshly prepared hepatocytes and of cryopreserved hepatocytes after removal of Me,SO and resuspension in KR (Groups AKR and H-KR) increased over the incuba-
incubation
time
in min
148
DE LOECKER ET AL.
102030
60
90
120 180 Incubation time in men
FIG. 4. Incorporation of L-[U-‘4C]isoleucine into the proteins of fresh and cryopreserved hepatocytes. Fresh hepatocytes (Wroup A-KR) or hepatocytes cryopreserved in KR (0) containing 1.8 M of Me,SO after removal of the cryoprotectant (Group H-KR) by dilution were incubated in KR (5 x 106cells/ml) in the presence of 0.1 &i of L-[U-“CJisoleucine at 37°C over 180 min. In another series of experiments fresh hepatocytes (U-Group A-KR ~180) and cryopreserved hepatocytes (o--Group I-I-KR ~180) after the removal of Me+ were preincubated without radioactivity for 180 min in KR at 37°C. Subsequently 0. l PCi of L-[U-‘4C]isoleucine was added and incubation was continued for another 180 min during which the amino acid incorporation was evaluated. Each incorporation value expressed as disintegrations per min @PM) per I x 106of originally viabk hepatocytes is the mean of eight experiments zSEM. Cryopreserved hepatocytes (H-KR) showed a significantly lower incorporation of leucine at 60 min (P < 0.001 compared to fresh A-KR). Preincubation for I80 min considerably reduced activity in both fresh and cryopreserved groups subsequently tested for 180 min.
tion period of 180 min, the rate of incorporation progressively leveled off after the first hour. After 180 min of incubation the total radioactivity incorporated by cryopreserved hepatocytes (Group H-KR) amounted to 56% of the fresh samples (Group A-KR). Supplementary addition of 140 mmol of glucose (Merck) to the KR did not affect the rate of protein synthesizing activity. When fresh (Group A-KR ~180) and cryopreserved cells (Group H-KR p180), preincubated for 180 min, were subsequently subjected to a further incubation for up to 180 min in the presence of L-[U-14C]isoleucine, the observed incorpo-
ration of radioactivity was similar for both cell samples and progressed at a much lower level. In Fig. 5 the membrane transport activities of fresh and cryopreserved hepatocytes are shown. The uptake of the amino acid analog, u-amino[l-‘4C]isobutyric acid (a measure of amino acid membrane transport activity) by freshly prepared hepatocytes (Group A-KR) rapidly increased up to a peak value which was reached after 60 min of incubation. Upon prolonged incubation an equally fast de8000 i
I-* 1020304D
= 60
90
n 120 180 Incubation time ID mm
FIG. 5. The uptake of a-amino[l-‘4C]isobutyric acid by fresh and cryopreserved hepatocytes. Fresh hepatocytes (CGroup A-KR) and hepatocytes cryopreserved (O---Group H-KR) in KR containing 1.8 M of Me,SO after the removal of the cryoprotectant by dilution were incubated in KR (5 X lo6 cells/ml) in the presence of 0.1 &i of a-amino[l-14C]isobutyric acid over I80 min. In a further experiment the freshly prepared hepatocytes (Wroup A-KR ~180) and the cryopreserved hepatocytes (U--Group H-KR ~180) after removal of Me,SO were preincubated for I80 min without radioactivity and after the addition of 0.1 &i of u-amino[l-‘4C]isobutyric acid incubation was continued for another 180 min. Each value expressed as DPM per I x lo6 of originally viable hepatocytes is the mean of eight experiments zkSEM. Maximum uptake was seen between 40 and 90 min of incubation, and 60 min was chosen as a convenient point for comparison. Again cryopreserved hepatocytes (H-KR) showed significantly depressed uptake compared to that of controls (A-KR) (P < 0.001). Also as before, preincubation for 180 min (A-KR ~180 and H-KR ~180) diminished uptake greatly.
CRYOPRESERVED
cline in intracellular radioactivity occurred, which after 120 min progressively leveled off. The cryopreserved hepatocytes frozen in 1.8 M of Me,SO, after the progressive removal of the cryoprotectant by dilution (Group H-KR), also showed a peak uptake value of the amino acid analog which was attained after 30 min of incubation. Further incubation progressively reduced the accumulated radioactivity as was observed with the fresh hepatocytes, although again at a lower level of activity. After an initial preincubation for 180 min at 37°C of both freshly preparedhepatocytes (Group A-KR ~180) and cryopreserved cells after removal of 1.8 M of Me,SO (Group H-KR p180), the subsequent incubation for a further 180 min in the presence of 0.1 @Zi of cy-amino[ l-‘4C]isobutyric acid resulted in identical low values similar to nonspecific background radioactivity.
HEPATOCYTES
149
the recuperated hepatocytes after thawing (on the basis of the dye exclusion reaction) remained high, there was a marked decline in viability upon further incubation of the cryopreserved hepatocytes. The beneficial protective actions of the addition of sorbitol before dilution, which was observed with nonfrozen cells, was not apparent after thawing frozen hepatocytes. Since both fresh and cryopreserved hepatocytes incubated in a buffer medium with intracellular electrolyte composition retain viability to a much greater extent, even over longer periods of time, we surmise that the membrane (Naf ,K+)-ATPase pump mechanism is protected by the intracellular buffer composition (6, 7). Under these circumstances, however, viability values obtained with the dye exclusion method cannot be compared with the direct evaluation of metabolic parameters because membrane transport and protein synthesis are considerably inhibited DISCUSSION by the intracellular electrolyte composition of the incubation buffer. It should be During incubation in the KR buffer, which is characterized by an extracellular pointed out here that other workers have electrolyte composition, a progressive slow commented on the beneficial results obdecline in viability of freshly prepared he- tained when using potassium-rich solutions patocytes occurs, while in the presence of during addition and removal of cryoproMe,SO or of Me,SO together with sorbitol, tectants (4, 19, 30). In addition, we previviability is extremely poor. However, cells ously reported that the contractile reinitially exposed to Me,SO or to Me,SO sponses of smooth muscle after cryopreserand sorbitol, which are washed before be- vation were best preserved when freezing ing incubated in fresh buffer, retain a con- was carried out in a potassium-rich intrasiderable degree of dye-excluding ability. cellular buffer (3 1). Thus the importance of Cells exposed to Me,SO together with sor- these types of buffer in cryopreservation bitol before incubation show a higher de- generally is worthy of more research in the gree of viability upon subsequent incuba- future. Our present results show that the assesstion compared with cells exposed to Me,SO ment of viability may also depend on the alone. The removal of the cryoprotectant different parameters examined. Indeed the Me,SO in the presence of the extracellular sorbitol was beneficial as observed in pre- viability values expressed in terms of the vious studies with erythrocytes (6,7). It has trypan blue exclusion test or the ability to been noted previously that after the freeze- metabolize fluorescein diacetate are usually thaw procedure, followed by dilution of the substantially higher than when metabolic cryoprotectants and resuspension in fresh parameters such as gluconeogenesis, glycomedium, the initial number of hepatocytes gen synthesis, urea synthesis, bilirubin conmay already be reduced by around 30% jugation, ability to accumulate 99mT~ (12). Although the immediate survival of HIDA, and particularly protein synthesis
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DE LOECKER
are evaluated (13, 16, 18, 22, 29). This sug gests a lack of correlation between the dye exclusion tests and the metabolic tests; the latter probably more accurately represent metabolic recovery. Reservation of protein synthesizing activity and membrane transport may not always run parallel. In the present system, however, the viability values obtained by the dye exclusion are additionally illustrated and confirmed by the uptake of ol-amino[ l-L4C]isobutyric acid which is a measure of amino acid transport activity and by the incorporation of L-[U-‘~CIisoleucine into the protein. Indeed the initial increase in uptake of the amino acid analog in fresh as well as in cryopreserved hepatocytes was followed by a sharp decline. This is an indication of cell membrane damage and similarly the protein synthesizing activity leveled off as incubation progressed. According to both parameters, the metabolic activities of the cryopreserved hepatocytes were recorded at a lower level than the fresh cells. Dehydration induced by freezing leads to cell shrinkage and the ensuing changes in membrane mechanical properties may damage membrane structures resulting in vesiculation and -even loss of membrane material (10, 15, 17, 27, 29, 32, 36, 37). Apart from morphological anomalies, permeability changes modifying membrane transport functions are concomitant with an inhibited metabolic activity (9). As both fresh and cryopreserved hepatocytes, after an initial preincubation period of 180 min, are unable, upon subsequent incubation, to concentrate a-aminoisobutyric acid, while amino acid incorporation into the proteins still occurs at a low base level, it appears that even in the absence of adequate membrane transport function some residual aspects of protein synthesizing activity may continue to take place. Although hepatocytes prove particularly
ET AL.
sensitive to cryopreservation, careful handling allows recovery of a considerable proportion of viable cells capable of demonstrating efficient membrane transport activity and protein synthesizing activity even in an incubation medium not containing hepatotrophic factors (8). ACKNOWLEDGMENTS
The authors are indebted to the FGWO (Belgian National Foundation for Medical Research) for a grant to the laboratories and to Mrs. F. De Wever and Miss N. Volders for their excellent technical assistance. REFERENCES
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