the bioenergetics of mitochondria after criopreservation

CRYOBIOLOGY 26, 333-340 (1989)

The Bioenergetics of Mitochondria after Cryopreservation

BARRY J. FULLER,* ALESSANDRO RUBINACCI,1- KAREL GEBOES,$ AND WILLIAM DE LOECKER

*Academic Department of Surgery, Royal Free Hospital School of Medicine, University of London, London NW3 2QE, England; fClinica Ortopedica, Universitci degli Studi di Milano, Istituto ScientiJico San Raffaele,

20132 Milan, Italy; and Afdeling Biochemie en #LaboratorYurn voor Histochemie en Cytochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium

The functional characteristics of rat liver mitochondria after cryopreservation with and without the addition of the cryoprotectant dimethyl sulfoxide (Me,SO) were evaluated. As criteria of functional integrity, polarographic measurements of substrate-linked oxygen consumption and luminescent assay of adenosine triphosphate (ATP) synthesis were considered before and after cryopreservation. The results demonstrated that mitochondrial damage after freezing was indicated by the polarographic studies but was not evident when ATP synthesis was considered. Me,SO present during cryopreservation was partially protective for mitochondrial substrate-linked oxygen consumption; however, simple exposure to and dilution from Me,.50 effected some changes in mitochondrial function. Q 1989 Academic Press, Inc.

Various studies have been undertaken over the past 2 decades on cryopreservation of mitochondria for fundamental research into the nature of freezing damage (1, 11). The recovery of mitochondria after freezing which retain characteristic functional capabilities (most notably the cou- pling of oxygen consumption and ATP synthesis) remains controversial. The early studies of Privitera et al. suggested that successful recovery of functional rat liver mitochondria from temperatures below -70C could be achieved in the absence of any added cryophylactic agents employing fast cooling by direct transfer into the cryo- genic bath (18). Dickinson er al. (4) cooled plant mitochondria in the presence of dimethyl sulfoxide (Me,!30 - 5% (v/v)) at in- termediate rates (-25C/min) and at fast rates by direct transfer to liquid nitrogen and reported little difference in substrate- linked oxygen uptake compared to that seen in fresh, unfrozen mitochondria. More recent studies by Tsvetkov et al. (22) and

Received December 21, 1987; accepted November 22, 1988

Petrenko and Subbota (17) indicated that a variety of functional changes occurred in liver mitochondria cooled rapidly in sucrose solutions without any additional cryoprotectant. Also, the studies from Fishbeins group (7) suggested that, while rapid cooling in the presence of Me,SO resulted in good preservation of ultrastruc- ture and enzymatic profiles of rat liver mitochondria, coupled respiration was de- stroyed. However, in these studies the oxygen consumption measurements were made with Me,SO still present in the system. The present experiments were de- signed to investigate the relationship between coupled respiration and ATP production in rat liver mitochondria following cryopreservation and dilution of the cryoprotectant in order to determine whether functionally useful organelles can be batch- stored and recovered for subsequent metabolic studies.

MATERIALS AND METHODS

(i) Mitochondrial Preparation Mitochondrial fractions were isolated

from the livers of fasting rats following the technique of Schnaitman and Greenawalt

333 001 l-2240/89 $3.00 Copyri&t 0 1989 by Academic Press, Inc. AII rights of reproduction in any form reserved.

334 FULLER ET AL.

(20) with minimal modifications. Mitochon- dria were finally suspended in 0.25 M sucrose at pH 7.4. Transmission electron mi- croscopy was considered as criteria of structural integrity. In preliminary studies, the retention of typical inner compartment features in all fresh and cryopreserved fractions indicated that gross ultrastructural damage could be excluded as was observed in other systems (9). Thus, polarographic measurements of substrate-linked oxygen consumption and ATP synthesis were evaluated as criteria of mitochondrial functional integrity (15).

(ii) Polarographic Oxygen Uptake Studies

Substrate-linked oxygen consumption and ADP:O ratios were measured as described previously by Estabrook (6). For this purpose a closed reaction chamber fit- ted with a Clark-type oxygen electrode (Rank Brothers, Bottisham, Cambridge, UK) was used. The incubation medium (2.2 ml) contained 220 mM mannitol, 70 m&f sucrose, 5 rr& Tricine (Merck), 5 n&f KC1 (Merck), 10 m&f KH,PO, (Carlo Erba), 15 m&f MgCl, (Merck). The solution was fully equilibrated with air. To this 0.2 ml of mitochondrial suspension was added to give a final protein concentration of 2-3 mg/ml. Oxygen consumption after the addition of succinate (6 mmol) and ADP (200 pmol) was recorded on a chart recorder (DBD Ankersmit, Belgium) set at 1-mV full scale deflection. From the traces of respiratory control ratio (RCR) (ratio of state III to state IV respiration) and ADP:O ratio were calculated (6). Measurements were made at 20-22C. Preparations which did not show a good respiratory control were discarded.

(iii) ATP Synthesis by Luminescence

ATP synthesis by liver mitochrondria in- cubated as under section (ii) was followed by a multiple time point analysis. For this, immediately and at 1, 2, and 3 min after the addition of ADP (1 r&f) to the incubation medium, 200~cl.1 aliquots of the experimen-

tal mixture were removed for assay. ATP was extracted in 2 ml of 0.9 M trichloroace- tic acid containing 4 mM EDTA and further diluted x103 in Tris-EDTA, pH 7.5. ATP concentration was measured by the luminescence method using firefly luciferase (LKB-Wallach Test Kit). The luciferin/ luciferase reagent was added to each sample and the luminescence was evaluated by a 1250 LKB luminometer (Wallac, Turku, Finland).

(iv) Cryopreservation

For these studies, undertaken on mitochrondria in sucrose alone, the final mitochondrial suspension was pipetted into polypropylene tubes (Nunc cryotubes, GIBCO Europe, Gent, Belgium; maximum volume, 3 ml) at a volume of 1.5 ml and cooled to -3C in a refrigerated bath. Ice crystallization was induced by clamping the tubes in surgical forceps cooled to - 196C which produced crystallization without further significant cooling, and latent heat of ice formation was allowed to dissipate over 10 min. By this time, the temperature had returned to - 3C as measured in a dummy tube treated similarly and containing a thermocouple attached to a digital thermometer. The tubes were then transferred to liquid nitrogen, producing a cooling rate of 150-200C/min (measured on a sample tube into which was inserted a copper/ constantan thermocouple attached to a digital thermometer-Comark). Rewarming was achieved by placing the tubes directly into a water bath at 37C until all the ice had just disappeared.

For studies on cryopreservation with Me,SO, a final concentration of 1.5 M was chosen as this was known to protect some metabolic functions of intact liver cells (11). A problem remained for the method of re- moval of this agent after cryopreservation. The normal method of a centrifugal washing step was inappropriate, since this would necessitate an additional centrifugation, which could damage mitochondrial func-

MITOCHONDRIAL CRYOPRESERVATION 335

tion. It was considered, however, that as much Me,SO as possible should be removed in case the agent itself affected mitochondrial function. The problem was overcome by adding Me,SO to a final concentration of 1.5 M to the mitochondria- containing supernatant after the first pre- parative step (2 x 600g spins). To achieve this, Me,SO was added dropwise (over 4 min) and with stirring at 4C to achieve a final concentration of 1.5 M. This partially purified suspension was then used for cryopreservation in Nunc tubes as before. The samples were cooled to -7C and ice nu- cleation was again induced using precooled forceps. A control tube containing the thermocouple was used to follow the thermal history. After dissipation of latent heat of ice formation (at 10 min), the tubes were transferred to liquid nitrogen. The samples (both sucrose alone and sucrose plus Me,SO) were maintained at - 196C for a minimum of 1 hr before rewarming in a water bath at 37C, giving a warming rate of IOO-20OWmin. Upon thawing, the mitochondrial fraction containing Me,SO was then spun at lO,OOOg, discarding the supernatant containing most of the Me,SO. The final mitochondrial pellet was resuspended in fresh 0.25 M sucrose, dropwise (over 4 min) at 4C and used for the functional as- sessment. This method resulted in an esti- mated remaining Me,SO content of 0.1 M in the organelles.. Studies undertaken during the experiments to assess the effects of freezing dilute versus concentrated suspensions of mitochondria in sucrose alone showed no difference in RCR (data not shown) and thus we were happy to proceed with this method of handling Me,SO- exposed organelles after cryopreservation. Control mitochondria were assayed for functional integrity immediately after preparation. Cryopreserved mitochondria were assayed immediately after recovery from - 196C and final centrifugation. Statistical evaluations were made using Students t test for unpaired data.

RESULTS

(i) Functional Characteristics of Freshly Isolated Mitochondria

Substrate-linked oxygen consumption and ATP synthesis by freshly isolated rat liver mitochondria indicated that the organelles retained good functional characteristics. In Fig. 1A a typical recorder tracing of oxygen consumption which was well controlled after addition of succinate is shown. There was an immediate rapid increase in coupled oxygen uptake, in a reproducible fashion, after addition of ADP. The mean RCR values for fresh mitochondria (3.30 -+ 0.63) and ADP:O ratios (2.24 + 0.37), given in Table 1, are within the previously published range for freshly isolated liver mitochondria (22). ATP synthesis by these organelles was found to be 0.68 + 0.44 ~mol/min/mg protein (Table 1).

(ii) Effect of Exposure to Me@

Figure 1B presents a typical graph of oxygen uptake by the mitochondria from the same preparation which had been exposed to 1.5 M Me,SO and subsequently washed, as described above. In general the pattern was that of functional change, with less well-controlled substrate-linked respiration

I

41.2pM02

1 min \ ---*

FIG. 1. Typical pattern of oxygen consumption by control rat liver mitochondria (MTCH); RCR = 3.2; ADP:O = 2.04 (graph A). Oxygen consumption after exposure to 1.5 M Me,SO and subsequent dilution is shown in graph B; RCR = 1.75; AlIP: = 2.50. Num- bers on the graphs are measured stope values.

336 FULLER ET AL.

Respiratory Control (RCR), ADP:O, and ATP Synthesis in Three Groups of Mitochondria

RCR ADP:O ATP

synthesis ATP synthesis

after oligomycin

Control Frozen (sucrose alone) Frozen (sucrose + 1.5 M Me,SO)

3.30 f 0.63 2.24 * 0.31 0.68 -+ 0.44 0.04 * 0.02 1.67 + 0.33* 2.61 2 0.51 0.56 * 0.48 0.21 k 0.11* 2.20 t o/w 2.54 2 0.31 0.87 a 0.67 0.08 f 0.02*

Note. The three groups of mitochondria are control, freshly isolated mitochondria (n = 5); mitochondria recovered after cryopreservation in sucrose alone (n = 5); and mitochondria recovered after cryopreservation in 1.5 M Me,SO (n = 5, except for ATP synthesis data when n = 4). Values are means ? SDS. ATP synthesis is expressed as pmol/min/mg protein.

* denotes statistically significant (P < 0.05) difference from control data.

(RCR, 1.88 compared to 3.2 for control) and lower efficiency in the coupled oxygen consumption during ADP phosphorylation (ADP:O of 2.50 compared to control value of 2.04). A similar picture was observed using mitochondria from another preparation. RCR was reduced from 3.0 to 2.54 after Me,SO exposure and washing; ADP:O increased from 2.13 to 3.06 after Me,SO exposure.

In preliminary experiments Me,SO at ti- nal concentrations of 1 and 5% (v/v) was added to the measuring chamber during oxygen uptake studies. These concentrations were chosen to cover the range of residual Me,SO remaining after washing. There was an immediate increase in oxygen consumption, indicating a change in respiration toward the uncoupled state (i.e., oxygen consumption rate increased in the absence of added ADP). The RCR changed from 3.2 in the control to 2.57 in the presence of 1% Me,SO and to 2.2 in the presence of 5% Me,SO. An example of the type of trace produced after addition of Me,SO is shown in Fig. 2.

(iii) Functional Characteristics of Cryopreserved Mitochondria

Figure 3 represents typical graphs of oxygen consumption studies using mitochondria recovered from cryopreservation without added Me$O. Again, compared to freshly isolated mitochondria there was evidence of functional change toward uncou-

pled respiration. Similarly, Fig. 4 illustrates the pattern of oxygen consumption after cryopreservation in the presence of 1.5 M Me,SO, which also shows functional com- promise after freezing. However, having stated this it will be evident that the mitochondria cryopreserved in the presence of Me,SO do retain the ability to respond to ADP addition by increased uptake of oxygen in a reproducible fashion so that gross damage and uncoupling (as seen for example by adding dinitrophenol) were not evident (13). The RCR values were both lower than those seen in fresh mitochondria (Ta- ble 1). For mitochondrial frozen in sucrose

1 ADP

Me+0

FIG. 2. Pattern of oxygen consumption in a mitochondrial sample after addition of Me,.90 to the cu- vette. On addition of Me,SO (1% (v/v) final concentration) at the point indicated, a change in oxygen uptake (in state IV) was noted, tending toward an uncoupled respiration.

MITOCHONDRIAL CRYOPRESERVATION

1 min +

FIG. 3. Typical pattern of oxygen consumption by control rat liver mitochondria (MTCH); RCR = 3.2; ADP:O = 2.04 (graph A). Oxygen consumption for mitochondria after cooling to - 196C and rewarming in a solution of 0.25 M sucrose alone is shown in graph B; RCR = 1.6; ADP:O = 3.35.

alone the mean RCR was 1.67 + 0.16, which was significantly lower than that for fresh mitochondria (P < 0.01). In fact Me,SO did seem to prevent some of the damage which was indicated by the decreased RCR, since the value for this group (mean of 2.20) was significantly higher than that for the group cryopreserved in sucrose alone (RCR mean of 1.67) at a significance level of P < 0.05.

In terms of efficiency of oxygen consumption during ADP phosphorylation (ADP:O), there was no difference between the cryopreserved groups (means of 2.61 + 0.51 frozen without Me,SO and 2.54 f 0.31 frozen in the presence of 1.5 M Me,SO). Both groups tended to have higher values than those seen in fresh mitochondria, and indeed higher than those theoretically possible using succinate as substrate (ADP:O of 2), which is a surprising finding. When ATP synthesis was examined, no differ- ences were evident between fresh mito-

1 min

FIG. 4. Typical pattern of oxygen consumption by control rat liver mitochondria (MTCH); RCR = 3.2; ADP:O = 2.04 (graph A). Oxygen consumption for mitochondtia which have been cooled to - 196C in a solution of 0.25 M sucrose plus 1.5 M Me,SO and subsequently rewarmed is shown in graph B; RCR = 2; ADP:O = 2.6.

chondria (0.68 + 0.44 p,mol/min/mg protein) and those cryopreserved in sucrose alone (0.56 -t 0.48 pmoYmin/mg) or with added Me,SO (0.87 _+ 0.67 p.moYmin/mg; Table 1).

A further group of experiments on ATP synthesis was performed in the presence of oligomycin, known to inhibit the ATP- synthase (16). In fresh mitochondria, as ex- pected, oligomycin decreased ATP synthesis by about 90%. However, in the mitochondria recovered from cryopreservation in sucrose alone, inhibition was only about 60%, while in those cryopreserved in Me,SO, oligomycin inhibition was similar to that seen in controls.

DISCUSSION

In these studies we have demonstrated that rat liver mitochondria recovered from cryopreservation were able to respond in a qualitatively similar fashion in terms of oxygen consumption after addition of sub- strates (succinate) and phosphate acceptor (ADP) when compared to the response of

338 FULLER ET AL.

fresh, control organelles. However there was a significant alteration in the quantita- tive response of cryopreserved mitochondria, which showed in general a decrease in efficiency of substrate-linked oxygen uptake. The values for RCR were lower than those in fresh mitochondria (suggesting that respiration was less well controlled in the absence of added substrate after freezing). The presence of Me,SO during cryopreservation did in fact provide some protection of mitochondrial RCR, which was higher than that seen in organelles cooled in sucrose alone and was also observed in other systems (2).

There have been few reports comparing mitochondrial cryopreservation with and without Me,SO (8). One problem in this type of study is that the Me,SO must be removed, and the most common approach to this is to perform centrifugal washes. However, mitochondria are sensitive to damage during centrifugation and additional centrifugation steps are best avoided. Moreover the exposure to Me,SO was damaging by itself. This caused a significant change in substrate-linked oxygen uptake even in the absence of freezing. We feel that this may be due to alteration of the permeability characteristics of the inner mitochondrial membrane, perhaps due to the presence of trace levels of Me,SO remaining after washing. In other experiments we demonstrated that addition of low concentrations of Me,SO (-0.1 M) to the organelles undergoing oxygen uptake studies caused an immediate decrease in efficiency of the process. These concentrations of Me,SO were similar to those calculated to be present in the mitochondria after washing. Perhaps these actions of Me,SO in the absence of freezing could partly explain some of the changes in cell function noted in the presence of Me,SO by other workers (3, 12).

We were puzzled by the values of ADP:O displayed by the two groups of cryopreserved mitochondria. In both, ADPO val-

ues were similar to or tended to be higher than those seen in controls. This was in contrast to the RCR values which suggested that respiration in cryopreserved mitochondria was to a significant degree uncoupled. We feel that the large consumption of oxygen by the population of cryopreserved organelles which were in the uncoupled state tended to make the measurements of ADP-stimulated oxygen consumption difficult to measure accurately. Also in cases where ADP:O tended to be greater than that for control organelles, this apparent contradiction might be answered by considering that only a few mitochondria in the suspension exhibited coupled respiration after freezing, thus consuming less oxygen per aliquot of suspension in state III. It is this lower oxygen consumption which when used in the ADP:O ratio calculation produced an apparent increase in the value. ADP:O ratio is a good index of function when the respiratory control ratio is 3 or more. However, problems arise when the RCR is only slightly above 1 (23) and thus ADP:O in this situation is not valid. We display the data to draw attention to this artifact in our studies on cryopreserved mitochondria.

It is interesting that the ATP synthesis in both groups of cryopreserved mitochondria was similar to that in fresh organelle fractions even though the oxygen consumption characteristics were changed. This appar- ently conflicting evidence could be ex- plained or understood by considering that ADP, as required by the reaction conditions in the ATP-synthesis studies, was present in concentrations much higher than those used in oxygen uptake studies; thus the thermodynamic pressure on ATP- synthase was increased and an equivalent ATP production was achieved (16). Indeed, ATP-ase related phenomena in sealed membrane vesicles were not affected by freezing per se (19), suggesting that the enzyme moiety could retain function and di- rectional transport across the, membrane af-

MITOCHONDRIAL CRYOPRESERVATION 339

ter freezing. It might also be considered that higher concentrations of ADP may stimulate the adenylate kinase system which may produce ATP from direct con- densation of ADP molecules without in- volving the electron transport chain (5). If this latter system were operational, block- ing the ATP synthase with oligomycin would have a smaller inhibitory effect on ATP production quantitatively dependent on the amount of damage to the primary ATP-synthesizing system, i.e., the proton circuit. Indeed, when the mitochondria were better protected by the addition of Me,SO, the accessory production of ATP (e.g., possibly the adenylate kinase system) was comparatively less prominent. In addition it is worthy to note that under certain conditions the measured activity of the ATP-synthase reaction and the acceptor control index may vary independently of one another (14). This is especially the case when the respiratory control ratio is only slightly above 1 (23). This was the case in our cryopreserved organelles.

The unexpectedly high ATP production after cryopreservation, in the face of al- tered substrate-linked oxygen consumption, may be one of the explanations of the apparent successful cryopreservation of rat liver mitochondria originally reported by Grieff and co-workers (18). In their system, functionality was measured by incorpora- tion of inorganic phosphate during phosphorylation, which would produce data similar to our ATP synthesis results. Only the relatively more recent polarographic methods for measuring substrate-linked oxygen uptake are sensitive enough to dem- onstrate small but significant changes in inner membrane permeability and/or enzyme organization. It is likely that the noted functional changes result from the type of alter- ations in the inner mitochondrial membrane described previously by Tsvetkov er al. (22).

Another point of minor interest is that the rat liver mitochondrial fractions in the pres-

ence of Me,SO appear to have survived cryopreservation reasonably well at rates which are known to be damaging (-200Wmin) to the parent hepatocytes (10). Under these conditions virtually no active metabolism can be measured in isolated hepatocytes, and after ultrastructural examination the mitochondria can barely be recognized, showing gross disruption immediately on thawing. This would argue that the environment around the organelles in situ is less able to protect during fast cooling. One obvious difference is that during cryopreservation of the isolated organelles sucrose is present at relatively high concentrations, and the demonstrated membrane-protective effects of this carbo- hydrate may be important. However, since this large sugar cannot penetrate the inner mitochondrial membrane, total protection is not achieved. Another possibility is that the cytoplasm might inhibit mitochondrial water movement and favor formation of in- tramitochondrial ice although at present we have no evidence for this.

In summary, we can say that cryopreservation of mitochondria for batch storage and subsequent use in substrate-linked oxygen uptake studies remains unsatisfactory at the present. However, the organelles did show qualitatively similar responses to the added substrate and phosphate acceptor, and gross disruption is not suspected. Me,SO provided some additional protection over sucrose alone, but the agent by itself will affect mitochondrial function. The weight of evidence from our own and previous studies suggests that hepatic mitochondria are not easy to cryopreserve in a fully functional state as has been discussed in the past. Our results also raise questions for further study into the effects of cryopreservation on mitochondrial function. In particular it will be interesting to try and establish the route of ATP synthesis in the organelles which exhibit low RCR, includ- ing the role of adenylate kinase in this production. It is apparent that much more

340 FULLER ET AL.

work is required before a complete under- standing of low temperature storage of mitochondria is achieved.

ACKNOWLEDGMENTS

The authors are indebted to the Belgian National Foundation for Medical Research (F.G.W.O.) for a grant to the laboratory, to the Nationale Loterij for a research grant, and to Mrs. F. De Wever for her valu- able technical assistance.

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