CRYOBIOLOGY

28, 436-444 (1991)

Cryopreservation of Isolated Hepatocytes: Intracellular Ice Formation under Various Chemical and Physical Conditions CHERYL L. HARRIS,* MEHMET TONER,* ALLISON HUBEL,* ERNEST G. CRAVALHO,* MARTIN L. YARMUSH,t,$ AND RONALD G. TOMPKINSS,’ *Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139; Massachusetts Institute of Technology, Department of Mechanical Engineering, Cambridge, Massachusetts 02139; fRutgers University, Department of Chemical and Biochemical Engineering, Piscataway, New Jersey 08854; and .#Surgical Service, Massachusetts General Hospital, Department of Surgery, Harvard Medical School, Boston, Massachusetts 02114

Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 4OOWmin in isotonic solution. At SO”C/min and below, none of the hepatocytes underwent IIF; whereas at lSO”C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of - 7.7”C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. “TrlF from isothermal temperature experiments was - - 5°C as opposed to -7.X for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until - - 20°C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me,SO) on IIF was also quantified. 5oT,rF decreased from - 7.7”C in the absence of Me,SO to - 16.&X in 2.0 M Me,SO for a cooling rate of 4OO”Umin. However, the cooling rate (between 75 and 4OO”CYmin) did not significantly affect 5oT,,F (-8.7”C) in 0.5 M Me,SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes. 0 1991 Academic

Press. Inc.

Although the cryopreservation of hepatocytes has been the objective of numerous studies, optimal cryopreservation methods have not been developed. Several investigators have published cryopreservation methods for both animal and human hepatocytes; however, in all cases, biochemical functions were altered and viability was significantly decreased (1, 2, 8, 9, 11, 23). In most of these studies, the cellular viability was related to enzymatic activity in the short term. The observed metabolic activities of thawed cells appear to be contribuReceived August 20, 1990; accepted December 10, 1990. ’ To whom correspondence should be addressed at: Massachusetts General Hospital, Trauma Services, Boston, MA 02114. 436 001 l-2240/91 $3.00 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

tions of only a relatively small number of surviving intact cells, a number that decreases rapidly during subsequent culture. None of these studies have investigated the fundamental physicochemical parameters involved in the freezing of hepatocytes; however, this fundamental understanding is clearly required in order to develop successful cryopreservation protocols. Intracellular ice formation (IIF) is an important physicochemical parameter in cryobiology because of a strong correlation between IIF and cellular death (14). Maximum survival after a freeze-thaw protocol to - 196°C can be obtained if IIF is avoided and the cooling process occurs as rapidly as possible minimizing the damaging effects of the extracellular ice on the cell (14). At suboptimal cooling rates, cellular injury may

INTRACELLULAR

ICE

FORMATION

result from long time exposure to high electrolyte concentrations, excessive cell dehydration, and mechanical effects of the external ice (14). On the other hand, at supraoptimal cooling rates cellular injury occurs from IIF (14). Therefore, it is very important for the conditions resulting in IIF to be determined for any cell type under consideration for cryopreservation. The objective of this study was to characterize the IIF behavior of isolated rat hepatocytes under various freezing conditions with the aid of a cryomicroscopy system. The initial work was performed in the absence of cryoprotective additives (CPAs) to facilitate the evaluation of the complex physicochemical processes occurring during freezing. Further data were collected in the presence of dimethyl sulfoxide (Me,SO) to quantify the effect of Me,SO on the IIF behavior of isolated rat hepatocytes.

OF

HEPATOCYTES

437

50 ml of Dulbecco’s modified Eagle’s medium (DMEM, Hazelton, Lenexa, KS), and 12.5 ml of cell suspension was added to 10.8 ml of Percoll (Pharmacia, Piscataway, NJ) and 1.2 ml of 10x concentrated DMEM. The mixture was centrifuged at 50g for 5 min and the cell pellet was washed twice with DMEM. Routinely, 200 to 300 million cells were isolated from an 8-g liver, with viability ranging from 90-98% using trypan blue exclusion dye. The cells were resuspended in high-glucose DMEM (4.5 g/liter glucose, Hazelton) prior to use. Cryomicroscopy system. The apparatus used to cool and observe the hepatocytes consisted of a Zeiss Universal light microscope (Carl Zeiss, West Germany), a video camera (Ikegami ITC 62, Japan), a color monitor (Panasonic Sl300, Japan), a video cassette recorder (Panasonic 6300), a specially designed cryostage, a system consisting of a computer-based temperature conMATERIALS AND METHODS troller and a video image processing system Hepatocyte isolation. The animals and (Interface Techniques, Cambridge, MA). isolation techniques used to obtain isolated The cryomicroscopy system is described in rat hepatocytes have been described in de- detail elsewhere (4). IIF during freezing tail by Koebe et al. (9). Hepatocytes were was manifested as a sudden and marked inobtained from 2-month-old, female Lewis crease in opacity (29). Direct correlation of rats (200 g) by a modified procedure of Se- IIF with time and temperature was accomglen (27). Animals were anesthetized with plished by electronic superposition of these ether. The liver, weighing roughly 8 g, was data on the video image. Timing signals were derived from the video chain (0.1 s perfused in situ with 300 ml of Ca*+-free intervals) which provided the software with Krebs Ringer bicarbonate buffer, containing 5.5 mM glucose and 20 mM Hepes, pH an accurate clock for generating the tem7.4, at 47 ml/min. The perfusate was mainperature protocol by updating the display tained at 37°C and was equilibrated with synchronously with the camera. The fastest 95% O2 and 5% CO*. The liver was subse- cooling rate used in this study was quently perfused in vivo with 0.05% colla400”C/min resulting in -0.67”C change in temperature during 0. l-s intervals. genase (Type IV, Sigma, St. Louis, MO) Cryomicroscopy and statistics. A 5-t.~1 solution containing 5 mM Ca*+ for 10 min in a recirculation system. The resulting cell sample of the hepatocyte suspension was suspension was filtered through two nylon positioned on the cryostage window with a meshes (Small Parts, Miami, FL), with grid microliter syringe under a cover slip (12 X sizes of 210 and 62 pm. The cell pellet was 12 mm*) and sealed with silicone grease. The thickness of the sample was between collected by centrifugation at 17 g for 3 min. Hepatocytes were further purified by a 50 and 150 pm. A 40X objective (Ph2 40/ modified procedure of Kreamer et al. (10). 0,75, Carl Zeiss) and a 1.25X photo-ocular (Carl Zeiss) were used. Thermal gradients Briefly, the cell pellet was resuspended into

438

HARRIS

in the field of view were determined with distilled water and the maximum temperature difference recorded was 0.25”C over a field of 1 mm (32). Only hepatocytes located within 200 pm of the thermocouple junction were monitored for IIF experiments to minimize errors introduced by temperature gradients across the cryostage window. Cells were used in less than 4 h after isolation. The isolated cells were continuously held on ice during experimental testing. In a typical experiment, 4 to 10 cells were observed. Results from 3 to 5 replicates were pooled together for the final analysis. Mann-Whitney statistics for unpaired, nonparametric data at a significance level of (Y = 0.05 were used to compare various experimental observations in the rest of this study (13). RESULTS

Effects of cooling rate on IIF in the absence of CPAs. The kinetics of IIF in isolated rat hepatocytes were determined for cooling rates between 20 and 4OO”C/min in the presence of external ice. From 3°C the specimen was cooled at a constant predetermined rate to a final temperature of -40°C with extracellular ice covering the hepatocytes at - - 1°C. At a cooling rate of 400”C/min, the cumulative incidence of IIF was 0% at - - 5°C and increased rapidly to 100% by - - 13°C (Fig. 1). Similar behavior was obtained for cooling rates greater than SO”C/min. For cooling rates less than SO”C/min, no IIF was observed. The temperature at which 50% of hepatocytes underwent IIF (50Tnr) was obtained from Fig. 1 at different cooling rates between 75 and 40OVmin. “TnF as a function of the cooling rate is shown in Fig. 2. The 5oTur for cooling rates of 75, 100, 125, 150, and 400”C/min were -6.7 + 1.2, -8.6 + 0.7, -8.0 + 1.9, -7.5 ? 1.0, and -7.7 ? 1.8”C (mean + SDM), respectively. The average 5oTuF and the standard deviation of the mean for all cooling rates was -7.7 + 0.7”C. It was observed that the 5oTnF did

ET

AL.

TEMPERA TUt?E,

“C

FIG. 1. Cumulative incidence of IIF in isolated rat hepatocytes as a function of temperature.

not vary significantly with cooling rate. Although it was not statistically significant, a noticeable depression in “TnF occurred in the range of cooling rates for the maximum cumulative incidence of IIF to increase from 0 to 100%. The 5oT,1r decreased slightly for cooling rates between 75”C/min (- 6.7”C) and lOO”C/min (- 8.6”C); whereas, between 1OO”C/min (- 8.6”C) and lSO”C/min ( - 7.5”C) the “TnF increased. For cooling rates higher than 15O’Umin ( -7.5”(Z), the “Trrr remained constant.

-16

t

COOLING

RATE,

“C/mn

FIG. 2. Maximum cumulative incidence of IIF in isolated rat hepatocytes as a function of cooling rate and temperature at which 50% of hepatocytes underwent IIF as a function of the cooling rate. Numbers in parentheses refer to the number of cells used for each cooling rate. Final temperature was -40°C. Figure redrawn from data in Fig. 1.

INTRACELLULAR

ICE FORMATION

Although 5oTnF was independent of the cooling rate, the maximum cumulative incidence of IIF in isolated rat hepatocytes cooled to -40°C showed a strong dependence on the cooling rate as shown in Fig. 2. A transition zone was identified where the maximum cumulative incidence of IIF increased from 0% at SO”C/min to 100% at 15OWmin. Hepatocytes with IIF lost the intactness of their plasma membrane at the light microscopy level (macroscopic discontinuties along their membrane was observed) and they were osmotically inactive after freeze-thaw cycle. Since the cells could not be recovered from the cryomicroscope stage, no viability tests were run after a freeze-thaw cycle. Effects of isothermal holding on ZZF in the absence of CPAs. To determine the effect of an isothermal subzero temperature period on IIF, a two-step freezing protocol was used. From 3”C, the specimen was cooled at a rate of SOOWmin to a final preset temperature of - 1.6, -3.7, -5.2, or -7.5”C and held at the preset temperature for -2 min. Extracellular ice covered the hepatocytes at - - 1°C. The cumulative incidence of IIF during a constant subzero temperature holding period is presented in Fig. 3 for each of several different holding temperatures. At -7.5”C, 95% of the hepatocytes had IIF within the first second. It was observed that the lower the holding temperature, the greater the IIF at any given time. This result indicates that the IIF behavior of isolated rat hepatocytes was strongly influenced by the subzero holding temperature as demonstrated by the maximum cumulative incidence of IIF in Fig. 4. The maximum cumulative incidence of IIF increased from 0 to 100% as the temperature decreased from - 1.6 to -7.5”C. The cumulative incidence of IIF for a constant cooling rate of 4OOWmin is also shown for comparison. IIF occurred at slightly higher temperatures for the constant temperature experiments than the rapid cooling rate experiments. These results demonstrate that

OF HEPATOCYTES

439

SECONDS

FIG. 3. Maximum cumulative incidence of IIF in isolated rat hepatocytes suspended in DMEM as a function of time at subzero isothermal holding temperatures. Numbers in parentheses indicate the number of cells used for each condition.

both time and temperature are important in determining the IIF behavior of isolated hepatocytes. Effects of seeding extracellular ice on ZZF. The effect of the extracellular iceseeding temperature, Tseed, on the maximum cumulative incidence of IIF was investigated for cooling rates of between 50 and 15OWmin (Table 1). The formation of

.i

(221 /I *J 0

-2

/’ !

2‘

,j

I -8

I -10

TEMPERATURE

“C

-4

-6

I

I

-12

-14

FIG. 4. Cumulative incidence of IIF in isolated rat hepatocytes as a function of holding temperature used in subzero isothermal experiments shown in Fig. 3. Numbers represent hepatocytes used for each data point. The dotted line represents data from 4OO’Wmin experiments given in Fig. 1.

440

HARRIS

TABLE 1 Ice Seeding Temperature, Tseed, on the Maximum Cumulative Incidence of IIF at Different Cooling Rates”

Effect of Extracellular

Cooling Rate

T seed “C

SOWmin

-1.3 -1.4 -1.6 -1.7 -1.8 -1.9 -2.0 -2.1 -2.3 -2.7 -2.9 -3.7 -3.8 -4.5

75”Clmin -

0 0

0

-

-

25 29 33 -

100 100 100

o Hepatocytes suspended in DMEM

t0

10%

at Tseed

1OOWmin

-

1SOWmin

-

31 36 37 42

100 100 100

-

-

solution.

extracellular ice was initiated at temperatures ranging between - 1 and - 6°C and the cells were then cooled down to -40°C at constant rates. The transition zone for the maximum cumulative incidence of IIF to increase from 0 to 100% was a strong function of Tseed. In general for the cooling rates studied, lower seeding temperatures resulted in a higher maximum cumulative incidence for IIF. The maximum cumulative incidence of IIF increased from 0% at Tseed = - 2.0”c

ET AL.

=

-

3.7”c

for a cooling rate of SO”C/min. The transition zone for IIF moved to higher temperatures for higher cooling rates. For a cooling rate of lSO”C/min, the maximum cumulative incidence of IIF was 100% for Tseed< - 15°C. These results underlined the importance of precisely controlling Tseedfor reliable cryopreservation protocols. IIF in absence of extracellular ice. To determine the effect of the presence of extracellular ice formation on IIF, cells were cooled in the absence of extracellular ice. Both the extracellular solution (DMEM) and the hepatocytes were cooled until the extracellular ice spontaneously nucleated

at - 19.3”C. IIF did not occur in any of the hepatocytes until immediately after the external ice formed at - 19.3”C. All the cells had undergone IIF following the spontaneous extracellular ice formation at - 19.3”C. This temperature was significantly different than the observed IIF temperatures ( - 7.7”C) in the presence of extracellular ice suggesting that external ice is an important factor in the initiation of IIF. Effects of Me,SO on ZZF. Concentrations of 0.25, 0.5, 1.0, and 2.0 M Me,SO in DMEM were used to investigate the effect of CPAs on the kinetics of IIF for hepatocytes at a cooling rate of 4OOYXnin (Fig. 5). Me,SO concentrations higher than 0.5 M were added in a step-wise manner at ambient temperature with a 5-min equilibration time between each 0.5 M Me,SO addition. All data were obtained using cells isolated on the same day. The range of temperatures observed for the control data (i.e., no Me,SO) fell within the allowable range of nucleation temperatures presented in Fig. 1. With increasing concentrations of Me,SO, “TIIF decreased significantly. The values for “TIIF were -6.4”C in control,

INTRACELLULAR

JEMPE/?ATlJI?E,

ICE FORMATION

“17

FIG. 5. Cumulative incidence of IIF in isolated rat hepatocytes as a function of temperature in various concentrations of Me,SO. The cooling rate for all the experiments was 4OOWmin. Me,SO was added to the cells with 5-min steps of 0.5 M.

in 0.25 M, - 1OS”C in 0.5 M, - 11.9”C in 1.0 M, and - 163°C in 2.0 M. These results are shown in Table 2 together with the equilibrium freezing point (T,,) and the homogenous nucleation temperature (Thorn) of the cytoplasm. Values of 50 TIIF, Teq, and Thorn decreased with increasing molarity of Me$O. It is notewor-7.7”C

OF HEPATOCYTES

441

thy that the depression of the value of “TIIF is slightly greater than the depression of Teq as a function of Me,SO concentration. Similarly the difference between Teqand Thornis slightly increased by increasing the Me,SO concentration. The effect of the cooling rate on IIF in 0.5 M Me,SO was determined for cooling rates between 50 and 400Wmin as shown in Fig. 6. Me,SO was added in one step and equilibrated for 5 min prior to experiments. The ‘OTlp were -8.2 ? l.l”C for 75”C/min, -7.6 + 1.2”C for lOO”C/min, -9.2 + 1.6”C for lSO”C/min, and -9.7 + 1.6”C for 400Wmin (mean * SDM). No IIF was observed at SOWmin. The average 5oTIIF in the presence of 0.5 M Me,SO for all the cooling rates was -8.7 2 0.8”C (mean & SDM). This was not statistically different than the 5oTIIF of -7.7 + 0.7”C (mean * SDM) in the absence of Me,SO. A noticeable peak in ‘OTIp was observed in the cooling rates for the transition zone from 0 to 100% incidence of IIF. However, this peak was not statistically significant. The maximum cumulative incidence of

TABLE 2 Effect of Me,SO Concentration on the “Tur, T,,” (Equilibrium Freezing Temperature), and Thomb (Homogeneous Nucleation Temperature) of the Cytoplasm for Isolated Rat Hepatocytes Molar&y M 0.00 0.25 0.50 1.00 2.00

‘“Tm “C

-6.4 -7.7 -10.5 -11.9 - 16.8

+ 2 k ” ”

1.1 1.3 2.3 1.5 2.0

T 0;

T hoIn “C

-0.5 -1.0 -1.4 -2.4 -4.2

-37.8 -38.7 - 39.6 -41.5 -45.2

0 Equilibrium freezing temperature of the cytoplasm estimated from Teq = -(0.5 + 1.858 A4) relationship in which -0.5”C refers to the equilibrium freezing point of the cell cytoplasm in the absence of Me,SO. b Homogeneous nucleation temperature of the cell calculated assuming the cytoplasm has the same icenucleation characteristics as an electrolyte-Me,SO droplet of equivalent size and concentration (22).

-16

COOLING

RA JE ~ “Urnin

FIG. 6. Maximum cumulative incidence of IIF in isolated rat hepatocytes as a function of cooling rate and temperature at which 50% of hepatocytes underwent IIF as a function of the cooling rate. Hepatocytes were suspended in 0.5 M Me,SO solution. Numbers in parentheses refer to the number of cells used for each cooling rate. Final temperature was - 50°C.

442

HARRIS

IIF in the presence of 0.5 M Me,SO is shown as a function of cooling rate in Fig. 6. The maximum cumulative incidence of IIF in 0.5 M Me,SO increased with increasing cooling rates. A transition zone was observed where the maximum cumulative incidence of IIF increased from 0% at SO‘Unin to 100% at 15OWmin. This transition zone in the presence of Me,SO was not statistically significantly different than the transition zone observed in the absence of Me,SO presented in Fig. 2. This effect was not determined for higher concentrations of Me-SO. DISCUSSION

Intracellular ice formation has been quantified experimentally in isolated rat hepatocytes in order to develop a fundamental understanding of the response of these cells to freezing. The optimum cooling rate, i.e., the fastest cooling rate without IIF, was found to be -5O”C/min in DMEM. The average 5oTIIF in DMEM was -7.7”C. These observations were altered only slightly in the presence of Me,SO. Results from this study suggest that multistep freezing protocols in the presence of cryoprotective agents will be required to cryopreserve isolated hepatocytes. Furthermore, since the behavior of IIF is dependent on both time and temperature, these parameters should be taken into account in the design of multistep cooling protocols with one or more isothermal holding periods. The maximum cumulative incidence of IIF increased from 0 to 100% by increasing the cooling rate from 50 to 15OWmin; therefore, the maximum survival would be expected to occur at cooling rates near 5OWmin. At higher cooling rates, the cells more likely will be damaged from IIF. On the other hand, at slower cooling rates, the cells will be injured by long time exposure to damaging extracellular ice effects (14). This transition zone is cell-type specific and has been observed for other cell types (12,

ET AL.

15, 17, 24-26, 28, 32). This cell specific IIF behavior could be attributed to the differences in the plasma membrane permeability to water, surface-to-volume ratio, and icenucleation parameters (30, 31). 5oTUF as a function of the cooling rate was also determined. Hepatocytes had an overall average 5oTIIF of -7.7”C between 50 and 400Wmin which is consistent with the observations from other cells in the absence of CPAs (3, 6, 7, 15-22, 24-26, 28, 32). These observations are in agreement with a recent theory of IIF proposed by Toner et al. in which the presence of external ice alters the plasma membrane making it an energetically favorable site for icenucleation (30). The effect of subzero isothermal temperatures on the cumulative incidence of IIF suggests that IIF is a time- and temperature-dependent phenomenon. During the subzero isothermal periods, the cumulative incidence of IIF consistently increased with time and the maximum cumulative incidence of IIF occurred more rapidly at the lower subzero temperatures. Similar behavior has been observed for mouse oocytes and correlated with the heterogeneous nucleation theory (30, 31). Mouse oocytes, however, had a much slower response (-10 s) than hepatocytes (-1 s). The time to reach maximum cumulative incidence of IIF for Drosophilia melanogaster embryos was also more than 30 times slower than hepatocytes (19, 21). This study demonstrates the importance of precisely controlling Tseed for hepatocyte cryopreservation protocols. In general, lower seeding temperatures resulted in a higher maximum cumulative incidence of IIF. An increased intracellular water content and undercooling (i.e., the difference between the equilibrium freezing temperature of the cytoplasm and its actual temperature) provide conditions favorable for IIF at a given cooling rate. This behavior was predicted for mouse oocytes on theoretical

INTRACELLULAR

ICE

FORMATION

grounds using IIF model (31). A similarly dramatic increase in IIF as a function of T seed has also been demonstrated experimentally for erythrocytes (3, 5) and granulocytes (26). In the presence of Me,SO at 4OO”C/min, 5oTrIF decreased with increasing concentrations of Me,SO (Fig. 5). Much lower 5oTIIF have been observed in the presence of penetrating CPAs with all the other cell types (17-19, 28, 32). In general, CPA solutions lower “TIIF to a range between -20” and - 60°C depending on the cell type. In addition, 5oTIIF usually decreases (18, 19, 28, 32) with decreasing cooling rates with the exceptions of hepatocytes, fibroblasts (17), and D. melanogaster (19, 21). Since the IIF temperatures are not significantly lowered with either increasing concentrations of Me$O or decreasing cooling rates, multistep freezing protocols may be required to depress the IIF temperatures. Multistep freezing protocols may be defined based on the combined competing criteria of minimizing cell exposure time to solution effects while avoiding the critical states associated with IIF. A possible strategy is to use rapid cooling steps interrupted with isothermal holding periods to achieve enough cellular dehydration to prevent IIF while minimizing the total freezing time. The exact protocol will be a function of the time and temperature dependence of IIF as well as membrane water permeability characteristics of isolated hepatocytes (31). Further experimental and analytical investigations are necessary to develop optimal multistep freezing protocols for hepatocytes prior to any empirical attempts.

OF

2.

3.

4. 5. 6. 7.

8.

9.

10.

11.

12.

ACKNOWLEDGMENTS

The authors express their gratitude to J. C. Y. Dunn and M. L. Sterling for their support and help. This research was supported in part by a grant from The Whitaker Foundation, Mechanicsburg, PA. REFERENCES 1.

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