CRYOBIOLOGY
29, 478-484 (1992)
Freezing Preservation of the Mammalian Cardiac Explant VI. Effect of Thawing Rate on Functional Recovery QINGYAN ZHU, JACK R. LAYNE, JR.,* MELINDA CLAYDON, GEORGE L. HICKS, JR., AND TINGCHUNG WANG Department
of Surgery,
University of Rochester, Rochester, Nazareth College, Rochester,
New York 14642; and *Department New York 14610
of Biology,
This study investigated the effect of thawing rate on the preservation of frozen isolated rat hearts. The hearts were flushed with a hyperosmotic cardioplegic solution, CP-14/EtOH (1.15 Osm/kg), frozen at a rate of 0.18”Cihr for 6 h to - 3.2”C. Thereafter, the hearts were thawed at rates ranging from 0.08 to l.l”C/min for 1 to 14 min until the heart temperature reached - 2.l”C, the melting point (MP) of the flush solution; then they were held at - 1°C for 11 to 24 min so that the total thaw time was 25 min. Post-thaw function was assessed by working reperfusion and expressed as percentage of unstored control function. Cardiac output (CO) and other hemodynamic performance showed biphasic responses to the thaw rate. At 0.08”Cimin rate, CO recovered to 29.1 -t 4.1 mlimin (40.8 2 5.8% of control). Thawing at 0.13”Clmin enhanced the recovery of CO to 60.5 ? 4.9%. Between 0.13 and 0.34”C/min, recovery was statistically insignificant. Faster thawing at 0.59 and l.l”C/min caused progressively less recovery. Overall, 0.13”Cimin offered the highest recovery. In conclusion, function in slowly frozen heart is intimately affected by the thawing rate; there was an optimal intermediate thawing rate and both too slow and too fast thawing were detrimental. o 1992 Academic PT~SS. I~C.
Successful freezing, storage, and reanimation of the adult mammalian heart was recently reported by us (1-3, 21). This scheme depends on several facets of the natural freeze tolerance of some vertebrate species-slow freezing at high subzero temperature, moderate concentrations of cryoprotectants, and limited supercooling followed by inoculative freezing (18). More recently, by adding EtOH to the storage solution, we achieved freezing preservation of the heart at - 3.4”C (20). In order to obtain better function recovery and longer storage time for the cardiac explant, a fundamental understanding of the effects of storage conditions on cardiac viability is necessary. From the literature, it is known that thawing rate intimately affects the survival of single cells or organs after cryopreservation (4-7, 14, 16, 17, 19) and the dynamics of recovery in freeze-tolerant Received December 3, 1991; accepted January 20, 1992.
frogs after a freezing bout (8). In the present study, we examined the effect of thawing rate on functional recovery in the isolated rat heart frozen at - 3.4”C for 6 h. The optimal thawing rate was found to be 0.13”Ci min. MATERIALS
Freezing storage of the cardiac explant.
Male Sprague-Dawley rats (300 to 350 g) were anesthetized with sodium pentobarbital (65 mg/kg, ip) and anticoagulated with heparin (2.50 units, iv). The heart was excised and immediately immersed in ice-cold Krebs-Henseleit buffer (KHB), which contained (in mM): 118 NaCl, 11 glucose, 25 NaHCO,, 4.7 KCl, 1.2 MgSO,, 1.2 KH,PO,, 0.5 Na,-EDTA, and 2.5 CaCl,. The aorta was cannulated and the heart was retrograde perfused at 70 mm Hg for 9 min with KHB equilibrated with 95%0,/5%CO, at 36.5”C (Fig. 1). The perfusion was continued for 2 min at 60 mm Hg with a cardioplegic solution (CP-14/EtOH) saturated
478 001l-2240/92 $5.00 Coovrieht 0 1992 bv Academic Press. Inc. Ail rights of reproduction m any form reserved.
AND METHODS
THAWING
RATE
AND
THE
VIABILITY
OF THE
FROZEN
/
KHB KHB
Flush
70mmHg 9 min 36.5T
CP Flush 6OIIUllHg
I min 36.5"C
1 min 15°C
Supercool
Freezing
6 hours -3.4"C
Reperfusion
’
Thawing Langendorff 70mmHg
25 min 60 min -3.4"C
479
HEART
I 0.5 1" 195°C
II -1-z
15 mitl 36.5T
Working IlmmHg 20 min 36.5"C /
I. Illustrated experimental protocol depicts flushing, supercooling, freezing, thawing, and reperfusion of the isolated rat heart. FIG.
with 95%0,/5%CO, at 36.X. The composition of CP-14/EtOH was (in mM): 110 NaCl, 14 KCl, 7 glucose, 10 mannitol, 15 MgSO,, 1.2 KH,PO,, 5 Na-Hepes, 0.15 CaCl,, 4.7% (v/v) EtOH with a pH of 7.5 (22”(Z), and osmolality of 1100 + 25 mOsm/ kg water. During the second minute of cardioplegic flush, the heart was immersed and topically cooled with ice-cold CP-14/EtOH. Because warming and oxygenation of the flush solution caused the evaporation of EtOH, the osmolality of CP-14/EtOH was constantly monitored using a freezing point depression osmometer and maintained at 1100 mOsm by EtOH supplementation. After cardioplegic flush, the hearts were placed individually in a 50-ml plastic centrifuge tube on CP-lWEtOH-moistened gauze (5.1 x 5.1-cm eight-ply gauze sponge, Johnson & Johnson, New Brunswick, NJ) situated at the bottom of the tube. Temperature inside the tubes was maintained at -3.4 + O.l”C by immersing the tubes in a refrigerated circulating bath. A 1.5 x 3-cm strip of CP-lWEtOH-moistened Whatman No. 1 filter paper was draped over the heart, which was allowed to supercool to - 3.4”C during a 60-min period. Freezing was initiated by placing a small ice crystal at - 20°C on the filter paper and the hearts were frozen for 6 h. During supercooling and freezing, a 3-cm-long foam plug was inserted into the tube to reduce the air space in the tube and to provide better temperature control. Under this condition, the cooling rate, as estimated from the decline of ventricular temperature during freezing, was approximately O.l8”C/h. Ventricular
temperature at the end of freeze was -3.2”C 2 0.06”C (n = 16) and tissue ice content was 38.4 rt 1.1% (n = 4) of total tissue water. After freezing, the hearts were defrosted in two stages, Thaw I and II (Fig. 1). At the onset of Thaw I, freezing tubes with the hearts were moved to a bath set to the intended thawing temperature, including -0.5, +0.5, +2.5, +4.5, +9.5, and + 19.5”C. Twenty milliliters of CP-14/EtOH at the designated temperature was poured into the freezing tube to warm the hearts. We reasoned that when ventricular chamber temperature reached the melting point of CP-14/EtOH, thawing was likely completed. Therefore, during Thaw I, the hearts were warmed to the melting point (- 2.1”C) of CP-14/EtOH, irrespective of the thawing temperature. The duration of Thaw I (min) = 1.l”C/thawing rate (‘C/ min), where l.l”C was the difference between ventricular temperature at the end of freezing and the melting point of CP-14/ EtOH (Table 1). Thaw II was carried out by transferring the hearts to tubes containing 20 ml CP-14/EtOH precooled to - 1°C and kept in a - 1°C bath until the total thawing time reached 25 min. This step was implemented to ensure complete thaw and to compensate for the varied lengths of Thaw I at different thawing rates. Determination of the thawing rates at different temperatures. In a preliminary experiment, we calibrated the thawing rate as a function of the thawing temperature. After the hearts (n = 4 per group) were flushed with CP-14/EtOH, a 2-cm segment
480
ZHU ET AL.
atrium were cannulated. Then the perfusion was switched to the working mode with a left atria1 perfusion pressure of 15 cm H,O and an afterload of 70 mm Hg (Fig. 1). Thawing Thaw I Thaw I Thaw II Heart rate (HR, beats/min), aortic and cortemperature rate duration duration onary flow (AF and CF, ml/min), cardiac (“C/mitt) (min) (min) c-3 output (CO, the sum of AF and CF), and 0.08 k 0.01 -0.5 14 11 systolic and diastolic pressures (SP and DP, +o.s 0.13 k 0.01 16 9 mm Hg) were monitored at lo-min intervals f2.5 0.22 t 0.02 5 20 for 30 min. External work (g-m/min) was +4.5 0.34 -+ 0.03 3 22 calculated according to Neely et al. (15). +9.5 2 23 0.59” Coronary vascular resistance (CVR) (mm + 19.5 1.11” 1 24 Hg-mm/ml) was calculated from DP and CF Note. Data are presented as mean 2 SEM (n = 4 (CVR = DP/CF). The function of the per group). Total duration of thawing (Thaw I + Thaw freshly isolated unstored heart perfused II) was 25 min for all groups. a Thawing rates at +9.5” and + 19.s”C were calcu- with KHB in working mode for 45 min lated by extrapolation of data obtained at the lower served as the control. Function of the temperatures. stored heart at the end of 30 min working reperfusion was calculated as a percentage of polyethylene tubing (PE 240) was in- of the control function and reported as perserted via the aorta into the left ventricle. A centage of recovery. Both KHB and CP-141 thin-wire thermocouple probe (Type T, EtOH were passed through 0.22~km memgauge 36, Omega Engineering, Inc., Stam- brane filter discs after preparation and reford, CT) was guided through the PE tubing circulated during perfusion through 3-p,m and secured in place to monitor the ventric- in-line filter discs. All chemicals were eigrade or ACS ular chamber temperature using a tempera- ther cell-culture-tested ture recorder (Model H22, Omega Engi- (American Chemical Society) grade from the neering, Inc., Stamford, CT). Since this Sigma Chemical Company (St. Louis, MO). Tissue ice determination. Tissue ice conprocedure damaged the hearts, they were not used in functional assessments. Subse- tent of the frozen heart was determined usquent freezing and thawing steps were performed as already described. The thawing temperatures studied were -0.5, +0.5, +2.5, and +4S”C. Average thawing rate was estimated from the time interval for the ventricular temperature to change from - 3.2”C (ventricular temperature at the end of 6-h freeze) to - 2.1”C (melting point of CP-14/EtOH). As shown in Fig. 2 and Table 1, between - 0.5 and + 4.5”C, thawing rate (“Urnin) correlated linearly with the thawing temperatures. From these data thawing rates at + 9.5 and + 19.5”C were calculated by extrapolation. FIG. 2. Thawing rate (“Urnin) as a function of the Assessment of the cardiac function. The thawing temperature (“C). Data plotted represent thawed heart was reperfused retrograde at mean * SE (n = 4 per point). The linear relationship 70 mm Hg with KHB for 15 min during can be described as thawing rate = 0.1 + 0.0518 X which the pulmonary artery and the left (thawing temperature), ? = 0.995. TABLE 1 Thawing Rates at Different Thawing Temperature and the Corresponding Duration of Thaw I and Thaw II for Hearts Frozen at - 3.4”C for 6 h
THAWING
RATE AND THE VIABILITY
481
OF THE FROZEN HEART
recovery was significantly less than the 0.13Umin group. CVR (Fig. 3D) was elevated in all groups; but those thawed at the optimal rates showed less increase. Nevertheless, the difference was not statistically significant. These observations demonstrated that the thawing rate was critical to the recovery of function in frozen rat hearts. Moreover, thawing at O.l3”C/min offered the best overall return of cardiac RESULTS function. We also compared the effect of changing After freeze-thawing, HR in all stored the duration of Thaw I on recovery. The hearts returned to levels not significantly different from the controls and, therefore, hearts were thawed at 0.13YYmin for 9 min was not influenced by the thawing rate. to - 2°C or 25 min to + 1°C without expeOther hemodynamic function showed a bi- riencing Thaw II. The percentage recovery phasic response to the thawing rate (Table 2 of HR, AF, CF, CO, SP, work, and CVR in and Fig. 3). For ease of explanation, car- the 9-min group were 93.1 + 3.3, 46.0 ? diac output (CO) will be used as an index of 4.2, 62.0 k 4.3, 50.8 -+ 3.8, 71.1 + 1.7, 59.4 cardiac function, although the other param- + 1.8, and 123.6 ? 9.2%, respectively. Reeters, including AF (Fig. 3A), CF (Fig. 3B), covery in the 25-min group was 99.5 -+ 8.0, SP (Fig. 3C), CVR (Fig. 3D), and work fol- 51.1 + 4.8, 64.5 ? 4.6, 55.2 + 4.3, 72.9 ? lowed essentially the same trend. At the 2.3, 65.4 * 4.6, and 124.1 + 9.2%, respecthawing rate of O.O8”C/min, the recovery of tively. These functions were not signifiCO was 40.8 ? 5.8% of control. As thawing cantly different from those thawed at rate was elevated to O.l3”C/min, CO recov- O.l3”C/min (Thaw I) for 9 min and held at ery reached 60.5 + 4.9% of control. Be- - 1°C (Thaw II) for 16 min. This observatween groups thawed at 0.13, 0.22, and tion indicated that Thaw II was unneces0.34”C/min, recovery was similar and sta- sary and the heart could be thawed at + 1°C tistically nondifferent. Function declined for as long as 25 min without detrimental progressively when thawing rate was in- effects on the recovery or reperfused as creased further; at 0.59 and 1.1l”C/min the soon as 9 min after the onset of thawing. ing calorimetry and expressed as a percentage of total tissue water frozen (9, 21). Statistical analysis. Data analysis was performed using a personal microcomputer. Differences among the various experimental groups were tested by the analysis of variance. Significant difference between groups (P < 0.05) was detected by the Fisher’s least significant difference test.
TABLE 2 Effect of Thawing Rate on Poststorage Recovery of Hemodynamic Performance of Isolated Rat Heart at the End of 30 min Working Reperfusion Rate
(“C/min) 0.08 0.13 0.22 0.34 0.59 1.11
Temp (“C)
n
-0.5 +0.5 f2.5 +4.5 +9.5 +19.5
8 9 6 6 6 9
HR 337 298 313 270 287 216
+ t t + + f
CF
AF 2-l 15 25 15 19 23
18.0 29.6 23.5 24.3 20.1 13.1
+ + -c ir -c t
3.4* 2.1 2.3 1.5 3.4* 1.8*
11.1 13.6 12.7 12.8 10.9 9.9
+ T + f + -c
0.9 1.0 0.7 1.1 0.8 0.6*
co 29.1 43.2 36.2 37.1 30.9 23.0
f k + + f 2
SP 4.1* 3.5 2.9 2.2 3.8* 2.4*
94 111 102 106 99 94
t c 2 + + f
Work 3* 3 3* 3 4* 4*
29.2 48.8 38.3 31.7 31.2 22.6
-t k + It ? -t
4.9* 5.2 3.8* 2.5 4.6* 2.6*
CVR 5.75 5.02 5.13 4.19 5.72 6.34
2 k + + + ?
0.52 0.23 0.21 0.45 0.48 0.46*
Note. Differences between groups were detected by ANOVA and tested by Fisher precision least significant difference tests. Means ? SE are presented. Hemodynamic function of the control hearts was (n = 28): HR, 296 2 6 beats/min; AF, 50.3 ? 0.9 ml/min; CF, 21.3 2 0.7 ml/min; CO, 71.5 2 1.2 mumin; SP, 142 2 2 mm Hg; work, 95.5 2 2.4 g-m/min; CVR, 3.65 2 0.12 mm Hg-min/ml. * P < 0.05 vs O.l3”C/min group.
482
ZHU ET AL.
80 ~...._~ 60 70
50 40 30 20 IO
i 0 0.0 0.2
04
0.6 0.X
1.0 1.2
00
0.2
0.4 0.6 0.8
1.0 I2
0.0 0.2
0.4 0.6 0.8
1.0 1.2
0.0 0.2
0.4 0.6 0.8
1.0 1.2
Thawing rate, “Clmin FIG. 3. Percentage recovery of hemodynamic function in frozen/thawed hearts as a function of thawing rate. Recovery in A, aortic flow (AF); B, coronary flow (CF); C, systolic pressure (SP); and D, coronary vascular resistance (CVR) after 30 min of working reperfusion were calculated as percentage of control function. Values for control function are listed in the legend for Table 2. Data plotted represent means ? SE. **P < 0.01 vs hearts thawed at 0.13YYmin and YIP< 0.01 vs hearts thawed at 0.22 and 0.34Wmin.
been made by others (17). Izumi et al. (7) found that islets isolated from slowly We demonstrated a biphasic response of thawed pancreata at room temperature had function in freeze-stored hearts to thawing better insulin-releasing activity than those rates. Hearts thawed at rates ranged from isolated from rapidly thawed pancreata at 0.13 to 0.34”C/min displayed similar func- 37°C. Mouse embryos frozen at 0.2 to 2°C tional recovery, although the O.l3”C/min min survived best with a rewarming rate of group was overall the best among all hearts. 4 to 25”C/min (22). Ram spermatozoa frozen at 2”C/min sustained more damage Both slower (O.O8”C/min) and faster (0.59”Clmin or higher) rates led to less re- when thawed fast in 20°C water than slowly covery. Understandably, “slow” and in 20°C air (5). Function of rabbit kidneys “fast” rates must be different from system stored at - 80°C at a cooling rate of l”C/h to system. It is generally recognized that was found to be dependent on the warming when cells are frozen at a rapid rate which rate during thawing: l”C/min was better promotes the formation of intracellular ice, than l”C/h or 20”C/min (16). Resumption of vital functions in freeze-tolerant wood frogs rapid thawing improves cellular viability for 24 h was (4). When frozen at slow cooling rate and after being frozen at -2.K intracellular ice is not evident, rapid thaw- also thawing rate dependent (8). Frozen ing is more injurious than slow thawing, ir- frogs thawed in 6.5 to 8.5”C air at a rate of respective of the cell systems. Gao et al. (6) O.l2”C/min and those thawed in 23 to 25°C reported that when frozen at a cooling rate air at 0.26”C/min rate had the similar surof O.S”C/min, human erythrocytes showed vival rate, however. Freezing-thawing process is known to the least hemolysis if rewarmed at OS”C/ min. Miller and Mazur (14) observed that cause cell damage by concentrating the exwhen cooling rate was slow (0.27”C/min tra- and intracellular solutes during freezing and 1.7”C/min), the human red cells sur- and a following solute dilution and cell vival was high with slower warming rates swelling during thawing (11, 18). During (0.06-l”C/min); similar observation have freezing at a slow rate, cells shrink and deDISCUSSION
THAWING
RATE AND
THE VIABILITY
hydrate due to efflux of cellular water. During thawing, the extracellular ice melts and water reenters the cells. Thawing at too fast a rate will not allow enough time for reequilibration of water and ions. It induces rapid changes in solute concentrations and causes osmotic stress to cells leading to cell swelling and damage (10). These mechanisms may account for our results with the supraoptimal thawing rates. Certainly other factors like cooling rates, temperature, duration of storage, kind and concentration of cryoprotectants interact with thawing rate and affect the outcome of freezing storage (6, 14, 19), these factors will be considered and examined in further optimization of the freezing protocol for the cardiac explant. By adding EtOH, a cryoprotectant, to the CP-14 solution we increased its osmolality to 1.1 Osm/Kg water, thus lowering the freezing point of the stored heart to -2.l”C. Our previous study (20) demonstrated that 4.7% EtOH in CP solution had no obvious toxic effect to the heart during a 12-h storage period. In contrast, EtOH could prevent edema, reduce energy depletion, and promote functional preservation by inhibiting Na+/K+ ATPase and Nat/ Ca2+ exchanges, and dehydrating myocardium (23). The incomplete return of the cardiac function in the present study may be the result of ischemia, freezing-thawing damage, and unoptimized solution composition. ACKNOWLEDGMENT
This study was supported in part by Grant HL45129 from NIH. REFERENCES
1. Banker, M. C., Layne, J. R., Jr., Hicks, G. L., Jr., and Wang, T. Freezing preservation of the mammalian cardiac explant. II. Comparing the protective effect of glycerol and polyethylene glycol. Cryobiology 29, 87-94 (1992). 2. Banker, M. C., Layne, J. R., Jr., Hicks, G. L., Jr., and Wang, T. Freezing preservation of the mammalian cardiac explant. III. Tissue dehydration and cryoprotection by polyethylene glycol. J. Heart Lung Transpl., in press.
OF THE FROZEN
HEART
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3. Banker, M. C., Layne, J. R., Jr., Hicks, G. L., Jr., and Wang, T. Freezing preservation of the mammalian cardiac explant. IV. Functional recovery after &hour freezing. Curr. Surg. 48, 428-430 (1991). 4. Farrant, J. General observations on cell preservation. In “Low Temperature Preservation in Medicine and Biology” (M. J. Ashwood-Smith and J. Farrant, Eds.), pp. l-18. Univ. Park Press, Baltimore, 1980. 5. Fiser, P. S., Fairfull, R. W., and Marcus, G. J. The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws. Cryobiology 23, 141-149 (1986). 6. Gao, D. Y., Lin, S., Kornblatt, J. A., and Guttman, F. M. A study of the separate effects of influence factors and their coupled interactions on cryoinjury of human erythrocytes. Cryobiology 26, 3X5-368 (1989). 7. Izumi, R., Koyama, F., Konishi, K., Sekino, H., Katoh, O., Ohhori, I., Hashimoto, T., Hirosawa, H., Shimizu, K., Ueno, K.. and Miyazaki, I. Effects of thawing temperature on the viability of islets isolated from a cryopreserved pancreas. Transpl. Proc. 19, 1345-1347 (1987). 8. Layne, J. R., Jr., and First, M. C. Resumption of physiological functions in the wood frogs (Rana sylvatica) following freezing. Am. J. Physiol. 261, R134-R137 (1991). 9. Layne, J. R., Jr., and Lee, R. E., Jr. Seasonal variation in freeze tolerance and ice content of the tree frog Hyla versicolor. J. Exp. ZOO/. 249, 133-139 (1989). 10. Mazur, P. Freezing of living cells: Mechanisms and implications. Am. J. Physiol. 247, C125Cl42 (1984). 11. Mazur, P., Leibo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury. Exp. Cell Res. 71, 345-355 (1972).
12. Meryman, H. T., and Williams, R. J. Basic principles of freezing injury to plant cells: Natural tolerance and approaches to cryopreservation. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 1348. CRC Press, Boca Baton/Ann Arbor/Boston, 1991. 13. Meryman, H. T., Williams, R. J., and Douglas, M. St. J. Freezing injury from “solution effects” and its prevention by natural or artificial cryoprotection. Cryobiology 14, 287-302 (1977). 14. Miller, R. H., and Mazur, P. Survival of frozenthawed human red cells as a function of cooling and warming velocities. Cryobiology 13, 404414 (1976).
IS. Neely, J. R., Liebermeister, H., Battersby, E. J.,
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and Morgan, H. R. Effect of pressure development on oxygen consumption by the isolated rat heart. Am. J. Physiol. 212, 804-814 (1967). 16. Pegg, D. E., Jacobsen, I. A., Diaper, M. P., and Foreman, J. The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys. Cryobiology 21, 52%535 (1984). 17. Rapatz, G., Luyet, B., and MacKenzie, A. Effect of cooling and rewarming rates on glycerolated human erythrocytes. Cryobiology 12, 293-308 (1975). 18. Storey, K. B. Life in a frozen state: Adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am. J. Physiol. 258, R559-R568 (1990). 19. Taylor, M. J., and Benton, M. J. Interaction of cooling rate, warming rate, and extent of permeation of cryoprotectant in determining survival of isolated rat islets of Langerhans during cryopreservation. Diabetes 36, 5-5 (1987).
20. Wang, T., Banker, M. C., Claydon, M., Hicks, G. L., Jr., and Layne, J. R., Jr. Freezing preservation of the mammalian cardiac heart. V. Cryoprotection by ethanol. Cryobiology, 29, 47w77 (1992). 21. Wang, T., Connery, C. P., Batty, P. R., Hicks, G. L., Jr., DeWeese, J. A., and Layne, J. R., Jr. Freezing preservation of adult mammalian heart at high subzero temperatures. Cryobiology 28, 171-176 (1991). 22. Whittingham, D. G. Principles of embryo preservation. In “Low Temperature Preservation in Medicine and Biology” (M. J. Ashwood-Smith and J. Farrant, Eds.), pp. 65-84. Univ. Park Press, Baltimore, 1980. 23. Wikman-Coffelt, J., Wagner, S., Wu, S., and Parmley, W. Alcohol and pyruvate cardioplegia. J. Thorac. Cardiovasc. Surg. 101, 509-516 (1991).
29, 478-484 (1992)
Freezing Preservation of the Mammalian Cardiac Explant VI. Effect of Thawing Rate on Functional Recovery QINGYAN ZHU, JACK R. LAYNE, JR.,* MELINDA CLAYDON, GEORGE L. HICKS, JR., AND TINGCHUNG WANG Department
of Surgery,
University of Rochester, Rochester, Nazareth College, Rochester,
New York 14642; and *Department New York 14610
of Biology,
This study investigated the effect of thawing rate on the preservation of frozen isolated rat hearts. The hearts were flushed with a hyperosmotic cardioplegic solution, CP-14/EtOH (1.15 Osm/kg), frozen at a rate of 0.18”Cihr for 6 h to - 3.2”C. Thereafter, the hearts were thawed at rates ranging from 0.08 to l.l”C/min for 1 to 14 min until the heart temperature reached - 2.l”C, the melting point (MP) of the flush solution; then they were held at - 1°C for 11 to 24 min so that the total thaw time was 25 min. Post-thaw function was assessed by working reperfusion and expressed as percentage of unstored control function. Cardiac output (CO) and other hemodynamic performance showed biphasic responses to the thaw rate. At 0.08”Cimin rate, CO recovered to 29.1 -t 4.1 mlimin (40.8 2 5.8% of control). Thawing at 0.13”Clmin enhanced the recovery of CO to 60.5 ? 4.9%. Between 0.13 and 0.34”C/min, recovery was statistically insignificant. Faster thawing at 0.59 and l.l”C/min caused progressively less recovery. Overall, 0.13”Cimin offered the highest recovery. In conclusion, function in slowly frozen heart is intimately affected by the thawing rate; there was an optimal intermediate thawing rate and both too slow and too fast thawing were detrimental. o 1992 Academic PT~SS. I~C.
Successful freezing, storage, and reanimation of the adult mammalian heart was recently reported by us (1-3, 21). This scheme depends on several facets of the natural freeze tolerance of some vertebrate species-slow freezing at high subzero temperature, moderate concentrations of cryoprotectants, and limited supercooling followed by inoculative freezing (18). More recently, by adding EtOH to the storage solution, we achieved freezing preservation of the heart at - 3.4”C (20). In order to obtain better function recovery and longer storage time for the cardiac explant, a fundamental understanding of the effects of storage conditions on cardiac viability is necessary. From the literature, it is known that thawing rate intimately affects the survival of single cells or organs after cryopreservation (4-7, 14, 16, 17, 19) and the dynamics of recovery in freeze-tolerant Received December 3, 1991; accepted January 20, 1992.
frogs after a freezing bout (8). In the present study, we examined the effect of thawing rate on functional recovery in the isolated rat heart frozen at - 3.4”C for 6 h. The optimal thawing rate was found to be 0.13”Ci min. MATERIALS
Freezing storage of the cardiac explant.
Male Sprague-Dawley rats (300 to 350 g) were anesthetized with sodium pentobarbital (65 mg/kg, ip) and anticoagulated with heparin (2.50 units, iv). The heart was excised and immediately immersed in ice-cold Krebs-Henseleit buffer (KHB), which contained (in mM): 118 NaCl, 11 glucose, 25 NaHCO,, 4.7 KCl, 1.2 MgSO,, 1.2 KH,PO,, 0.5 Na,-EDTA, and 2.5 CaCl,. The aorta was cannulated and the heart was retrograde perfused at 70 mm Hg for 9 min with KHB equilibrated with 95%0,/5%CO, at 36.5”C (Fig. 1). The perfusion was continued for 2 min at 60 mm Hg with a cardioplegic solution (CP-14/EtOH) saturated
478 001l-2240/92 $5.00 Coovrieht 0 1992 bv Academic Press. Inc. Ail rights of reproduction m any form reserved.
AND METHODS
THAWING
RATE
AND
THE
VIABILITY
OF THE
FROZEN
/
KHB KHB
Flush
70mmHg 9 min 36.5T
CP Flush 6OIIUllHg
I min 36.5"C
1 min 15°C
Supercool
Freezing
6 hours -3.4"C
Reperfusion
’
Thawing Langendorff 70mmHg
25 min 60 min -3.4"C
479
HEART
I 0.5 1" 195°C
II -1-z
15 mitl 36.5T
Working IlmmHg 20 min 36.5"C /
I. Illustrated experimental protocol depicts flushing, supercooling, freezing, thawing, and reperfusion of the isolated rat heart. FIG.
with 95%0,/5%CO, at 36.X. The composition of CP-14/EtOH was (in mM): 110 NaCl, 14 KCl, 7 glucose, 10 mannitol, 15 MgSO,, 1.2 KH,PO,, 5 Na-Hepes, 0.15 CaCl,, 4.7% (v/v) EtOH with a pH of 7.5 (22”(Z), and osmolality of 1100 + 25 mOsm/ kg water. During the second minute of cardioplegic flush, the heart was immersed and topically cooled with ice-cold CP-14/EtOH. Because warming and oxygenation of the flush solution caused the evaporation of EtOH, the osmolality of CP-14/EtOH was constantly monitored using a freezing point depression osmometer and maintained at 1100 mOsm by EtOH supplementation. After cardioplegic flush, the hearts were placed individually in a 50-ml plastic centrifuge tube on CP-lWEtOH-moistened gauze (5.1 x 5.1-cm eight-ply gauze sponge, Johnson & Johnson, New Brunswick, NJ) situated at the bottom of the tube. Temperature inside the tubes was maintained at -3.4 + O.l”C by immersing the tubes in a refrigerated circulating bath. A 1.5 x 3-cm strip of CP-lWEtOH-moistened Whatman No. 1 filter paper was draped over the heart, which was allowed to supercool to - 3.4”C during a 60-min period. Freezing was initiated by placing a small ice crystal at - 20°C on the filter paper and the hearts were frozen for 6 h. During supercooling and freezing, a 3-cm-long foam plug was inserted into the tube to reduce the air space in the tube and to provide better temperature control. Under this condition, the cooling rate, as estimated from the decline of ventricular temperature during freezing, was approximately O.l8”C/h. Ventricular
temperature at the end of freeze was -3.2”C 2 0.06”C (n = 16) and tissue ice content was 38.4 rt 1.1% (n = 4) of total tissue water. After freezing, the hearts were defrosted in two stages, Thaw I and II (Fig. 1). At the onset of Thaw I, freezing tubes with the hearts were moved to a bath set to the intended thawing temperature, including -0.5, +0.5, +2.5, +4.5, +9.5, and + 19.5”C. Twenty milliliters of CP-14/EtOH at the designated temperature was poured into the freezing tube to warm the hearts. We reasoned that when ventricular chamber temperature reached the melting point of CP-14/EtOH, thawing was likely completed. Therefore, during Thaw I, the hearts were warmed to the melting point (- 2.1”C) of CP-14/EtOH, irrespective of the thawing temperature. The duration of Thaw I (min) = 1.l”C/thawing rate (‘C/ min), where l.l”C was the difference between ventricular temperature at the end of freezing and the melting point of CP-14/ EtOH (Table 1). Thaw II was carried out by transferring the hearts to tubes containing 20 ml CP-14/EtOH precooled to - 1°C and kept in a - 1°C bath until the total thawing time reached 25 min. This step was implemented to ensure complete thaw and to compensate for the varied lengths of Thaw I at different thawing rates. Determination of the thawing rates at different temperatures. In a preliminary experiment, we calibrated the thawing rate as a function of the thawing temperature. After the hearts (n = 4 per group) were flushed with CP-14/EtOH, a 2-cm segment
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atrium were cannulated. Then the perfusion was switched to the working mode with a left atria1 perfusion pressure of 15 cm H,O and an afterload of 70 mm Hg (Fig. 1). Thawing Thaw I Thaw I Thaw II Heart rate (HR, beats/min), aortic and cortemperature rate duration duration onary flow (AF and CF, ml/min), cardiac (“C/mitt) (min) (min) c-3 output (CO, the sum of AF and CF), and 0.08 k 0.01 -0.5 14 11 systolic and diastolic pressures (SP and DP, +o.s 0.13 k 0.01 16 9 mm Hg) were monitored at lo-min intervals f2.5 0.22 t 0.02 5 20 for 30 min. External work (g-m/min) was +4.5 0.34 -+ 0.03 3 22 calculated according to Neely et al. (15). +9.5 2 23 0.59” Coronary vascular resistance (CVR) (mm + 19.5 1.11” 1 24 Hg-mm/ml) was calculated from DP and CF Note. Data are presented as mean 2 SEM (n = 4 (CVR = DP/CF). The function of the per group). Total duration of thawing (Thaw I + Thaw freshly isolated unstored heart perfused II) was 25 min for all groups. a Thawing rates at +9.5” and + 19.s”C were calcu- with KHB in working mode for 45 min lated by extrapolation of data obtained at the lower served as the control. Function of the temperatures. stored heart at the end of 30 min working reperfusion was calculated as a percentage of polyethylene tubing (PE 240) was in- of the control function and reported as perserted via the aorta into the left ventricle. A centage of recovery. Both KHB and CP-141 thin-wire thermocouple probe (Type T, EtOH were passed through 0.22~km memgauge 36, Omega Engineering, Inc., Stam- brane filter discs after preparation and reford, CT) was guided through the PE tubing circulated during perfusion through 3-p,m and secured in place to monitor the ventric- in-line filter discs. All chemicals were eigrade or ACS ular chamber temperature using a tempera- ther cell-culture-tested ture recorder (Model H22, Omega Engi- (American Chemical Society) grade from the neering, Inc., Stamford, CT). Since this Sigma Chemical Company (St. Louis, MO). Tissue ice determination. Tissue ice conprocedure damaged the hearts, they were not used in functional assessments. Subse- tent of the frozen heart was determined usquent freezing and thawing steps were performed as already described. The thawing temperatures studied were -0.5, +0.5, +2.5, and +4S”C. Average thawing rate was estimated from the time interval for the ventricular temperature to change from - 3.2”C (ventricular temperature at the end of 6-h freeze) to - 2.1”C (melting point of CP-14/EtOH). As shown in Fig. 2 and Table 1, between - 0.5 and + 4.5”C, thawing rate (“Urnin) correlated linearly with the thawing temperatures. From these data thawing rates at + 9.5 and + 19.5”C were calculated by extrapolation. FIG. 2. Thawing rate (“Urnin) as a function of the Assessment of the cardiac function. The thawing temperature (“C). Data plotted represent thawed heart was reperfused retrograde at mean * SE (n = 4 per point). The linear relationship 70 mm Hg with KHB for 15 min during can be described as thawing rate = 0.1 + 0.0518 X which the pulmonary artery and the left (thawing temperature), ? = 0.995. TABLE 1 Thawing Rates at Different Thawing Temperature and the Corresponding Duration of Thaw I and Thaw II for Hearts Frozen at - 3.4”C for 6 h
THAWING
RATE AND THE VIABILITY
481
OF THE FROZEN HEART
recovery was significantly less than the 0.13Umin group. CVR (Fig. 3D) was elevated in all groups; but those thawed at the optimal rates showed less increase. Nevertheless, the difference was not statistically significant. These observations demonstrated that the thawing rate was critical to the recovery of function in frozen rat hearts. Moreover, thawing at O.l3”C/min offered the best overall return of cardiac RESULTS function. We also compared the effect of changing After freeze-thawing, HR in all stored the duration of Thaw I on recovery. The hearts returned to levels not significantly different from the controls and, therefore, hearts were thawed at 0.13YYmin for 9 min was not influenced by the thawing rate. to - 2°C or 25 min to + 1°C without expeOther hemodynamic function showed a bi- riencing Thaw II. The percentage recovery phasic response to the thawing rate (Table 2 of HR, AF, CF, CO, SP, work, and CVR in and Fig. 3). For ease of explanation, car- the 9-min group were 93.1 + 3.3, 46.0 ? diac output (CO) will be used as an index of 4.2, 62.0 k 4.3, 50.8 -+ 3.8, 71.1 + 1.7, 59.4 cardiac function, although the other param- + 1.8, and 123.6 ? 9.2%, respectively. Reeters, including AF (Fig. 3A), CF (Fig. 3B), covery in the 25-min group was 99.5 -+ 8.0, SP (Fig. 3C), CVR (Fig. 3D), and work fol- 51.1 + 4.8, 64.5 ? 4.6, 55.2 + 4.3, 72.9 ? lowed essentially the same trend. At the 2.3, 65.4 * 4.6, and 124.1 + 9.2%, respecthawing rate of O.O8”C/min, the recovery of tively. These functions were not signifiCO was 40.8 ? 5.8% of control. As thawing cantly different from those thawed at rate was elevated to O.l3”C/min, CO recov- O.l3”C/min (Thaw I) for 9 min and held at ery reached 60.5 + 4.9% of control. Be- - 1°C (Thaw II) for 16 min. This observatween groups thawed at 0.13, 0.22, and tion indicated that Thaw II was unneces0.34”C/min, recovery was similar and sta- sary and the heart could be thawed at + 1°C tistically nondifferent. Function declined for as long as 25 min without detrimental progressively when thawing rate was in- effects on the recovery or reperfused as creased further; at 0.59 and 1.1l”C/min the soon as 9 min after the onset of thawing. ing calorimetry and expressed as a percentage of total tissue water frozen (9, 21). Statistical analysis. Data analysis was performed using a personal microcomputer. Differences among the various experimental groups were tested by the analysis of variance. Significant difference between groups (P < 0.05) was detected by the Fisher’s least significant difference test.
TABLE 2 Effect of Thawing Rate on Poststorage Recovery of Hemodynamic Performance of Isolated Rat Heart at the End of 30 min Working Reperfusion Rate
(“C/min) 0.08 0.13 0.22 0.34 0.59 1.11
Temp (“C)
n
-0.5 +0.5 f2.5 +4.5 +9.5 +19.5
8 9 6 6 6 9
HR 337 298 313 270 287 216
+ t t + + f
CF
AF 2-l 15 25 15 19 23
18.0 29.6 23.5 24.3 20.1 13.1
+ + -c ir -c t
3.4* 2.1 2.3 1.5 3.4* 1.8*
11.1 13.6 12.7 12.8 10.9 9.9
+ T + f + -c
0.9 1.0 0.7 1.1 0.8 0.6*
co 29.1 43.2 36.2 37.1 30.9 23.0
f k + + f 2
SP 4.1* 3.5 2.9 2.2 3.8* 2.4*
94 111 102 106 99 94
t c 2 + + f
Work 3* 3 3* 3 4* 4*
29.2 48.8 38.3 31.7 31.2 22.6
-t k + It ? -t
4.9* 5.2 3.8* 2.5 4.6* 2.6*
CVR 5.75 5.02 5.13 4.19 5.72 6.34
2 k + + + ?
0.52 0.23 0.21 0.45 0.48 0.46*
Note. Differences between groups were detected by ANOVA and tested by Fisher precision least significant difference tests. Means ? SE are presented. Hemodynamic function of the control hearts was (n = 28): HR, 296 2 6 beats/min; AF, 50.3 ? 0.9 ml/min; CF, 21.3 2 0.7 ml/min; CO, 71.5 2 1.2 mumin; SP, 142 2 2 mm Hg; work, 95.5 2 2.4 g-m/min; CVR, 3.65 2 0.12 mm Hg-min/ml. * P < 0.05 vs O.l3”C/min group.
482
ZHU ET AL.
80 ~...._~ 60 70
50 40 30 20 IO
i 0 0.0 0.2
04
0.6 0.X
1.0 1.2
00
0.2
0.4 0.6 0.8
1.0 I2
0.0 0.2
0.4 0.6 0.8
1.0 1.2
0.0 0.2
0.4 0.6 0.8
1.0 1.2
Thawing rate, “Clmin FIG. 3. Percentage recovery of hemodynamic function in frozen/thawed hearts as a function of thawing rate. Recovery in A, aortic flow (AF); B, coronary flow (CF); C, systolic pressure (SP); and D, coronary vascular resistance (CVR) after 30 min of working reperfusion were calculated as percentage of control function. Values for control function are listed in the legend for Table 2. Data plotted represent means ? SE. **P < 0.01 vs hearts thawed at 0.13YYmin and YIP< 0.01 vs hearts thawed at 0.22 and 0.34Wmin.
been made by others (17). Izumi et al. (7) found that islets isolated from slowly We demonstrated a biphasic response of thawed pancreata at room temperature had function in freeze-stored hearts to thawing better insulin-releasing activity than those rates. Hearts thawed at rates ranged from isolated from rapidly thawed pancreata at 0.13 to 0.34”C/min displayed similar func- 37°C. Mouse embryos frozen at 0.2 to 2°C tional recovery, although the O.l3”C/min min survived best with a rewarming rate of group was overall the best among all hearts. 4 to 25”C/min (22). Ram spermatozoa frozen at 2”C/min sustained more damage Both slower (O.O8”C/min) and faster (0.59”Clmin or higher) rates led to less re- when thawed fast in 20°C water than slowly covery. Understandably, “slow” and in 20°C air (5). Function of rabbit kidneys “fast” rates must be different from system stored at - 80°C at a cooling rate of l”C/h to system. It is generally recognized that was found to be dependent on the warming when cells are frozen at a rapid rate which rate during thawing: l”C/min was better promotes the formation of intracellular ice, than l”C/h or 20”C/min (16). Resumption of vital functions in freeze-tolerant wood frogs rapid thawing improves cellular viability for 24 h was (4). When frozen at slow cooling rate and after being frozen at -2.K intracellular ice is not evident, rapid thaw- also thawing rate dependent (8). Frozen ing is more injurious than slow thawing, ir- frogs thawed in 6.5 to 8.5”C air at a rate of respective of the cell systems. Gao et al. (6) O.l2”C/min and those thawed in 23 to 25°C reported that when frozen at a cooling rate air at 0.26”C/min rate had the similar surof O.S”C/min, human erythrocytes showed vival rate, however. Freezing-thawing process is known to the least hemolysis if rewarmed at OS”C/ min. Miller and Mazur (14) observed that cause cell damage by concentrating the exwhen cooling rate was slow (0.27”C/min tra- and intracellular solutes during freezing and 1.7”C/min), the human red cells sur- and a following solute dilution and cell vival was high with slower warming rates swelling during thawing (11, 18). During (0.06-l”C/min); similar observation have freezing at a slow rate, cells shrink and deDISCUSSION
THAWING
RATE AND
THE VIABILITY
hydrate due to efflux of cellular water. During thawing, the extracellular ice melts and water reenters the cells. Thawing at too fast a rate will not allow enough time for reequilibration of water and ions. It induces rapid changes in solute concentrations and causes osmotic stress to cells leading to cell swelling and damage (10). These mechanisms may account for our results with the supraoptimal thawing rates. Certainly other factors like cooling rates, temperature, duration of storage, kind and concentration of cryoprotectants interact with thawing rate and affect the outcome of freezing storage (6, 14, 19), these factors will be considered and examined in further optimization of the freezing protocol for the cardiac explant. By adding EtOH, a cryoprotectant, to the CP-14 solution we increased its osmolality to 1.1 Osm/Kg water, thus lowering the freezing point of the stored heart to -2.l”C. Our previous study (20) demonstrated that 4.7% EtOH in CP solution had no obvious toxic effect to the heart during a 12-h storage period. In contrast, EtOH could prevent edema, reduce energy depletion, and promote functional preservation by inhibiting Na+/K+ ATPase and Nat/ Ca2+ exchanges, and dehydrating myocardium (23). The incomplete return of the cardiac function in the present study may be the result of ischemia, freezing-thawing damage, and unoptimized solution composition. ACKNOWLEDGMENT
This study was supported in part by Grant HL45129 from NIH. REFERENCES
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