Survival of Canine Kidneys After Treatment with Dimethylsulfoxide, Freezing at -8oO’C, and Thawing by Microwave
FRANK M. GUTTMAN,z JACQUES LIZIN, PIERRE ROBITAILLE, ASD CLAIRE TURGEON-KNAACK HERVfi BLANCHARD, Dcpartmcnt.P
of Surgery, Pathology, ad Pediatrics, Pediatric Rcscarcl~ Ccnte,; Snintc-Jzrstinc
According to Karow (24), an anticipated demand of 12,000 transplantable kidneys will be required in 4 years in the U.S.A. In 1974 approximately 14,000 patients wcrc maintained on chronic dialysis and 2,500 patients received transplants. Kountz (25) has noted that the total numbrr of transplants in the U.S. is not increasing significantly: in 1971, 1660; in 1972, 2164; and in 1973, 2207. At the same time SOOO-10,000 patients per year become caudidatcs for treatment of end-stage renal disease. Since the mortality rate of renal transplantation after 5 years is not greater than that of chronic dialysis (25)) more doctors and patients will opt for transplantation. In childrcn, transplantation is the therapy of choice. It is therefore obvious that, while at present there is a great unmet need for more transplantation, this need will becon~e more and more acute \\itli time. This prcscntly unmet need can only bc rcsolvcd through our understanding of freeze-thaw injury ai~d the ability to crcatc Received April 14, 1976. 1 Supported by Medical Researcll Council, Grant No. hlA 4916, aud Canadian Air Liquidc Cie. 2 Address for reprints: Lady Davis IJlStitUtP Jewish General Hospital, 5750 Ci,tc dcs Ncig~s. Montreal, H3T 1E2, Canada. 3 Supported by Justine-Lacostc-B(~aul)icn Foundation.
GpyriRht All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
organ banks (22, 23, 28, 30, 32). To quote T. Starzl, “There is no way that widespread and efficient utilization of human organ homografts will ever be possible without major new developments in organ preservation which will allow banking for weeks or months” (34). At present, unicellular systems such as human and bull spermatozoa (23)) lymphocytes (2)) and bone marrow, red blood cells (21) and tissue culture stem cells (11) may be frozen with a suitable cryoprotective agent to -196°C and kept for months and years at any convcnicnt in a bank to bc used time. \VhoIc organs arc more difficult to freczc possibly because the variety of cells and bulk of tissue does not allow for a uniform freczc, nor for a uniform thaw (5, 6, 7, 10). For several years Lehr am1 associates (3, 19, 20, 27) and Guttman et al. ( 12-16, 29) using diffcrcnt techniques have \vorkcd on the problems of freezing the small bowel with some s~~cccss. In the latest scrics of csperiments, Guttmnn et al. have rcportcd a SOY; survival of bowel segments frozen dowu to -76°C (16). 13arncr and Schcnk (4) rcportcd on the successful freezing of spleen. Early efforts to freeze the kidney have failed (5, 17, 18). In 1972, Dietzman et (11. (9) reported prcwrvation of canine kidneys at -22°C.
At this temperature, it is most likely that the kidneys wcrc not completely frozen.
Adult mongrel dogs of both sexes were used weighing from 15 to 25 kg. Prcopcrative baseline studies of hemoglobin and creatininc were carried out. The dogs were observed from 10 to 14 days in our animal quarters prior to experiment on regular rations. After fasting overnight, preoperntive medications of dchydrobenzpcritol, 3 mg; fentanyl, 0.1 mg; and diazepam, 10 mg were given. Anesthesia was maintained with fluothane and a mechanical volullle respirator (Harvard). A midline incision was performed. One kidney was removed after priming the animal with mannitol (100 ml of a 20% solution) and furoscmide (40 mg). Perfusion The removed kidney was flushed with: (a) Perfudex (Pharmacia, Sweden), (b) modified Collins’ solution (8) and (c) modified Sacks’ solution for a period of 15 min at a temperature of 4°C (see Table 1). Additives to each solution (per liter) were heparin (2000 IU), hydrocortisonc (100 mg), isoproterenol (1 mg), procaine (10 mg), and sodium bicarbonate to produce a pH of 7.4. Collins’ solution was modified by substituting a small quantity of MgCl, (4 meq/liter) for MgSOI, which was necessary because of precipitation of magnesium phosphate. After this initial flush, the kidneys were perfused with the same solutions containing I.4 M DMSO (dimethylsulfoxide) for 30 min at 4°C. Control kidneys were reimplanted in the groin with an end-to-end arterial anastomosis, end-to-side venous anastomosis, and a tunneling uretero-vesical anastomosis. Freezing The kidney was placed in a Linde BF4 biological freezer (see Fig. 1). Cold he-
FOR ORGAN FREEZING
Freerlng chamber . . . . . ..m.......mma.............. I
of pre\ious technique in freezing is shown here. The organ is placed in FIG. 1. Modification the freezing chamber (Linde BF4). Two copper coils within the chamber are connected to the He tank and N, tank. At the other end, the helium line is led into an intra-arterial catheter. A feedback thermistor (A-C) controls the solenoid valve allowing for the flow of liquid nitrogen into the chamber. El, EL, and Es rcpresrnt thermistors implnntcd into the kidney at different depths. The N, gas has n high flow rntc (80 liter/min) which permits the feedback system to operate regularly by stirring the gases in the chamber.
of 8-9 liters/inin circulated throughout the kidney via the arterial cannula in the mamwr \vc have dcscribed in past publications on the intestint ( 12). Thermistor probes wwe placed at depths of 1, 2, and 2.5 cm in the kidney (Fig. 2). The kidney \vas frozen at a rate of l”C/min to -2SOC and thc11 111orc rapidly 2-3”C/min to -80°C. The cold agents (helium and nitrogen gas) thus reach the kidney tissue simultaneously through an intra-arterial route and by surface contact. lium
at n flo\v
Thntcing The kidney was kept at -80°C for 15 min after each thermistor had registered -80°C and then was placed in a Toshiba microwave oven producing 2450 mIIz at 1.3s kW, after removal of the thermistor probes. Thawing to 0°C \vas carried out taking about 90 SW with manual rotation of the kidney in four positions every 20 xc.
In a preliminary study, 13 experiments were carried out as outlined, except that thawing by conduction with immersion of the kidney in a 40°C saline bath was attcmptecl. Prior to autotraiisplal~tation a S-10 lniii fimh of the kidney was carried out with the initial perfusion fluicl (without IlllSO). Aut0tmns~~lantfltion
The kidney nas then autografted into the groin of the donor dog in a standard manner. Mannitol (20 g), furosemide (40 mg), and 500 ml of 0.45 NaCl in 5% glucose w-erc given intravenously. Serum crcatinine was followed at 3-day intervals. One to two months postoperatively, the normal contralateral kidney was removed. ,4t this time, biopsies of the experimental kidney were taken for light and clcctron microscopic analysis.
-. Prrfudex Prrfudex Perfudrx Collins Sacks
Perfudex + DMSO (1.4 Y) Collins + DRISO (1.4 nf) Sacks + DAIS0 (1.4 x) Collins + DRlSO (1.4 m) Harks + DMSW (1.4 M)
Tots1 a Solution precipitatrd b Acute tubular nwrosis
1 3 4 2 1 11
1 3 20 lb 1 s
during perfusion. of perfused kidney.
The study was carried out in 40 dogs. The control group in which the kidney was not frozen comprised 11 dogs. There were two technical deaths in the control group (Table 2) and one acute tubular necrosis; the other kidneys functioned well. Creatinine levels were within normal limits (1.0 2 0.14 mgcjo). The histologic examination of these control kidneys rcvealed apical vacuolization of the proximal convoluted tub&s, which has been seen in other causes of toxic damage to the kidney and is probably an osmotic effect clue to the furosemide and mannitol (7, 35). Other specimens showed recovering tubular necrosis. The experimental group was originally composed of 29 dogs. There were 13 early ‘C
deaths due to anesthetic prob1~~ms. I.;IS~‘IIlnr anastomic Icakngc, and Carl? postopcrative pneui~~onia. Some of thcsc frozenthawed kidneys appeared to bc of good quality on autopsy, but they \verc cscludcd from the study because they still had both kidneys at the time of death. Thus the experimental group was finally composed of 16 dogs, eight of which survived for a prolonged period (12-14 months) on a single frozen-thawed kidney. The results are outlined in Tables 3 and 4. The subgroups are very small because of the pilot nature of this study. All experimental dogs arc included in Table 3, but only the eight survivors arc listed in Table 4. Failure was defined as inability of the dog to survive on a sole kidney, or when at contralatcral nephrectomy the kidney was obviously not viable. When conduction thawing in a saline bath was attempted only one of 13 kidneys survived. However, this kidney supported life for 18 months following the experiment. The creatinine was 1.2 mgCjo at sacrifice. DISCUSSION
Recently Kubota et nl. (26) claimed a 60% success rate after cooling canine kidneys to -22°C; at -75”C, 18% were said
FIG. 2. The temperatures registered by three thermistors the kidney. The range of difference is from 3 to 5°C.
of 1, 2, and 2.5 cm in
Frozen-Thawed Initial flush
Perfudex Perfudex Perfudex Collins Sacks Sacks None
Perfudex + I~;1150 (1.4 M) Collins + DMSO (1.4 hf) Sacks + D&IS0 (1.4 M) Collins + DMSO (1.4 M) Sacks + D&180 (1.4 M) Sacks + DMSO (1.6 M) Sacks f DMSO (1.6 nf) 20
z 0 1 5
1 1 2 1C 0 3 0
a Technical death includes early pneumonia, bilateral renal infraction, sepsis, uretero-vesical stricture, and vascular anastomotic problems. * Failure = atrophic, absent, or inadequate kidney (incapable of susiaiuing life) found at the time of exploration for contralateral nephrectomy. c This kidney produced a fair quantity of urine G months after freezing b11t had a low P:ZlI clearance of 10.23 ml/min and an inulin clearance of 5.43 ml,/miu.
to be “successful,” but it is certain that at -22°C the organ was not completely frozen, and none of the kidneys cooled to -75°C had life-sustaining function. Others have demonstrated the sporadic ability to freeze whole organs: the spleen (4), the heart (24)) although not below -22”C, and the intestines (15, 28). In 1967, Halasz et al. (17, 18) reported that four of 38 kidneys thawed from -60°C supported host dogs as the sole kidney. Two of these kidneys were thawed with a microwave oven, one by diathermy at 27 mHz and 450 W, and one by conduction (water bath). One of our longest survivors (18 months) was also thawed by conduction, the only kidney to survive SO thawed in this series. Holst et al. (20) found that microwave heating can injure kidneys after reaching 0-4°C. At temperatures of 4”C, 2450 mHz applied to a nonfrozen kidney as a single 5-set burst at a power level sufficient to warm the standard model (80 ml of water) at a rate of 67”C/min gave renal necrosis after reimplantation. They found that dog kidneys could be thawed at 2450 mHz (900 W) producing a change of -55 to 10°C in about 1 min, but most did not function.
Of 30 kidneys, 11 produced good urine initially, but they were not life-sustaining as in our series. At the cellular level Georgiev and Matercva (11) have reported that rat renal slices may be frozen to -79 or to -196°C after protection with 2.1 or 4.2 M glycerol, thawed, and survive even after 30 days. TABLE
fj” 1.8 0.9 14 (s)h 1 .(i nc 1.5 7 (s) 1.1 9 (s) 0.x 12 (s)d t .o 11 (s) 0.9 %e .___--__~~__ a I)ied during anesthesia for metabolic studies. b (s) = Sacrifice. c Strangulated hernia. d This dog gave birth to a normal litter of five puppies 10 months after the experiment. e J%ophageal perforation during tube feeding for metabolic studies. 7
24 2.5 26 :m S.5 4s 44
I 1 1 1 1 :i 3
Il’l~csc slicxls sho\v 110r11ral Iiiitotic wproduction. Thus the t11:ijor problem is prol)ably in the mliformity of the diffusion of cryoprotcctant (7, 35) and the uniformit) of freezing and thawing. While Schimmcl, and Robertson and Jacob (32) in 1964, demonstrated that freezing using the organs’ internal radiator, i.e., the circulatory system, is effective in distributing cold throughout the organ, not many rcscarchrrs use these techniques; Schimmcl used liquid nitrogen aAd Kobcrtson and Jacob cold helium. Henclerson and Bickis ( 15) demonstrated that helium is not harmful to renal slices in oitro (12). Guttman in collaboration with others ( 12) adapted the use of helium through the mcscnteric artery to freeze the intcstinc with a 50% success rate. It would seem only logical that this is a technique superior to conduction cooling, since it is intrinsically far more uniform. Conduction heating seems to be theoretically less miiform than microwave irradiation for thawing (31). In the prc-
liniinary slutl! \\‘(‘ rol111(10111!-onca sI11.ii\ ~11 \vith conductiwi thawing. ‘I’lrc power 0C the microwave oven used in this stud! was higher than that reported by most workers ( 1.35 kW) (36). MJe do not have an automatic rotator but relied on expcrionce and gross judgment iu turning the kidney manually on a platform every 1520 WC for four periods. WC found, as did Hoist et al. (20), that great damage occurs when the kidney is warmed past 0°C. Espcricncc led us to stop the heating when we judged ( and then mcasurcd with thermistors) that the kidney was at 0 to 6°C. There was some variation from cspcrimcnt to experiment which did account for some of the failures, in that the ureter and renal pelvis jvere sometimes macroscopically burnt. Increasing the concentration of DhISO to 1.6 KI did not change the results in a small number of dogs (Table 3). The difference between the two intracellular solutions and the one estraccllular solution arc not apparent in the smnll numbers
FIG. 3. Photomicrograph taken 6 months following the freeze-thaw experiment. Cryoprotection was by modified Sacks solution containing 1.6 hc DhlSO. The dog was sacrificed 11 months after the cxpcrimcnt. x 50.
PK. 4. A higher mcllt
photomicrograph of a kidney is rlormnl. % 310.
of slll,group.s. [email protected]
’lla c’t (/I. ( 1) have shown that a low-Cl-, high-Kl, and low-Nat solution which has a high osmolality is the lcast damaging to renal cortical cell slices and causes sonic> cell shrinkage, but the least loss of K’ and entry of Na+ into the cell. Sacks (33) dcmonstratcd that an intracellular solution, modificxl from Collins’ formulation by incrcnsing ihc osmolality and replacing the sulfate ion by chloride , gave superior results. ‘il’e have previously noted (unpublished data) that a dense precipitate forms in Collins’ solution with our additives and we attribute this to the magnesium precipitating out as a phosphate salt. Conseqllently wc modified both Sacks’ and Collins’ solutions bvi adding only 4 meq/liter of hlgC1,. In our small subgroups thcrc would not seem to be much difference but this should bc confirmed in additional studies. \T:e believe that our 50% survival rntc is very encouraging. Obviously, 15 min is not a sufficient preservation time and \vork is in progress to extend this period.
Forty canine kidneys wcrc the subject of this pilot study where control groups perfused with Pcrfudcs plus DhlSO (1.4 nr), modified Collins’ solution nith DMSO (1.4 XI) and modified Sacks’ solution with DAIS0 (1.4 M) shon~~l little toxicity and life-sustaining conservation, In the rxpcrimental group, 16 kidneys were frozen for 15 min to -SO”C, thawed by microwave illumination, and rcimplanted. Of the 16 dogs, eight survived 2-14 months on their single kidney. The technique of inducing freezing by using intra-arterial cold helium and thawing with high-power microwave illumination gave an overall success rate of SOc/o long-term life-sustaining survival. REFERENCES 1. Acquatella, Perez-Gonzalez, hl., hIorales, G. hl., and Whittembury, G. Ionic rind histological changes in the kidney after perfusion and storage for transplantation. Trans$~ntation 14, 480-489 ( 1972 ). 2. Ashood-Smith, hl. J. Low temperature prcservation of mouse lymphocytes with dimcthylsulfoxidc. Blood 23, 494-501 (1961).
3. Barchi, R., Lundy, B. S., Hamilton, R., and Lehr, H. B. Frozen preserved canine intestine; ultrastructural change in the mucosa of frozen preserved canine ileum. Cryobiology 6, 211-226 ( 1969). 4. Barner, H. B., and Schenk, E. A. Autotransplantation of the frozen-thawed spleen. Arch. PuthoE. 82, 267-271 ( 1966). 5. Bloch, J. H., Longerbeam, J. K., Manax, R. C. PreW. G., Hilal, S., and Lillehei, servative solutions for freezing whole organs in vitro. Trans. Amer. Sot. Artif. Intern. Organs 9, 139-147 ( 1963). 6. Burns, C. P., Burdett, E. C., and Karow, A. M. Thawing of rabbit kidneys from -79°C with 2450 MHz radiation. Cryobiology 12, 577 ( 1975). 7. Cady, B., Barner, H. B., Rivers, R. J., Jr., Haynes, L. L., and Watkins, E., Jr. Glycerolization of the canine kidney. 11. Pathological patterns. Cryobiology 3, 306-317 (1967). 8. Collins, G. hl., Hartley, L. C. J., and Clunie, G. J. A. Kidney preservation for transporExperimental analysis of optimal tation. perfusate composition. Brit. J. Szrrg. 59, 187-189 (1972). 9. Dietzman, R. H., Rebelo, A. E., and Lillehei, R. C. Long-term success following freezing of canine kidneys. Surgery 74, 181-189 (1973). 10. Filo, R. S., Bell, R. T., Small, A., and Sell, K. W. Current status of organ freeze preservation. Tissue Bank Symposium 13-15 August, 1975. 11. Georgiev, I., and Matereva, G. Survival and growth of kidney tissue in cultures in vi00 after freezing and preservation at low temperature. C.R. Acad. Bulg. Sci. 20, 621624 (1967). 12. Guttman, F. M., Khalessi, A., Huxley, B. W., Lee, R., and Savard, G. Whole organ preservation. I. A technique for in viva freezing canine intestine using intraarterial helium and ambient nitrogen. Cryobiology 6, 32-36 ( 1969). 13. Guttman, F. M., Khalessi, A., and Berdnikoff, G. Whole organ preservation. II. A study of the protective effect of glycerol dimethylsulfoxide and both combined, while freezing canine intestine employing an in vice technique. Cryobiology 6, 339-346 ( 1970). 14. Guttman, F. M., Sangbhungdhu, K., Berdnikoff, G., Makita, T., and Sandbom, E. B. Dimethylsulfoxide glycerol and inositol as cryophylactic agents in preserving frozen
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