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Distribution and removal of glycerol by vascular albumin perfusion in rabbit kidneys
CRYOBIOLOGY
15, 302-311
(19%)
Distribution
and
Removal of Glycerol by Vascular Perfusion in Rabbit Kidneys1
Albumin
IB A. JACOBSEN Laboratory
of Nephrology, Institute of Pathology, Odmse Unitienity, D&5000
Odense C, Dawnark
Most mammahan celIs wiI1 survive freezing only in the presence of a cryoprotectant (17) and when cooled and thawed at optimum rates ( 12, 9). These rates are highly dependent on the type of cell in question and on the concentration of the cryoprotectant used; generally the concentration and rate are inversely related (9). This presents at least two major problems in the attempt to transfer the principles of cryopreservation ‘of cell suspensions to freezing of whole organs; namely, the addition and removal of high concentrations of cryoprotective agents ‘and the physical difficulties of cooling and rewarming large amounts of tissue at high rates. One of these probIems can only be avoided by solving the other, that is low (and Iess tissue-damaging) concentrations of cryoprotectants are likely to be &ective if a rapid and uniform change in temperature can be achieved, whereas slow cooling and rewarming is likely to be compatible with surviva1 only if high concentrations can be distributed in the organ without tissue damage. Our approach to kidney freezing and thereby long-term preservation has been the latter.
Received June 16, 1977; accepted September 7, 1977. 1 Supported by The Danish Medical Research Council.
‘X)11-22<0/78/0153-0302$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form resewed.
As previously shown (7) rabbit kidneys can be perfused with up to 4 M glycerol dissolved in a perfusate of extracellular electrolyte composition made hypertonic with mannitol and containing gelatine polypeptides (Haemaccel) :as a colloid. After autotransplantation these kidneys showed only temporary functional impairment with survival of the animals subjected to immediate contralateral nephrectomy and return to a functional capacity not different from that of unperfused control grafts. A major problem in these glycerol perfusions was rising vascuIar resistance during the last ,one-third of gradna1 dilution, probably due to delayed permeation of glycerol through the cell membranes. Consequently the aim of the present experiments was to study if this development of resistance could be diminished by using a different and probab,ly more effective colloid in the perfustate and by decreasing ‘the glycerol concentration more slowly during the last lthird of deglycerolization. Furthermore, the change of glycerol concentration in the perfusate does not necessarily imply the same variation of gIycero1 content in the renal tissue, High tissue concentrations mare likely to be essential for successful freezing at a Iater stage, because the cryoprotective agent must be brought into contact with the cells to exert its effect, even if penetration into the
GLYCEROLIZATION TABLE Composition
of
1
the HAP-2 Perfusate
Composition Na+
K+
Amount
131 3.8 1.0 1.7 86 25 1.0 0.6 0.4
mmol/liter mmol/liter
OF RABBIT
KIDNEYS
303
The pH of the perfustates was adjusted to 7.0 measured at 30°C by equilibration with 576 CO, in oxygen. Perfusion Apparatm
The perfusion circuit, shown in Fig. 1, consisted of a central perfusate reservoir, mmol/liter mmol/liter in which the perfusate was continuously ClHCOzmmoI/liter stirred and equilibrated with a gas mixmmd/liter 5042ture of 5% COZ and 95% 0, to maintain HPOPmmd/liter a constant pH. The perfusate was drawn mmol/liter Hap04 from this reservoir by a pump (WatsonMannitol 107 mmol/liter Carbenicillin 100 mg/liter Marlow MHRE 100) and passed through Human albumin 40 g/liter two 0.2%pm filters in paralfel (Acropor, 147 mm, Gelman Instruments), a heat excells has not been proven to be necessary change consisting of a bloodwarming bag ( Fenwal Laboratories) sandwiched be(15). Accordingly this study was an attween two cooled copper plates, an.d a tempt to look into the distribution of bubble trap to a T-piece can&a inserted glycerol in various parts of the kidney as a function of the perfusion procedure, by in the renal ,artery. The heat exchanger and measuring the freezing point depression of the kidney were situated in a thermally insulated encIosure. Temperatures in the the renal tissue at different stages of glycerolization to a maximum perfusate con- perfusate immediately before entering the centration of 3 M and subsequent de- kidney and in the insulated enclosure were continuously recorded ,and the perfusate glycerolization. temperature was kept constant at +lO”C. The kidney was supported by a stainlessMATERIALS AND METHODS steel cradle over a graduated reservoir Perfusion Fluids into which the venous effluent drained, so The composition of the basic perfusate that perfusate flow rate could be measured used as a vehicle for gIycero1 (HAP-Z) is at IO-min intervals by stopping the pump shown in Table 1. The electrolyte com- which returned the effluent to the central position and mannitof content was identical reservoir. The perfusion pressure was conwith that used in previous experiments (7), tinuously measured in the arterial cannula whereas the colloid was changed to human with a pressure transducer (Statham P 23 serum albumin (KABX), 40 g/liter. A Db) and the flow ,rate was adjusted to yield stock solution containing salts and man- a constant pressure of 40 mm Hg by the nitol was stored in the frozen state at means of .a pressure-flow feedback unit -2O”‘C ,and was filtered through a O-22-pm (19). A by-pass with a constant resistance Acropor filter ( Gelm.an Instruments >. AI- to flow was inserted between a point just bumin was added in the form of a 20% proximal to the ,arterial can&a and the solution immediately before use. central reservoir. This ‘diverted a part of the flow .directIy to the reservoir to ensure A perfusate containing 4.8 M glycerol was prepared by ,dissoIving the salts of a reasonably high flow through the circuit the stock solution in a minimum volume even when the vascular resistance in the kidney was high. Perfusate samples for of water to which 442 g/liter of glycerol was added, and the solution was diluted to glycerol estimations were obtained from the final composition with distilIed water. this by-pass. Mgz+ CW+
mmullliter
304
IB A. JACOBSEN
FIG. 1. Glycerolization circuit: P1, P2, Pa, and P4, perfusate pumps; CDR, reservoir for highglycerol perfusate or glycerol-free perfusate; TTF, Trendtrak Programmer; MR, centrltl perfusate reservoir; F, filter; BE, heat exchanger; BT, bubble trap; K, kidney; FM, flow-measuring reservoir; PT, pressure transducer; PM, pressure monitor; CPC, constant pressure corltrol unit; S, sample point.
The concentrated glycerol solution or the gIycerol-free perfusate was added to the central reservoir by a pump (WatsonMarlow MHRE ZOO} controlled by a Trendtrak Programmer (Leeds and Northrup) on which the program for volumes to be added per unit time to provide the desired variation of glycerol concentration in the central reservoir had been drawn with India ink. The volume of the central reservoir was 70 ml and that of the kidney circuit was 330 ml, The tota volume was kept constant at: 400 ml by withdrawing surplus fluid from the reservoir by a pump.
The changes in perfusate glycerol concentration were programmed to 0.025 M/ min during glycerolization to a maximum of 3 M. This concentration was kept constant for a ZO-min period of equilibration. Deglycerolization was performed at the same rate to a concentration of 1 M, after which the rate was decreased to 0.016 M/ min until the minimum concentration of approximately 0.1 M was reached. Rates of
addition of concentrated glycerol solution or glycerol-free perfusate were calculated from the following approximated equations: glycerolization
:
z = s+
v2(1 - e-a-tlu*) -M c
deglycerolization:
x = M
t
e*ti
“l&X - cat
where x = flow rate (milliliters per minchange ute), c = rate of concentration (molar per minute ), z)~= volume of the perfusate reservoir (milhliters ) , v2 = the total volume of the kidney circuit minus u1 ( milliliters ) , a = flowrate in the kidney circuit (milliliters per minute), M = glycerol concentration in the high-glycerol perfusate (molar), f = perfusion time (minutes), o = v + o2 and M,,, = maximum glycerol concentration in the perfusate. Renal vascular resistance during perfusion was standardized to a 10 g of kidney and calculated from the expression:
GLYCEROLIZATION
r---j ra
r-d L, I I
FIG. 2. Differential thermal analysis apparatus: S, tissue sample; R, reference; T, temperature reference; AT, differential temperature; TF, Thermos flask.
where P = perfusion pressure (millimeters of Hg), W = initial kidney weight (grams), 7 = perfusate viscosity (centipoise), and F = perfusate flow (milliliters per minute). Glycerol concentrations were measured at IO-min intervals by refractometry at +3O”C (Abbk refractometer). Perfusate viscosity as a function of glycerol concentration was measured at +lO”C using an Ubbelohde viscometer, and the observed values fitted, in the concentrations used, the equation
where x = glycerol concentration (molar), Thus perfusate viscosity could be calculated throughout the perfusion, Experimental Procedures
French lop-eared male rabbits weighing 2.5-3.5 kg were anaesthetized with Hypnorm (fentanyl citrate, 0.315 mg/ml, + fluanisone, 10 mg/ml) and BrietaI (sodium methohexital) as previously described (8). Before removal of the left kidney for perfusion the animal was pretreated with 5 mg of furosemide, 10 ml of 10% mannitol, and
OF RABBIT
KIDNEYS
305
4 mg/kg body weight of chlorpromazine intravenousIy, During the time of perfusion the abdomen was closed and a rapid recovery was produced by injection of nalorphine. After perfusion the right kidney was removed, and the perfused kidney was implanted by anastomosing the renal vessels to the aorta and the inferior vena cava, respectively. The ureter was implanted into the bladder, Postoperatively daily blood samples were collected for serum creatinine estimations, and weight loss was replaced with isotonic saline and dextrose intravenously (for details see 8). Endogenous creatinine clearance was measured 21 days after transplantation, As soon as possible after clamping the renal artery (average warm ischaemia was 3 min) the kidney was flushed at a controlled pressure of 60 mm Bg with 100 ml of WF 5 PD wash out solution at +4X (22), Twelve to fourteen minutes after clamping of the artery the kidney was connected to the perfusion circuit, and perfusion with HAP-2 was started. The program for glycerolization was started after 15 min of perfusion, and the glycerol concentration was increased at a rate of 0.025 M/min to a maximum of 3 M. Twenty minutes of perfusion was allowed at this concentration for equilibration of glycerol with the kidney tissue. Deglycerolization was initiated at a rate of 0.025 M/min until a perfusate concentration of approximately 1M was reached and was then completed at a rate of 0.016 M/min. The perfusion was continued after deglycerolization for 6090 min to allow the vascular resistance to return to 2-3 times the initial value. As controls, six kidneys were autotransplanted immediately after initial wash out, and three were grafted after 4 hr of perfusion in the glycerolization circuit with the basic HAP-2 perfusate. III both series postoperative serum creatinines and the endogenous creatinine clearance on Day 21 were measured.
306
II3 A. JACOBSEN
FIG. 3. Reproduction of a differential thermal recording. SampIe: Ureter glycerolized to 3 M. The arrows indicate the end of the endotherm and the corresponding alxolute tamperature (the small offset is due to the arrangement of recorder pens).
Six kidneys were glycerolized in the described manner to a maximum of 3 M and subsequently deglycerolized prior to transplantation. Function was estimated as in the control groups, Freezing Point Depression Glycerol
and
Distribution
Melting points of the kidney tissue were measured in samples of renal cortex, renal medulla, and the ureter after perfusion with the HAP-2 perfusate only, after glycerohation to approximately 3 M, and after deglycerolization from 3 M. Melting points of the corresponding perfusates were also determined. All melting points were measured in duplicates by differential therma analysis ( DTA ). The apparatus for thermal analysis is shown in Fig. 2. It consisted of a cylindrical brass block in which three holes were drilled parallel to the axis. The tissue sampie (200 mg) and reference samples ( 200 ~1) were inserted in these holes in thin-
walled cylindrical copper tubes closed with an insulating lid through which copperconstantan thermocouples passed to the centre of the samples. Frozen distilled water was used as a reference and for measuring the absolute temperature of the system. Melting ice in a Thermos flask was used as a reference for the absolute temperature. The differential temperature output was amplified and fed into a Kipp and Zonen BD9 two-channel recorder (full-scale deflection, I00 pV), and the absolute temperature output was recorded on the same instrument (full-scale deflection, 500 PV). The brass block was cooled to -35°C after which the sample and references were inserted into the holes and covered by an insulating lid. As a result the tissue sample was cooled at a rate of more than lOO”C/ min in the range of phase transition to the block temperature. The system was then left on the bench at room temperature, which caused the block temperature to rise
CLYCEROLIZATION
OF RABBIT TABLE
Control
Survival
616
Peak serum-creatinine (pmol/liter)
400
Creatinine clearance (ml/min)
f 234”
Kidneys
Control perfused
Glyorrolised
Glycerolised to +&I~ a
“02.&2th
313 283 i 3
6.9 & 0.7 (4)d
307
2
Function of Autotransplanted trrtnaphlts
KIDNEYS
7.0 f 1.8 (3)
.!ifi/6 721
5/B
f 133
5.8 AZ (4)
610
0.4
f 257
6.2 zk 2.49 (4)
a Previomly published in Ref. 7. * One rabbit died because of cerebral damage but with good graft function. EResults are given as mean f SD. d Values in parentheses are numbers of observations.
at an approximately constant rate of 06 0,7”C/min. The melting point was read as the end of the endotherm as shown in Fig. 3. The kidneys for DTA were dissected immediately after perfusion so that samples of the renal cortex, medulla, and the ureter were isolated. The tissue samples were subsequently cut into smaI1 pieces (approximately 1 mm3 each) using a scalpel and placed in the copper tube before freezing. One kidney was perfused for 100 min with the HAP-2 perfusate before DTA. Three kidneys were glycerolized to 3 M according to the described procedure including 20 min of equilibration before DTA. In two of these kidneys melting
5
ICI
points were determined immediately after perfusion, and in the third DTA was performed immediately as well as 10 hr after termination of glycerolization. In two experiments kidneys were glycerolized to 3 M and subsequently deglycerolized, using the same technique as in the transplantation experiments, before being subjected to DTA. In all experiments samples of the fina perfusate were collected for melting point determination. RESULTS
Transplantation Experiments
Post-transplantation function of controls and perfused kidneys is shown in Table 2
15
20
TIMEINDAYS
s 2
FIG. 4. Postoperative with 3 M glycerol,
serum creatinines
in six raGits
transplanted
with kidneys
perfused
IB
308
A. JACOBSEN
FIG. 5. Perfusion characteristics of kidneys glyycerolized The dashed lines show renal vascular resistance and the concentration.
with the previously obtained results of perfusions with a different colloid and different glycerolization rates (7) for comparison. One kidney glycerolized to 3 M with the HAP-Z per f usate did not function because of total cortical necrosis, and another rabbit died on Day 3 because of severe cerebral damage due to the anaesthetic but with good graft function. Peak creatinines were higher in the glyceroltreated group than in controls, but the ultimate function measured as creatinine clearance after 3 weeks was not different. Postoperative serum creatinines in rabbits transplanted with glycerol treated grafts are shown in Fig. 4, Perfusion characteristics of kidneys perfused with 3 M glycerol are shown in Fig. 5 together with the simultaneous variations of glycerol concentration in the parfusate. Vascular resistance remained low and constant until toward the end of dcglycerolization, when it temporarily increased to a mean of 15 times the initial value, after which it rapidly decreased to some 3 times the initial resistance. Kidney weight increased during perfusion to 143 i- 7% (mean k SD) of the initial weight. GE~Jc~TuZ Distribution
Experiments
-4 typical example of the DTA recordings obtained is reproduced in Fig. 3. An exo-
to and deglycerolized from 3 M, solid lines show perfusate glycerol
therm produced by the freezing of the tissue sample is seen. The arrows indicate the end of the melting endotherm and the corresponding absolute temperature of the sample. Continuation of the recording beyond a sample temperature of 0°C did not revea1 additional endotherms. Melting points measured in different parts of the kidney and in the corresponding perfusion fluids are shown in Fig, 6. The melting point of the basic HAP-2 pcrfusate was -O.Z”C and not different from the vaIues obtained in a kidney perfused with this perfusate alone. Also, meIting temperatures in kidneys perfused with 3.10 and 3.12 M glycerol were almost identical with the melting points of the corresponding perfusates ( -9.5 and -9.9”C). Similarly, kidneys deglycerolized from concentrations just over 3 M had melting points very close to those of the final perfusates. One kidney glycerolized to 3.24 M had a melting point of -10.8”C immediateXy after perfusion as compared with - IO.0 oC in the corresponding perfusate. The melting temperature of a sample of this kidney stored at +lO”C for 10 hr was -10.7”C and thus not different from the value measured immediately after perfusion. In summary the resuIts of the differential thermal analysis showed that the freezing points of various parts of the kidneys
GLYCEROLIZATION
OF
RABBIT
KIDNEYS
0
309
PEWSAtE
q RENAL CORlEX 0 RENAL MEDULLA q URETER
PERFUSATE o GLYCEROL CDNCENTRATtW [M@LA@
a
a
0
FIG, 6. Melting points of perfusates and corresponding - glycerolization and degIycerolization. were identical with those of the corresponding perfusates and did not change with additional time for glycerol equiIibration. DISCUSSION
One of the major problems in cryopreservatian of whole organs is the distribution of high concentrations of a cryoprotective agent. Attempts to achieve this by simple vascular perfusion have demonstrated damaging osmotic effects of even short exposures to modest concentrations (4-6, 20) resulting in rising vascular resistance and serious impairment of circulation after transplantation. Experiments undertaken by Pegg and Wusteman (20) have shown however that these osmotic effects can be greatly modified by a gradual addition of cryoprotectant to the perfusate and by removing it in the same manner before perfusion with isotonic media [e.g., blood). Also the incIusion of a slowIy penetrating solute such as mannitol in the perfusate proved to be beneficial. Using these principles, perfusion of rabbit kidneys with maximum concentrations of 4 M glycerol has been possible with only short-lasting, reversible impairment of function after transplantation (7). A major diversion from optima1
0
kidney tissue at different
stages of
perfusion characteristics in these experiments was a dramatic rise in vascular resistance during the last third of the deglycerolization phase. As such changes in resistance to flow couId affect distribution of the perfusate and thereby the removal of glycerol, an attempt was made in the present study to diminish the increase in vascular resistance. This was done by increasing the coIIoid osmotic effect of the perfusate by repIacing 1.7570 Haemaccel with 470 human serum albumin and by decreasing the rate of deglycerolization during the period of high resistance. The observations shown in Fig. 5 and Table 2 demonstrate that no significant beneficial effect was obtained by these measures; only the organ weight gain during perfusion, 43% as opposed to 64% was different (p < 0.01, rank sum test), and in fact post-transplant function was certainly not improved. The previous experiments (7) demonstrated that rabbit kidneys could survive perfusion with high concentrations of glycerol, but the important question concerning penetration and distribution of the cryoprotectant remained unanswered. The exact mechanisms by which compounds like glycerol exert their protective effect during freezing is not completely understood.
310
IB A.
JACOBSEN
Lovelock ( 10) has argued that penetration of glycerol into the cells is necessary for cryoprotection, but: the evidence on which this conclusion is based is equivocal, and in fact successfu1 freezing has been achieved in the presence of nonpenetrating agents such as PVP, sucrose, and HES (I, 9, 13) and after very short exposures to glycerol ( 14). The same considerations are likely to apply in the case of whole organs, but it is certain that penetration of the capillary membrane and diffusion through the extracellular space to establish contact with all cells is necessary for the protective effect of glycerol. The variations in melting point as a function of glycerol concentration in the perfusate indicate that this cryoprotectant is completely distributed throughout and equilibrated with the water phase of the organs even in the whole length of the ureter, which is essential for Iater freezing experiments. Equilibration of glycerol limited to the vascular and extracellular spaces would give the observed results, if the intracellular phase was not frozen during thermal analysis, or if the melting point of intracellular fluid was higher due to lack of glycerol. However, the cooling rate of more than lOO”C/min in the range of phase transition makes intracellular supercooling highly improbable, and, if intracellular ice had melted at a higher temperature than the perfusate and extracellular phase, an additional endotherm would have been expected. Furthermore, if intracelluIar equilibration with glycerol had not been complete, a higher melting point would have been observed after storage at +lO”C because of further diffusion over the ceI1 membrane; consequently the experimental data presented here indicate complete distribution in and removal from the total water of the kidney when using the described procedure. The present melting points of perfusates containing glycerol measured by DTA are in good agreement with the values found
by Luyet and Rasmussen ( 11) and Shepard et al. (21) using the same method if the effect of the additional solutes present in the HAP-2 perfusate is taken into consideration. The finding of complete equilibration corroborates the findings by Brada and Schloerb (3) who found complete equilibration of [‘*Cl glycerol concentrations up to 1.5 M with the total water phase of canine kidneys within 30 min (temperature is not stated). Beisang et al. (2) and McCollough et aL (16) also found complete equilibration after 10 min when perfusing canine kidneys with glycerol at +4 and +2O”C, respectively. However, the method by which these results were obtained, namely, measurements of A-V differences of cryoprotectant concentrations in the perfusate, is not entirely convincing. Pegg (18) perfused rabbit kidneys with 2 M glycerol at +5 and +37”C for 2 hr and found compIete equilibration at the higher temperature but only 73% equilibration with the kidney water at 5°C. The result of the present experiments, where glycerolization was performed at an intermediate temperature (+lO’C) and over a longer period of time ( 3 hr ) , does not necessarily disagree with the findings at +5”C. CompIete distribution and removal of 3 M glycerol gives theoretically two new possibilities for enhancement of renal preservation, nameIy, further suppression of tissue metabolism by cooling to -9°C without freezing and long-term storage by freezing at slow cooling rates. SUMMARY
A simple perfusion circuit for gradual glycerolization and deglycerolization of rabbit kidneys is described; it has been used to study vascular resistance and glycerol distribution during perfusion at +lO”C with a perfusate of extracellular composition.
GLYCEROLIZATION
An attempt was made to diminish the dramatic rise in vascuIar resistance during deglycerolization of rabbit kidneys, seen in previous experiments, by increasing the perfusate colloid osmotic pressure and decreasing the rate of change of cryoprotectaut concentration during the last third of removal. These modifications of the method did not improve perfusion characteristics or post-transplant function, Melting points of perfusates and different parts of the kidney were determined by differential thermal anaIysis before glycerolization, after glycerolization to 3 M and after subsequent deglycerolization. The variations of tissue melting temperatures were found to be identical with variations in the perfusate, thus indicating a complete distribution and removal of cryoprotective concentrations of glycerol. REFERENCES 1. Ashwood-Smith, M. J., Warby, C., Cnnnor, K. W., and Becher, G. Low-temperature preservation of mammalian ce1l.s in tissue cultures with PVP, Dextrans and HES. Cyobi&ogy 9, 441-449 ( 1972). 2. Beisang, A. A., Dietzman, R. H., Mayo, C. H., Graham, E. F., and Lillehei, R. C. Damage during perfusion procedures used to prepare whole organs for freezing. Cryobiology 5, 273-277 ( 1969 ) _ 3. Brada, D. R., and Schberb, P. R. Dynamics of glycerol addition to the kidney. Szlrg. Gynecol. mstet. 121, 1004-1008 ( 1965). 4. EgdahI, R. H., and Harris, R. In uivo tolerance of canine kidneys to glycerol perfusion. Trunsplun~. Bull. 6, 110-112 ( 1959). 5, Halasz, N. A., Seifert, N. L., and Orlofi, M. J. Whole organ preservation. I. Organ perfusion studies, Surgery 60, 368-372 (1966). 6. Jacobsen, I. A., Kemp, E., and Starklint, H. Glycerol used as a cryoprotectant in subzero preservation of rabbit kidneys. C~Qbiology 12, 123-129 ( 1975), 7. Jacobsen, I. A., Pegg, D. E., Wusteman, M. C., and Robinson, S. M. Transplantation of rabbit kidneys perfused with glycerol solutions at 10°C. Cryoh~ol~gy 15, 18-26 (1978). 8. Jacobsen, I. A. Kidney transplantation in the rabbit. Labaratvry Animals, in press.
OF
RABBIT
KIDNEYS
311
9. Leibo, S. P., Farrant, J., Mazur, P., Hanna, M. G., and Smith, L. H. Effects of frt-zing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of PVP, sucrose or glycerol. Cqobiology 6, 315332 ( 1970). 10. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 11, Z&36 (1953). Il. Luyet, B., and Rasmussen, D. Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of glycerol, ethylene glycol, sucrose and glucose. Biodynamics 10, l67-191 (1988). 12. Mazur, P. Causes of injury in frozen and thawed cells. Fad. PPOC. 24 (suppl. 15), 175-182 ( 1965 ). 13. Mazur, P., Farrant, J., Leibo, S. P., and Chu, E. H. Y. Survival of hamster tissue culture cells after freezing and thawing. Cyohiology 6, 1-9 (1969). 14. Mazur, P., Miller, R. H., and Leibo, S. P. Survival of frozen-thawed bovine red cells as a function of the permeation of glycerol and sucrose. J. Membrane Bid. 15, 137158 ( 1974). 15. Mazur, P,, and Miller, R. H. PermeahiIity of the human erythrocyte to glycerol in 1 and 2 M solutions at 0 and 20°C. Cryobiology 13, 507-522 ( 1976). 16. McCollough, W. B., Jacobs, J. R., Miller, S. H., and Halasz, N. A. The effect of hyperbaria on the glycerolization of the canine kidney. Cryohinlogy 6, 542-545 ( 1970). 17. Meryman, H. T. Review of biological freezing. ha “Cryobiology” {H. T. Meryman, Ed), pp. I-114. Academic Press, New York, 1966. 18. Pegg, D. E. Perfusion of rabbit kidneys with cryoprotective agents. CTyobioZogy 9, 411419 (1972). 19. Pegg, D. E., and Green, C. J, Renal prcservation by hypothermic perfusion. Cryobiology lo, 56-66 (1973). 20. Pegg, D. E., and Wusteman, M. C. Perfusion of rabbit kidneys with glycerol solutions at 5°C. Cryobiology 14, 168-178 ( 1977). 21. Shepard, M. L., Goldston, C. S., and Cocko, F. H. The I&O-NaCl-glycerol phase diagram and its application in cyrobiology. Cryobiology 13, 9-23 (1976). 22. Wusteman, M. C., Jacobsen, I. A., and Pegg, D. E. A new solution for initial perfusion of kidneys. Stand. J. UroE. NephroE., in press.
15, 302-311
(19%)
Distribution
and
Removal of Glycerol by Vascular Perfusion in Rabbit Kidneys1
Albumin
IB A. JACOBSEN Laboratory
of Nephrology, Institute of Pathology, Odmse Unitienity, D&5000
Odense C, Dawnark
Most mammahan celIs wiI1 survive freezing only in the presence of a cryoprotectant (17) and when cooled and thawed at optimum rates ( 12, 9). These rates are highly dependent on the type of cell in question and on the concentration of the cryoprotectant used; generally the concentration and rate are inversely related (9). This presents at least two major problems in the attempt to transfer the principles of cryopreservation ‘of cell suspensions to freezing of whole organs; namely, the addition and removal of high concentrations of cryoprotective agents ‘and the physical difficulties of cooling and rewarming large amounts of tissue at high rates. One of these probIems can only be avoided by solving the other, that is low (and Iess tissue-damaging) concentrations of cryoprotectants are likely to be &ective if a rapid and uniform change in temperature can be achieved, whereas slow cooling and rewarming is likely to be compatible with surviva1 only if high concentrations can be distributed in the organ without tissue damage. Our approach to kidney freezing and thereby long-term preservation has been the latter.
Received June 16, 1977; accepted September 7, 1977. 1 Supported by The Danish Medical Research Council.
‘X)11-22<0/78/0153-0302$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form resewed.
As previously shown (7) rabbit kidneys can be perfused with up to 4 M glycerol dissolved in a perfusate of extracellular electrolyte composition made hypertonic with mannitol and containing gelatine polypeptides (Haemaccel) :as a colloid. After autotransplantation these kidneys showed only temporary functional impairment with survival of the animals subjected to immediate contralateral nephrectomy and return to a functional capacity not different from that of unperfused control grafts. A major problem in these glycerol perfusions was rising vascuIar resistance during the last ,one-third of gradna1 dilution, probably due to delayed permeation of glycerol through the cell membranes. Consequently the aim of the present experiments was to study if this development of resistance could be diminished by using a different and probab,ly more effective colloid in the perfustate and by decreasing ‘the glycerol concentration more slowly during the last lthird of deglycerolization. Furthermore, the change of glycerol concentration in the perfusate does not necessarily imply the same variation of gIycero1 content in the renal tissue, High tissue concentrations mare likely to be essential for successful freezing at a Iater stage, because the cryoprotective agent must be brought into contact with the cells to exert its effect, even if penetration into the
GLYCEROLIZATION TABLE Composition
of
1
the HAP-2 Perfusate
Composition Na+
K+
Amount
131 3.8 1.0 1.7 86 25 1.0 0.6 0.4
mmol/liter mmol/liter
OF RABBIT
KIDNEYS
303
The pH of the perfustates was adjusted to 7.0 measured at 30°C by equilibration with 576 CO, in oxygen. Perfusion Apparatm
The perfusion circuit, shown in Fig. 1, consisted of a central perfusate reservoir, mmol/liter mmol/liter in which the perfusate was continuously ClHCOzmmoI/liter stirred and equilibrated with a gas mixmmd/liter 5042ture of 5% COZ and 95% 0, to maintain HPOPmmd/liter a constant pH. The perfusate was drawn mmol/liter Hap04 from this reservoir by a pump (WatsonMannitol 107 mmol/liter Carbenicillin 100 mg/liter Marlow MHRE 100) and passed through Human albumin 40 g/liter two 0.2%pm filters in paralfel (Acropor, 147 mm, Gelman Instruments), a heat excells has not been proven to be necessary change consisting of a bloodwarming bag ( Fenwal Laboratories) sandwiched be(15). Accordingly this study was an attween two cooled copper plates, an.d a tempt to look into the distribution of bubble trap to a T-piece can&a inserted glycerol in various parts of the kidney as a function of the perfusion procedure, by in the renal ,artery. The heat exchanger and measuring the freezing point depression of the kidney were situated in a thermally insulated encIosure. Temperatures in the the renal tissue at different stages of glycerolization to a maximum perfusate con- perfusate immediately before entering the centration of 3 M and subsequent de- kidney and in the insulated enclosure were continuously recorded ,and the perfusate glycerolization. temperature was kept constant at +lO”C. The kidney was supported by a stainlessMATERIALS AND METHODS steel cradle over a graduated reservoir Perfusion Fluids into which the venous effluent drained, so The composition of the basic perfusate that perfusate flow rate could be measured used as a vehicle for gIycero1 (HAP-Z) is at IO-min intervals by stopping the pump shown in Table 1. The electrolyte com- which returned the effluent to the central position and mannitof content was identical reservoir. The perfusion pressure was conwith that used in previous experiments (7), tinuously measured in the arterial cannula whereas the colloid was changed to human with a pressure transducer (Statham P 23 serum albumin (KABX), 40 g/liter. A Db) and the flow ,rate was adjusted to yield stock solution containing salts and man- a constant pressure of 40 mm Hg by the nitol was stored in the frozen state at means of .a pressure-flow feedback unit -2O”‘C ,and was filtered through a O-22-pm (19). A by-pass with a constant resistance Acropor filter ( Gelm.an Instruments >. AI- to flow was inserted between a point just bumin was added in the form of a 20% proximal to the ,arterial can&a and the solution immediately before use. central reservoir. This ‘diverted a part of the flow .directIy to the reservoir to ensure A perfusate containing 4.8 M glycerol was prepared by ,dissoIving the salts of a reasonably high flow through the circuit the stock solution in a minimum volume even when the vascular resistance in the kidney was high. Perfusate samples for of water to which 442 g/liter of glycerol was added, and the solution was diluted to glycerol estimations were obtained from the final composition with distilIed water. this by-pass. Mgz+ CW+
mmullliter
304
IB A. JACOBSEN
FIG. 1. Glycerolization circuit: P1, P2, Pa, and P4, perfusate pumps; CDR, reservoir for highglycerol perfusate or glycerol-free perfusate; TTF, Trendtrak Programmer; MR, centrltl perfusate reservoir; F, filter; BE, heat exchanger; BT, bubble trap; K, kidney; FM, flow-measuring reservoir; PT, pressure transducer; PM, pressure monitor; CPC, constant pressure corltrol unit; S, sample point.
The concentrated glycerol solution or the gIycerol-free perfusate was added to the central reservoir by a pump (WatsonMarlow MHRE ZOO} controlled by a Trendtrak Programmer (Leeds and Northrup) on which the program for volumes to be added per unit time to provide the desired variation of glycerol concentration in the central reservoir had been drawn with India ink. The volume of the central reservoir was 70 ml and that of the kidney circuit was 330 ml, The tota volume was kept constant at: 400 ml by withdrawing surplus fluid from the reservoir by a pump.
The changes in perfusate glycerol concentration were programmed to 0.025 M/ min during glycerolization to a maximum of 3 M. This concentration was kept constant for a ZO-min period of equilibration. Deglycerolization was performed at the same rate to a concentration of 1 M, after which the rate was decreased to 0.016 M/ min until the minimum concentration of approximately 0.1 M was reached. Rates of
addition of concentrated glycerol solution or glycerol-free perfusate were calculated from the following approximated equations: glycerolization
:
z = s+
v2(1 - e-a-tlu*) -M c
deglycerolization:
x = M
t
e*ti
“l&X - cat
where x = flow rate (milliliters per minchange ute), c = rate of concentration (molar per minute ), z)~= volume of the perfusate reservoir (milhliters ) , v2 = the total volume of the kidney circuit minus u1 ( milliliters ) , a = flowrate in the kidney circuit (milliliters per minute), M = glycerol concentration in the high-glycerol perfusate (molar), f = perfusion time (minutes), o = v + o2 and M,,, = maximum glycerol concentration in the perfusate. Renal vascular resistance during perfusion was standardized to a 10 g of kidney and calculated from the expression:
GLYCEROLIZATION
r---j ra
r-d L, I I
FIG. 2. Differential thermal analysis apparatus: S, tissue sample; R, reference; T, temperature reference; AT, differential temperature; TF, Thermos flask.
where P = perfusion pressure (millimeters of Hg), W = initial kidney weight (grams), 7 = perfusate viscosity (centipoise), and F = perfusate flow (milliliters per minute). Glycerol concentrations were measured at IO-min intervals by refractometry at +3O”C (Abbk refractometer). Perfusate viscosity as a function of glycerol concentration was measured at +lO”C using an Ubbelohde viscometer, and the observed values fitted, in the concentrations used, the equation
where x = glycerol concentration (molar), Thus perfusate viscosity could be calculated throughout the perfusion, Experimental Procedures
French lop-eared male rabbits weighing 2.5-3.5 kg were anaesthetized with Hypnorm (fentanyl citrate, 0.315 mg/ml, + fluanisone, 10 mg/ml) and BrietaI (sodium methohexital) as previously described (8). Before removal of the left kidney for perfusion the animal was pretreated with 5 mg of furosemide, 10 ml of 10% mannitol, and
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4 mg/kg body weight of chlorpromazine intravenousIy, During the time of perfusion the abdomen was closed and a rapid recovery was produced by injection of nalorphine. After perfusion the right kidney was removed, and the perfused kidney was implanted by anastomosing the renal vessels to the aorta and the inferior vena cava, respectively. The ureter was implanted into the bladder, Postoperatively daily blood samples were collected for serum creatinine estimations, and weight loss was replaced with isotonic saline and dextrose intravenously (for details see 8). Endogenous creatinine clearance was measured 21 days after transplantation, As soon as possible after clamping the renal artery (average warm ischaemia was 3 min) the kidney was flushed at a controlled pressure of 60 mm Bg with 100 ml of WF 5 PD wash out solution at +4X (22), Twelve to fourteen minutes after clamping of the artery the kidney was connected to the perfusion circuit, and perfusion with HAP-2 was started. The program for glycerolization was started after 15 min of perfusion, and the glycerol concentration was increased at a rate of 0.025 M/min to a maximum of 3 M. Twenty minutes of perfusion was allowed at this concentration for equilibration of glycerol with the kidney tissue. Deglycerolization was initiated at a rate of 0.025 M/min until a perfusate concentration of approximately 1M was reached and was then completed at a rate of 0.016 M/min. The perfusion was continued after deglycerolization for 6090 min to allow the vascular resistance to return to 2-3 times the initial value. As controls, six kidneys were autotransplanted immediately after initial wash out, and three were grafted after 4 hr of perfusion in the glycerolization circuit with the basic HAP-2 perfusate. III both series postoperative serum creatinines and the endogenous creatinine clearance on Day 21 were measured.
306
II3 A. JACOBSEN
FIG. 3. Reproduction of a differential thermal recording. SampIe: Ureter glycerolized to 3 M. The arrows indicate the end of the endotherm and the corresponding alxolute tamperature (the small offset is due to the arrangement of recorder pens).
Six kidneys were glycerolized in the described manner to a maximum of 3 M and subsequently deglycerolized prior to transplantation. Function was estimated as in the control groups, Freezing Point Depression Glycerol
and
Distribution
Melting points of the kidney tissue were measured in samples of renal cortex, renal medulla, and the ureter after perfusion with the HAP-2 perfusate only, after glycerohation to approximately 3 M, and after deglycerolization from 3 M. Melting points of the corresponding perfusates were also determined. All melting points were measured in duplicates by differential therma analysis ( DTA ). The apparatus for thermal analysis is shown in Fig. 2. It consisted of a cylindrical brass block in which three holes were drilled parallel to the axis. The tissue sampie (200 mg) and reference samples ( 200 ~1) were inserted in these holes in thin-
walled cylindrical copper tubes closed with an insulating lid through which copperconstantan thermocouples passed to the centre of the samples. Frozen distilled water was used as a reference and for measuring the absolute temperature of the system. Melting ice in a Thermos flask was used as a reference for the absolute temperature. The differential temperature output was amplified and fed into a Kipp and Zonen BD9 two-channel recorder (full-scale deflection, I00 pV), and the absolute temperature output was recorded on the same instrument (full-scale deflection, 500 PV). The brass block was cooled to -35°C after which the sample and references were inserted into the holes and covered by an insulating lid. As a result the tissue sample was cooled at a rate of more than lOO”C/ min in the range of phase transition to the block temperature. The system was then left on the bench at room temperature, which caused the block temperature to rise
CLYCEROLIZATION
OF RABBIT TABLE
Control
Survival
616
Peak serum-creatinine (pmol/liter)
400
Creatinine clearance (ml/min)
f 234”
Kidneys
Control perfused
Glyorrolised
Glycerolised to +&I~ a
“02.&2th
313 283 i 3
6.9 & 0.7 (4)d
307
2
Function of Autotransplanted trrtnaphlts
KIDNEYS
7.0 f 1.8 (3)
.!ifi/6 721
5/B
f 133
5.8 AZ (4)
610
0.4
f 257
6.2 zk 2.49 (4)
a Previomly published in Ref. 7. * One rabbit died because of cerebral damage but with good graft function. EResults are given as mean f SD. d Values in parentheses are numbers of observations.
at an approximately constant rate of 06 0,7”C/min. The melting point was read as the end of the endotherm as shown in Fig. 3. The kidneys for DTA were dissected immediately after perfusion so that samples of the renal cortex, medulla, and the ureter were isolated. The tissue samples were subsequently cut into smaI1 pieces (approximately 1 mm3 each) using a scalpel and placed in the copper tube before freezing. One kidney was perfused for 100 min with the HAP-2 perfusate before DTA. Three kidneys were glycerolized to 3 M according to the described procedure including 20 min of equilibration before DTA. In two of these kidneys melting
5
ICI
points were determined immediately after perfusion, and in the third DTA was performed immediately as well as 10 hr after termination of glycerolization. In two experiments kidneys were glycerolized to 3 M and subsequently deglycerolized, using the same technique as in the transplantation experiments, before being subjected to DTA. In all experiments samples of the fina perfusate were collected for melting point determination. RESULTS
Transplantation Experiments
Post-transplantation function of controls and perfused kidneys is shown in Table 2
15
20
TIMEINDAYS
s 2
FIG. 4. Postoperative with 3 M glycerol,
serum creatinines
in six raGits
transplanted
with kidneys
perfused
IB
308
A. JACOBSEN
FIG. 5. Perfusion characteristics of kidneys glyycerolized The dashed lines show renal vascular resistance and the concentration.
with the previously obtained results of perfusions with a different colloid and different glycerolization rates (7) for comparison. One kidney glycerolized to 3 M with the HAP-Z per f usate did not function because of total cortical necrosis, and another rabbit died on Day 3 because of severe cerebral damage due to the anaesthetic but with good graft function. Peak creatinines were higher in the glyceroltreated group than in controls, but the ultimate function measured as creatinine clearance after 3 weeks was not different. Postoperative serum creatinines in rabbits transplanted with glycerol treated grafts are shown in Fig. 4, Perfusion characteristics of kidneys perfused with 3 M glycerol are shown in Fig. 5 together with the simultaneous variations of glycerol concentration in the parfusate. Vascular resistance remained low and constant until toward the end of dcglycerolization, when it temporarily increased to a mean of 15 times the initial value, after which it rapidly decreased to some 3 times the initial resistance. Kidney weight increased during perfusion to 143 i- 7% (mean k SD) of the initial weight. GE~Jc~TuZ Distribution
Experiments
-4 typical example of the DTA recordings obtained is reproduced in Fig. 3. An exo-
to and deglycerolized from 3 M, solid lines show perfusate glycerol
therm produced by the freezing of the tissue sample is seen. The arrows indicate the end of the melting endotherm and the corresponding absolute temperature of the sample. Continuation of the recording beyond a sample temperature of 0°C did not revea1 additional endotherms. Melting points measured in different parts of the kidney and in the corresponding perfusion fluids are shown in Fig, 6. The melting point of the basic HAP-2 pcrfusate was -O.Z”C and not different from the vaIues obtained in a kidney perfused with this perfusate alone. Also, meIting temperatures in kidneys perfused with 3.10 and 3.12 M glycerol were almost identical with the melting points of the corresponding perfusates ( -9.5 and -9.9”C). Similarly, kidneys deglycerolized from concentrations just over 3 M had melting points very close to those of the final perfusates. One kidney glycerolized to 3.24 M had a melting point of -10.8”C immediateXy after perfusion as compared with - IO.0 oC in the corresponding perfusate. The melting temperature of a sample of this kidney stored at +lO”C for 10 hr was -10.7”C and thus not different from the value measured immediately after perfusion. In summary the resuIts of the differential thermal analysis showed that the freezing points of various parts of the kidneys
GLYCEROLIZATION
OF
RABBIT
KIDNEYS
0
309
PEWSAtE
q RENAL CORlEX 0 RENAL MEDULLA q URETER
PERFUSATE o GLYCEROL CDNCENTRATtW [M@LA@
a
a
0
FIG, 6. Melting points of perfusates and corresponding - glycerolization and degIycerolization. were identical with those of the corresponding perfusates and did not change with additional time for glycerol equiIibration. DISCUSSION
One of the major problems in cryopreservatian of whole organs is the distribution of high concentrations of a cryoprotective agent. Attempts to achieve this by simple vascular perfusion have demonstrated damaging osmotic effects of even short exposures to modest concentrations (4-6, 20) resulting in rising vascular resistance and serious impairment of circulation after transplantation. Experiments undertaken by Pegg and Wusteman (20) have shown however that these osmotic effects can be greatly modified by a gradual addition of cryoprotectant to the perfusate and by removing it in the same manner before perfusion with isotonic media [e.g., blood). Also the incIusion of a slowIy penetrating solute such as mannitol in the perfusate proved to be beneficial. Using these principles, perfusion of rabbit kidneys with maximum concentrations of 4 M glycerol has been possible with only short-lasting, reversible impairment of function after transplantation (7). A major diversion from optima1
0
kidney tissue at different
stages of
perfusion characteristics in these experiments was a dramatic rise in vascular resistance during the last third of the deglycerolization phase. As such changes in resistance to flow couId affect distribution of the perfusate and thereby the removal of glycerol, an attempt was made in the present study to diminish the increase in vascular resistance. This was done by increasing the coIIoid osmotic effect of the perfusate by repIacing 1.7570 Haemaccel with 470 human serum albumin and by decreasing the rate of deglycerolization during the period of high resistance. The observations shown in Fig. 5 and Table 2 demonstrate that no significant beneficial effect was obtained by these measures; only the organ weight gain during perfusion, 43% as opposed to 64% was different (p < 0.01, rank sum test), and in fact post-transplant function was certainly not improved. The previous experiments (7) demonstrated that rabbit kidneys could survive perfusion with high concentrations of glycerol, but the important question concerning penetration and distribution of the cryoprotectant remained unanswered. The exact mechanisms by which compounds like glycerol exert their protective effect during freezing is not completely understood.
310
IB A.
JACOBSEN
Lovelock ( 10) has argued that penetration of glycerol into the cells is necessary for cryoprotection, but: the evidence on which this conclusion is based is equivocal, and in fact successfu1 freezing has been achieved in the presence of nonpenetrating agents such as PVP, sucrose, and HES (I, 9, 13) and after very short exposures to glycerol ( 14). The same considerations are likely to apply in the case of whole organs, but it is certain that penetration of the capillary membrane and diffusion through the extracellular space to establish contact with all cells is necessary for the protective effect of glycerol. The variations in melting point as a function of glycerol concentration in the perfusate indicate that this cryoprotectant is completely distributed throughout and equilibrated with the water phase of the organs even in the whole length of the ureter, which is essential for Iater freezing experiments. Equilibration of glycerol limited to the vascular and extracellular spaces would give the observed results, if the intracellular phase was not frozen during thermal analysis, or if the melting point of intracellular fluid was higher due to lack of glycerol. However, the cooling rate of more than lOO”C/min in the range of phase transition makes intracellular supercooling highly improbable, and, if intracellular ice had melted at a higher temperature than the perfusate and extracellular phase, an additional endotherm would have been expected. Furthermore, if intracelluIar equilibration with glycerol had not been complete, a higher melting point would have been observed after storage at +lO”C because of further diffusion over the ceI1 membrane; consequently the experimental data presented here indicate complete distribution in and removal from the total water of the kidney when using the described procedure. The present melting points of perfusates containing glycerol measured by DTA are in good agreement with the values found
by Luyet and Rasmussen ( 11) and Shepard et al. (21) using the same method if the effect of the additional solutes present in the HAP-2 perfusate is taken into consideration. The finding of complete equilibration corroborates the findings by Brada and Schloerb (3) who found complete equilibration of [‘*Cl glycerol concentrations up to 1.5 M with the total water phase of canine kidneys within 30 min (temperature is not stated). Beisang et al. (2) and McCollough et aL (16) also found complete equilibration after 10 min when perfusing canine kidneys with glycerol at +4 and +2O”C, respectively. However, the method by which these results were obtained, namely, measurements of A-V differences of cryoprotectant concentrations in the perfusate, is not entirely convincing. Pegg (18) perfused rabbit kidneys with 2 M glycerol at +5 and +37”C for 2 hr and found compIete equilibration at the higher temperature but only 73% equilibration with the kidney water at 5°C. The result of the present experiments, where glycerolization was performed at an intermediate temperature (+lO’C) and over a longer period of time ( 3 hr ) , does not necessarily disagree with the findings at +5”C. CompIete distribution and removal of 3 M glycerol gives theoretically two new possibilities for enhancement of renal preservation, nameIy, further suppression of tissue metabolism by cooling to -9°C without freezing and long-term storage by freezing at slow cooling rates. SUMMARY
A simple perfusion circuit for gradual glycerolization and deglycerolization of rabbit kidneys is described; it has been used to study vascular resistance and glycerol distribution during perfusion at +lO”C with a perfusate of extracellular composition.
GLYCEROLIZATION
An attempt was made to diminish the dramatic rise in vascuIar resistance during deglycerolization of rabbit kidneys, seen in previous experiments, by increasing the perfusate colloid osmotic pressure and decreasing the rate of change of cryoprotectaut concentration during the last third of removal. These modifications of the method did not improve perfusion characteristics or post-transplant function, Melting points of perfusates and different parts of the kidney were determined by differential thermal anaIysis before glycerolization, after glycerolization to 3 M and after subsequent deglycerolization. The variations of tissue melting temperatures were found to be identical with variations in the perfusate, thus indicating a complete distribution and removal of cryoprotective concentrations of glycerol. REFERENCES 1. Ashwood-Smith, M. J., Warby, C., Cnnnor, K. W., and Becher, G. Low-temperature preservation of mammalian ce1l.s in tissue cultures with PVP, Dextrans and HES. Cyobi&ogy 9, 441-449 ( 1972). 2. Beisang, A. A., Dietzman, R. H., Mayo, C. H., Graham, E. F., and Lillehei, R. C. Damage during perfusion procedures used to prepare whole organs for freezing. Cryobiology 5, 273-277 ( 1969 ) _ 3. Brada, D. R., and Schberb, P. R. Dynamics of glycerol addition to the kidney. Szlrg. Gynecol. mstet. 121, 1004-1008 ( 1965). 4. EgdahI, R. H., and Harris, R. In uivo tolerance of canine kidneys to glycerol perfusion. Trunsplun~. Bull. 6, 110-112 ( 1959). 5, Halasz, N. A., Seifert, N. L., and Orlofi, M. J. Whole organ preservation. I. Organ perfusion studies, Surgery 60, 368-372 (1966). 6. Jacobsen, I. A., Kemp, E., and Starklint, H. Glycerol used as a cryoprotectant in subzero preservation of rabbit kidneys. C~Qbiology 12, 123-129 ( 1975), 7. Jacobsen, I. A., Pegg, D. E., Wusteman, M. C., and Robinson, S. M. Transplantation of rabbit kidneys perfused with glycerol solutions at 10°C. Cryoh~ol~gy 15, 18-26 (1978). 8. Jacobsen, I. A. Kidney transplantation in the rabbit. Labaratvry Animals, in press.
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9. Leibo, S. P., Farrant, J., Mazur, P., Hanna, M. G., and Smith, L. H. Effects of frt-zing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of PVP, sucrose or glycerol. Cqobiology 6, 315332 ( 1970). 10. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 11, Z&36 (1953). Il. Luyet, B., and Rasmussen, D. Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of glycerol, ethylene glycol, sucrose and glucose. Biodynamics 10, l67-191 (1988). 12. Mazur, P. Causes of injury in frozen and thawed cells. Fad. PPOC. 24 (suppl. 15), 175-182 ( 1965 ). 13. Mazur, P., Farrant, J., Leibo, S. P., and Chu, E. H. Y. Survival of hamster tissue culture cells after freezing and thawing. Cyohiology 6, 1-9 (1969). 14. Mazur, P., Miller, R. H., and Leibo, S. P. Survival of frozen-thawed bovine red cells as a function of the permeation of glycerol and sucrose. J. Membrane Bid. 15, 137158 ( 1974). 15. Mazur, P,, and Miller, R. H. PermeahiIity of the human erythrocyte to glycerol in 1 and 2 M solutions at 0 and 20°C. Cryobiology 13, 507-522 ( 1976). 16. McCollough, W. B., Jacobs, J. R., Miller, S. H., and Halasz, N. A. The effect of hyperbaria on the glycerolization of the canine kidney. Cryohinlogy 6, 542-545 ( 1970). 17. Meryman, H. T. Review of biological freezing. ha “Cryobiology” {H. T. Meryman, Ed), pp. I-114. Academic Press, New York, 1966. 18. Pegg, D. E. Perfusion of rabbit kidneys with cryoprotective agents. CTyobioZogy 9, 411419 (1972). 19. Pegg, D. E., and Green, C. J, Renal prcservation by hypothermic perfusion. Cryobiology lo, 56-66 (1973). 20. Pegg, D. E., and Wusteman, M. C. Perfusion of rabbit kidneys with glycerol solutions at 5°C. Cryobiology 14, 168-178 ( 1977). 21. Shepard, M. L., Goldston, C. S., and Cocko, F. H. The I&O-NaCl-glycerol phase diagram and its application in cyrobiology. Cryobiology 13, 9-23 (1976). 22. Wusteman, M. C., Jacobsen, I. A., and Pegg, D. E. A new solution for initial perfusion of kidneys. Stand. J. UroE. NephroE., in press.