Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~2~9
1 This invention relates to a perfusate for preserv-
ing an organ to be transplanted and preserving method
by the use of the same and, more particularly, to a method
for preserving an organ to be transplanted by using as
perfusate a mixture comprising a modified Ringer's solution,
used as basal salt solution, albumin and a perfluorocarbon
emulsion.
In recent years, the technical progress in
preserving an organ for transplantation is remarkable;
especially the progress in the technique for preserving
kidneys has ever promoted the propagation of cadaveric
kidney transplantation. Such a progress is much indebted
to the improvement of perfusate. For instance, the clinical
methods for preserving kidneys for transplantation are
broadly classified into two categories, the one being
the hypothermic storage (by immersion) [Lancet, 2, 1219-
1222 (1969)] and the other the hypothermic continuous
perfusion CLancet, 2, 536 (1967)]. In actual practice,
the former is used in short period preservation and the
latter in long period preservation. With further prepaga-
tion of cadaveric kidney transplantation, the long period
preservation, that is, perfusional preservation will
gain in importance.
In cadaveric kidney transplantation, since
most of the kidneys are donated by the individuals who
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1 ran into accidents, the necessary steps to be taken between
the time of emergence of a cadaveric kidney and the time
of transplantation include histcompatibility testings
between the cadaveric kidney and the patient, selection
of a competent recipient, transportion of the donated
kidney, and preparations for the transplantation. For
these reasons, it is necessary to preserve the donated
kidney as long as possible. In fact, as a part of studies
on the cadaveric kidney transplantation, many attempts
have heretofore been made to preserve the cadaveric
kidney for a long period of time. One of the primary
objects of these attempts is the development and improve-
ment of perfusates.
The ever spreading clinical use of the hypothermic
continuous perfusion originates from the research work
of Belzer et`al. [Lancet 536 (1967)]. They succeeded in
preserving a canine kidney for 72 hours by employing as
the perfusate a plasma freed from fibrinogen as far as
possible prepared by freezing and melting a cryoprecipitated
plasma. Since the publication of their report, chiefly
the cryoprecipitated plasma has been employed as a perfusate
for kldneys.
In 1974, Toledo-Pereyra et al. [Surg, Gynecol.
Obstet, 138, 901-905 (1974)] developed a new perfusate
of silica gel-treated cryoprecipitated plasma which is
free from ~-lipoprotein of fibrinogen still remained in
the conventional cryoprecipitated plasma, and in which
the level of insoluble fat fraction, i.e. triglycerids,
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1 has been reduced to one-third. The conventional cry-
oprecipitated plasma involved a great risk of emboli
formation during the perfusion, which is caused by the
residual fibrinogen, lipoprotein or neutral fat globules,
whereas the risk is greatly reduced with the cryopre-
cipitated plasma treated with silica gel, which has been
developed by Joledo-Pereyla et al. Such cryoprecipitated
plasmas, however, still have several problems. Firstly,
the preparative procedure has not been perfectly established
and the composition of the prepared plasma tends to
fluctuate, making it difficult to obtain preparations
of constant protein content and constant electrolyte
composition. Secondly, there exists still some fear of
fat emboli formation during perfusion due to incomplete
removal of the insoluble fat fraction. In order to reduce
the risk, it is necessary to use an expensive membrane
oxygenator. Thirdly, an important and essential problem
is the lack of an adequate inactivation treatment against
hepatitis virus, leading to the fear of possible infection
Nith hepatitis virus. Although the cryoprecipitated
plasma is an excellent perfusate for the preservation
of kidney, yet it has several defects as mentioned above.
It was reported that in order to overcome the
above difficulties associated with the cryoprecipitated
plasma, an attempt was made to use an albumin solution
as the perfusate and achieved a success in animal experi-
ment. Grundman et al [Transpl., 17, 299-305 (1974)]
perfused a canine kidney with a 6% albumin solution and
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1 successfully preserved the kidney for 96 hours. Their
perfusate was a Krebs Ringer's solution containing 6~ (W/V)
of human albumin. The canine kidney perfused for 96
hours at a low temperature was autotransplanted and
the graft survival was over 80%, indicating that the
albumin solution is an excellent perfusate comparable
favorably to the cryoprecipitated plasma.
The long-period preservation of a kidney
requires a perfusate having a certain colloid osmotic
pressure at low temperatures as well as an adequate
electrolyte composition and, in addition, a certain amount
of oxygen. When preserved at low temperatures, the kidney
consumes much less oxygen than when at room temperature,
yet requires a certain amount of oxygen in order to main-
tain its function. According to a classic study of Levy[American Journal Physiology 197, 1111-1114, (1959)],
the oxygen consumption of a kidney at 8 to 10C is
about 3 ~Q/g/minute which correspond to about 20% of
the consumption at room temperature. Accordingly, in
preserving a kidney it is a general practice to perfuse
it ~ith a perfusate while being oxygenated.
A saturated perfluoro organic compound (herein-
after referred to as PFC) is a liquid capable of dissolving
a considerable volume of oxygen and functions as an oxygen
carrier when used in the form of emulsion. Developmental
researches are in progress on an artificial blood which
utilizes the above-noted characteristic property of the
PFC emulsion. The PFC emulsion well functions as an oxygen
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1 carrier either in vivo or in vitro (in an excorporeal
system such as a perfusion system). Such a property
suggests that a PFC emulsion can be utilized to advantage
as an oxygen carrier in kidney preservation. Beside
being a good solvent for oxygen, the selected PFC is
biologically inert and presents no problem of direct
toxicity to ~issues. Therefore, when it is used as
perfusate in preserving a kidney, no problems concerning
to~icity will be aroused.
The studies on the preservation of a kidney
by the use of a PFC emulsion started quite recently and
originate from the work of Nakaya et al. (Proceedings of
Symposium on Perfluorochemical Artificial Blood, Kyoto
1976, 187-201). They examined the viability of kidney
in a biological way by perfusing a rabbit kidney with
FC-43 emulsion (a perfluorotributylamine emulsion) for
9 hours at room temperature. As compared with a control
group perfused with Ringer's solution, the kidney perfused
with the FC~43 emulsion well retained the mitochondrion
function and higher activity levels of glycolytic and
gluconeogenetic key enzymes, indicating that the oxygen
transportion by FC-43 exerts an effective influence on
the retention of functions by the perfused kidney. The
above-noted authors, however, directed their research
efforts mainly to the biological aspect, but not to the
transplantation of a preserved kidney. It is Bercowitz
who reported the results of transplantation of a canine
:: kidney preserved by perfusion containing a PFC emulsion
1 [J. Surg. Res., 20, 595-600 (1976)]. He perfused a canine
kidney for 24 hours at a low temperature with a P~C
emulsion containing albumin and a cryoprecipitated plasma.
The preserved kidney was then autotransplanted and the
graft survival rate was examined. The rate was found
to be 100% in all five cases, whereas the rate was 62%
in five control cases out of eight where the perfusa~e
contained no FC-43 emulsion, indicating significantly the
advantage of the FC-43 system.
The two reports, cited above, showed that the
PFC emulsion is an effective perfusate for preserving
a kidney at both low temperatures and room temperature.
However, the preservation periods described in both reports
are not sufficiently long to ascertain the practicability
of the preserving methods. Particularly, the FC-43 emulsion
containing albumin is not sufficiently stable to produce
a normalized preparation.
Under the aforementioned circumstances, the
present inventors conducted studies on the PFC-43 contain-
ing perfusate for the long-period preservation of an
organ, particularly kidney, for transplantation. Based
on the results of such studies, the present invention has
been accomplished.
The object of this invention is to provide
a PFC perfusate and a method for the effective long-period
preservation of organs for transplantation.
According to this invention, there are provided
a perfusate for preserving an organ for transplantation,
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1 comprising a modified Ringer's solution characterized
by containing potassium ion in a concentration of 8 to 20
meq/liter, albumin dissolved therein, and an emulsified
liquid saturated perfluorocarbon compound and a method
for the perfusional preservation of an organ for trans-
plantation by the use of said perfusate.
The basal Ringer's solution used in the modified
Ringer's solution of this invention is subject to no
special restriction but can be a customary one or that
modified by Locke, Tyrode, Earle, Hanks or Krebs. The
modification according to this invention consists in that
the potassium ion concentration is increased from the
ordinary level of 4 to 6 meq/liter to 8 to 20, preferably
8 to 15 meq/liter. Such an increased concentration was
adopted in order to approximate more closely the potassium
ion level in the extracellular fluid. By the choice of
such a concentration of potassium ion, it becomes possible
to bring about a leaping increase in the graft survival
rate of an organ, particularly kidney, preserved for
a long period by perfusion. The modified Ringer's solution
of this invention has an osmolarity of 290 to 300 m Osm/
liter and a pH of 7.1 to 7.7 at 20C. In Table 1 are
shown, for reference, salt compositions of some typical
Ringer's solutions modified according to this invention,
together with that of a normal plasma which is a prototype
of an ordinary Ringer's solution.
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Table 1. Examples of electrolyte composition
of modified Ringer's solution.
Unit: meq/liter
Modified Ringer's solution Normal
according to this invention plasma
1 2 _- 3
Na 112 142 116 142
~ 11 8 10 5
Ca 7 2 6 2
Cl- 118 103 15 103
HCO3 3 27 10 27
HPO4 2 2 101 2
Glucose33 139
Lactose 5 5
1 The perfusion fluid for preserving an organ
according to this invention is prepared by mixing together
an emulsion of PFC in the modified Ringer's solution,
albumin and the modified Ringer's solut~ion so that the
concentrations of PFC and albumin may assume respectively
the predetermined values.
The PFC emulsion used in the invention is
prepared from a liquid perfluorocarbon compound capable
of absorbing oxygen by a known method disclosed in,
for example, US Patent 3,958,014, 3,911,138, 3,962,439,
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1 or West German Patent Application DT-OS 2530586. These
patent specifications discloses that a stable emulsion
suitable for use as an artificial blood can be prepared
by emulsifying a perfluorocarbon compound with a phos-
pholipid as emulsifier (U.S. 3,958,014), a phospholipidand a C8 22 fatty acid as auxiliary agent (US Patent
3,962,439); or by using a polyoxyethylene-polyoxypropylene
copolymer (molecular weight 2,000 to 20,000) as nonionic
emulsifier (US Patent 3,911,138); or by using a poly-
oxyethylene alkyl ether or polyoxyalkyl allyl ether orcojointly using the above-noted fatty acids, nonionic
emulsifiers and the above-noted auxiliary emulsifying agent
(West German Patent Application DT-OS 2630589).
The perfluorocarbon compounds to be emulsified
are those not giving such adverse effects to 'che organs
or tissues, and are saturated perfluorocarbon compounds
preferably having as a whole 9 to 12 carbon atoms some
or whole of which form at least one saturated alicyclic
ring, heterocyclic ring together with hetero nitrogen
atom and/or oxygen atom, aliphatic tertiary amine together
with ni'rogen atom or aliphatic ether together with oxygen
`atom or atoms.
The first group of the perfluorocarbon compounds
used in the invention is a perfluoroalkane, perfluoro-
cycloalkane or perfluoro(alkylcycloalkane) which includes,for example, perfluoro(Cg 12-alkanes) such as perfluoro-
decane, and perfluorododecane; perfluoro(C3_5-all~ylcyclo-
hexanes) such as perfluoro(methylpropylcyclohexanes),
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1 perfluoro(butylcyclohexanes), perfluoro(trimethylcyclo-
hexanes), perfluoro(ethylpropylcyclohexanes) and
perfluoro(pentylcyclohexanes); perfluorodecalin,.
perfluoro(methyldecalins) and perfluoro(dimethyldecalins).
The second group is a perfluoro(alkylsaturated
heterocyclic compound) which includes, for example,
perfluoro(alkyltetrahydropyrans) such as perfluoro(butyl-
tetrahydropyrans), perfluoro(pentyltetrahydropyrans) and
perfluoro(hexyltetrahydropyrans); perfluoroalkyltetra-
hydrofurans) such as perfluoro(pentyltetrahydrofurans),
perfluoro(hexyltetrahydrofurans) and perfluor~(heptyl-
tetrahydrofurans); perfluoro(N-alkylpiperidines) such
as perfluoro(N-pentylpiperidines), perfluoro(N-hexyl-
piperidines) and perfluoro(N-butylpiperidine); and
perfluoro(N-alkylmorpholines) such as perfluoro(N-
pentylmorpholines), perfluoro(N-hexylmorpholines) and
perfluoro(N-heptylmorpholines).
The third group is a perfluoro(tert-amine) which
includes, for example~ perfluoro(tributylamine), perfluoro-
(diethylhexylamine), perfluoro(dipropylbutylamine) andperfluoro(diethylcyclohexylamine); and a perfluoro-
~dioxaalkane), that is, perfluoro(alkylene glycol dialkyl
ether), such as perfluoro(3,8-dioxa-2,9-dimethyldecane)
or perfluoro(tetramethylene glycol diisopropyl ether),
perfluoro(3,7-dioxa-2,8-dimethylnonane) or perfluoro-
(trimethylene glycol diisopropyl ether) and perfluoro-
(4,6-dioxa-5,5-dimethylnonane), or perfluoro(isopropylene
glycol di-n-propyl ether).
-- 10 _
,
1 The thus prepared emulsion of PFC in the modified
Ringer's solution has a particle size of 0.05 to 0.3 ~,
contains 10 to 50% (W/V) of PFC, 2.0 to 5% (W/V) of an
emulsifier and, if necessary, 0.1 to 1.0% (W/V) of
an auxiliary emulsifier, and can be stored in stable state
for a long period.
The present perfusate for preserving an organ
for transplantation is prepared, before using, by mixing
together the above PFC emulsion, a commercial bovine or
human albumine and the aforementioned modified Ringer's
solution so that the resulting fluid may contain 7.5 to
12.5% (W/V) of PFC, 1 to 8% (W/V) of albumin. If necessary,
pharmaceuticals such as procaine hydrochloride, hepar n,
phenoxybenzamine, insulin, dexamethasone ("Decadron"),
methylprednisolone, antlbiotics and urokinase can be
added. The limits of PFC concentration given above were
set up in order to ensure the gra~t survival after
transplantation of the organ preserved by perfusion of
the fluid~ The concentration of albumine within the
above-mentioned limits is suitable for adjusting the
colloid osmotic pressure so that ~he perfusion fluid
may be kept physiologically isotonic.
The preservation of an organ to be transplanted
is carried out in a customary way under oxygenation with
oxygen by means of a customary equipment. For instance,
400 ml of a 25% (W/V) PFC emulsion, 3~0 ml of the modified
Ringer's solution and 240 ml of a 25% (W/V) human albumine
solution in the modified Ringer's solution are mixed
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1 together to make a total of 1 liter. The resulting mixture
is fed to a perfusate reservoir and perfused tnrough the
organ for transplantation, while being oxygenated with
a mixed gas (95-98% oxygen and 5-2% carbon dioxide)
or oxygen alone by means of an oxygenator of the membrane
type or bubbling type. The rate of circulation of the
perfusate is 15 to 50 ml/g kidney/hour and the perfusate
was oxygenated with 95% 2-5% C2 at flow rate of 30 to
1500 ml/min. If necessary, the above-noted pharmaceuticals
can be added to the perfusate, without causing deteriora-
tion of the perfusate. In the perfusional preservation,
it is also possible to recycle, by means of a pulsatile
pump, the perfusate fed to the artery side reservoir of
an organ preservation equipment. In the long-period
preservation, it is desirable to refresh appropriately
the perfusate. The perfusate of this invention can be
used not only in perfusional preservation but also in
the field of bench surgery.
The method of this invention makes it possible
to preserve an organ very safely for a long period until
the time of transplantation. Since the graft survival
rate is very high, the present method will assure commer-
cial success of organ transplantation in the field of
practical medical treatment.
Illustrative examples of the methods of
preparing each component of the present perfusate and
the procedure of preparing the perfusate are described
below. The symbol "% (W/V)" referred to in the
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~%~
1 specifica~ion and claims of this application means the
amount proportion of a material by weight (gram) based
on 100 ml of the resulting liquid.
Preparation Example 1
Preparation of fluorocarbon emulsion:
In 8 liters of a modified Ringer's solution
(described later), was dissolved 300 g of a polyoxyethylene-
polyoxypropylene copolymer (molecular weight 8,350). After
addition of 3 kg of perfluorodecalin, the resulting
mixture was stirred in a mixer to form a coarse emulsion.
The coarse emulsion was fed to the reservoir of a jet-type
emulsifier (Monton-Gaulin type homogenizer) to circulate
the emulsion. Emulsification was carried out at
35 + 5C under a pressure of 200 to 500 kg/cm2. The
concentration of perfluorodecalin in the resulting fine
emulsion ~as 31.3~ (W/V) and the average particle diameter
was 0.09 to 0.1 ~, as measured by the method of centrifugal
sedimentation. After 6 months of storage of the emulsion
at 4C, coalescence of the particles was not noticed and
the average particle diameter remained substantially
unchanged.
Preparation of albumin solution:
A commercial preparation of human serum albumin
was dissolved in a concentration of 25% (W/V) in the
modified Ringer's solution described below.
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1 Preparation of modified Ringer's solution:
A modified Ringer's solution was prepared by
dissolving in distilled water, in terms of g/liter,
6.51 NaCl, 0.4 MgS04, o.8 KCl, 0.24 NaHC03, 0.14 Na2HP04,
6.o glucose, forming a solution of the following electrolyte
composition, in terms of meq/liter: 112 Na , 11 K ,
7 Mg , 118 Cl , 3 HC03 , 2 HP04 , 7 S04 , 33 glucose.
Mixing of component fluids:
A 570 ml portion of the modified Ringer's solution
and 330 ml of the fluorocarbon emulsion were mixed and
thoroughly stirred. The mixture was sterilized by heating
in a sterilizer at 115C for 12 minutes. The sterilized
mixture was mixed with 100 ml of the albumin solution
which had been passed through a bacterial filter. The
resulting mixture was stored in cool place at 1 to 10C
and used at need as a perfusion fluid for preserving an
organ for transplantation.
Preparation Example 2
Preparation of perfluorocarbon emulsion:
In 8 liters of the modified Ringer's solution,
was dissolved 330 g of polyoxyethylene octyl ether having
an average molecular weight of 3,500. To the solution,
were added 40 g of soybean phospholipid and 2 g of
potassium oleate. The mixture was stirred in a mixer to
form a suspension. The suspension was admixed with 3 kg
of perfluoro (tributylamine) and stirred in a mixer to form
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1 a coarse emulsion. The coarse emulsion was further
emulsified in the same manner as in Example 1. The
fine emulsion thus obtained was divided into vails and
sterilized by heating in a rotary sterilizer at 115C
for 12 minutes. The concentration of the perfluorocarbon
in the sterized emulsion was 29.7% (W/V). On storage
at 4C for 6 months, the emulsion showed no coalescence
of the particles.
Mixing of component fluids:
A 550 ml portion of the modified Ringer's solution
used in Preparation Example 1, 100 ml of the albumin
solution and 350 ml of the fluorocarbon emulsion obtained
above were mixed. The mixed fluid was stored in cool
place at 1 to 10C and used, when needed, as a perfusion
fluid for preserving an organ to be transplanted.
Preparation Example 3
The procedures in Preparation Example 1 were
repeated, except that perfluoromethylpropylcyclohexane
was used in place of the perfluorodecalin. The perfusion
fluid obtained was similar in properties to that obtained
in Preparation Example 1.
Preparation Example 4
The procedures in Preparation Example 1 were
repeated, except that each of the perfluorobutylcyclohexane,
perfluorotrimethylcyclohexane, perfluoroethylpropyl-
cyclohexane, perfluoromethyldecalin, perfluorohexyl
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1 tetrahydropyran, perfluoropentyltetrahydrofuran, perfluoro-
heptyltetrahydrofuran and perfluorodecane was used in
place of the perfluorodecalin. The perfusion fluids
obtained were similar to that obtained in Preparation
Example 1.
Preparation Example 5
The procedures in Preparation Example 1 were
repeated, except that each of the perfluoro N,N-dibutyl-
monomethylamine, perfluoro N,N-diethylpentylamine,
perfluoro N,N-dipropylbutylamine, perfluorotripropylamine,
perfluoro N,N-diethylcyclohexylamine, perfluoro N-pentyl-
piperidine, perfluoro N-hexylpiperidine, perfluoro N-
butylpiperidine, perfluoro N-pentylmorpholine, perfluoro
N-hexylmorpholine, and perfluoro N-heptylmorpholine was
used in place of the perfluorodecalin. The perfusion
fluids obtained were similar to that obtained in Preparation
Example 1.
Preparation Example 6
The procedures of Preparation Example 1 were
repeated, except that perfluorotributylamine was used in
place of the perfluorodecalin. The perfusion fluid obtained
was similar to that obtained in Preparation Example 1.
Preparation Example 7
The procedure of Preparation Example 1 was
repeated, except that a polyoxyethylene-polyoxypropylene
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1 copolymer having a molecular weight of 15,800 was used
in place of the copolymer having a molecular weight of
8,350. The perfusion fluid obtained was similar in
properties to that obtained in Preparation Example 1.
Below are shown examples of comparative experi-
ments to demonstrate by animal experiments the effective-
ness of the perfusional preserving fluids of this invention
in transplantation of the kidney preserved by perfusion.
The survival or death of the graft was determined by
observing the function of the grafted kidney after the
contralateral normal kidney had been removed. In the
tables, the term "early function" means that urination
was observed in two days after the contralateral
nephrectomy; the term "late function" means that no urina-
tion was observed in two days after the contralateralnephrectomy but afterwards the function of the kidney
gradually improved until urination was observed and the
animal under test has survived; the term "necrosis" means
the death of the tissues in localized areas of the
transplanted kidney before the contralateral nephrectomy
(the experiment was discontinued~; and "A.T.N." (acute
tubular necrosis) means that after the contralateral
nephrectomy the animal suffered from anuria, loss of
appetite and frequent vomiting until death due to acute
renal insufflciency.
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1 Experiment Example 1
Optimal PFC concentration in preserving fluid:
It is necessary and essential to determine
the PFC concentration most suitable for perfusion, because
with the increase in PFC concentration of an PFC emulsion,
the oxygen carrying capacity of the emulsion increases,
while, on the other hand, the viscosity increases consider-
ably at low temperatures. In order to determine the
optimal PFC concentration during perfusion, a series of
perfusion fluids with varied PFC concentrations were
prepared using as the basal saline solution a modified
Collin's solution (M.C.S.) [The Lancet, 2, 1219-1222
(1969)], which is said to be the most favorable for
hypothermic stcrage of organs and has an electrolyte
composition resemble to intracellular fluid. Varid amounts
of the fluorocarbon emulsion obtained in Preparation
Example 2 were added to M.C.S. so that the ultimate PFC
concentrations in the perfusion fluids thus obtained may
become 5, 7.5, 10, 12.5 and 15% (W/V). Experiments were
run to preserve canine kidneys for transplantation by
perfusion with the above fluids at 5 to 8C at the perfu-
sion flow rate of 15 to 18 ml/g/hour for 24 hours under
oxygenation with an oxygen-containing gas (95%02, 5%CO2)
at a flow rate of 300 to 500 ml/minute. By using five
dogs per group, the preserved kidneys were autotrans-
planted. On the 7th day after autotransplantation, the
contralateral normal kidney of each dog was excised and
the subsequent survival days of each dog were counted.
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1 When a dog survived for 4 weeks or longer, the graft
survival was assumed to be positive, because in such a
dog, after 2 weeks both BUN (blood urea nitrogen) and
creatinine levels remained normal and urination was
observed. From the data obtained, the optimal PFC
concentration in the perfusate was determined.
The experiment was run in a customary way in
accordance with the following procedural sequence:
Anesthesia: Pentobarbital sodium (30 mg/kg i.v.
injection) Endotracheal administration
of halothane and oxygen
Midline abdominal incision
Surgical isolation of left kidney
Cannulation (ureter~ renal artery and vein)
Initial wash out with cold M.C.S. or cold Ringer's
lactate solution
Connect to the perfusion circuit
Perfusional preservation with oxygenated
perfusate at 5 to 8C for 24 hours
~ or 72 hours
Wash ol lt (M.C.S. or Ringer's lactate solution)
Autotransplantation into femoral fossa
Delayed contralateral nephrectomy
_ ~9 _
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Table 2. Effect of PFC Concentration in
Perfusate on Graft Survival
7 r , ~
Dog No. Perfusates tS~lmvi(dalys) Survivay Remarks
1 Alive Early function
2 _ Necrosis
3 M . C . S . 5 2/ 5 A.T.N.
(Anuria)
4 . Alive Early function
4 A.T.N.(Anuria)
.
6 _ Necrosis
7 Alive Late function
8 5% PFC 5 3/5 A.T.N.(Anuria)
9 Alive Early function
Alive Late functlon
11 Alive Early function
12 Alive Early function
13 in M.C S. Alive 4/5 Early function
14 7 A.T.N.(Anuria)
Alive Early function
..
16 Alive Early function
17 5 A.T.N.(Anuria)
18 10% PFC Alive 4/5 Early function
in M.C.S.
19 Alive Early function
Alive _ Early functlon
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21 Alive Early function
22 Alive Early function
23 in M C S Alive 4/5 Early function
24 5 A.T.N.(Anuria)
Alive Early function
26 8 A.T.N.
(Hematuria)
27 Alive (Hema.)
28 in M C S. 4 A.T.N.(Anuria)
29 Alive 2/5 Early function
(Hema.)
A.T.N.(Anuria)
~ Alive: Survived for longer than 4 weeks.
*~ A.T.N. (Acute tubular necrosis)
1 The results obtained were as shown in Table 2.
As is seen from Table 2, a high rate of graft survival
was obtained at a PFC conce~tration of 7.5 to 12.5% (W/V).
If the PFC concentration exceeds 12.5% (W/V) and reaches
15% (W/V~ 9 hematuria was observed in all cases on
resumption of the blood flow after transplantation.
Two dogs who survived for over 2 weeks showed the sign
of hematuria for several days after contralateral
nephrectomy, then recovered and survived. From the above
- 10 results it was concluded that a perfusion fluid adjusted to
- 21 -
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1 a PFC concentration of 10~ (W/V) is most e~ective for
the perfusional preservation of an excised kidney.
Experiment Example 2
Basal salt solution:
Long-period low temperature perfusional
preservation experiments were carried out in order to
select a suitable basal salt solution. The salt solutions
examined were M.C.S. (described before), Ringer's solution
and modified Ringer's solution (M.R.S.) prepared in
Preparation Example 1. Perfusion fluids for preservation
were prepared by adding the perfluorocarbon emulsion
of Preparatoin Example 2 and the bovine serum albumin to
a salt solution so that the ultimate concentration may
become 10% (W/V) of PFC and 6% (W/V) of albumin. Perfusion
was carried out on canine kidneys (5 per group) at 5 to
8C for 72 hours following the procedure of Experiment
Example 1. After transplantation and resumption of the
blood flow, examinations were made on the movement of
blood through the kidney, color and tone of the kidney,
irination, and symptoms due to contralateral nephrectomy
per~ormed on the 7th day after transplantation. The
perfusate (2,000 ml in total volume~ was not refreshed
during 72 hours of perfusion but recycled at a rate of
15-18 ml/g/hour. Except for the cases where M.R.S. was
used as the basal salt solution, the results of trans-
plantation were very unfavorable and almost all of tne
test animals died due to acute kidney insufficiency in
- 22 -
.
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: : :
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1 10 days after the contralateral nephrectomy. On the
contrary, in the cases where M.R.S. was used as the basal
salt solution, all of the animals recovered uneventfully
without exception.
Experiment Example 3
Effect of albumin concentration in perfusate
on canine kidney:
The persusates used in this experiment were
10% PFC emulsion in modified Ringer's solution, which
was prepared by the use of PFC emulsion prepared in
Preparation Example 6, and contained different concentra-
tions of human albumin. The kidneys were perfused for
72 hours at 5 to 8C under oxygenation with the oxygen-
containing gas used in Experimental Example 1 at a flow
rate of 300 to 500 ml/min and then autotransplanted as
in Experiment Example 1. The perfusion flow rate was
maintained between 15 and 18 ml/g/hour during perfusion.
The contralateral nephrectomy was carried out one week
later after autotransplantation.
The results were as shown in Table 3.
- 23 -
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Table 3
- Concentra- Kidney weight After contralateral
Dog tion of gain during nephrectomy
No. albumin perfusion
(%) (%)Function Survival
1 73.5No functionDied
2 O 49.4No functionDied
3 53.12 days Survived
4 38,42 days Survived
2 52.9No functionDied
6 49.8No functionDied
7 37.6No functionDied
8 4 20.3 Early function Survived
9 41.i Early function Survived
13.7 Early function Survived
11 6 ~ 30.6 Early function Survived
12 34.4 Early function Survivied
_ _
13 11.9 Early function Survived
14 8 22.8 Early function Survived
3 5 No function Died
1 Experiment Example 4
Long-period low temperature perfusion of kidney
for transplantation:
Transplantation experiments on canine kidneys
- 24 -
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1 out in a manner similar to that in Experiment Example 1.
Each kidney was preserved for one week by perfusion at
5 to 8C and a perfusion flow rate of 15 to 18 mg/g/hour
under oxygenation with the gas at a flow rate of 300 to
500 ml/min. The perfusion fluid was prepared by using
the M.R.S. of Preparation Example 1 as the basal salt
solution and adding thereto bovine serum albumin and
the PFC emulsion of Preparation Example 2 or a PFC emulsion
prepared in the same manner as in Preparation Example 2,
except that perfluorodecalin was used in place of the
perfluorotributylamine. The concentrations of albumin
and PFC in the perfusion fluid were 6% (W/V) and 10% ~W/V),
respectively. After one week of the perfusional perserva-
tion, the kidneys were autotransplanted as in Experiment
Example 1. The contralateral nephrectomy was carried
out after one week from the autotransplantation and
symtoms were observed. The results were as shown in
Table 4. The plasma levels of creatinine and BUN of
the survived animals became normal in 3 weeks.
- 25 -
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Table 4
Ex- Kidney weightAfter contralateral
ample Perfusategain duringnephrectomy .
No perfusionFunction ¦ Survival .
1 . 34. 3Early function ¦ Survival¦
. . .
2 M. R. S .46 . 9 No function Died
albumin
6% (W/V) 20 1 Late f`unction Survival
3 Perf'luoro- . 2 days
tribut yl-
4 amine 18 7 Late function Died
10% (W/V) . 3 days
33. 5 Early function 5~ ~i a:
I
6 19.3¦ Early function ¦ Survival
7 M . R . S .40 1 Late fUnction ¦ Died
albumin . 3 days l
_ 6% (W/V) _ _ .
8 Perfluoro-40 . 9 Early function Survived
de calin .
10% (I~/V)
9 23. 5 Early function Survived .
21. 6 No function ¦ Died
Experiment Examp le 5
Using canine kidneys, transplantation experi-
ments were conducted similarly to Experiment Example 1.
Each kidney was preserved at 18 to 22C for 12 or 24
5 hours by perfusion at a perfusion flow rate of 40 to
50 ml/g/hour under oxygenation with the mixed gas
(95~02 ~ 5%C02) at a flow rate of 300 to 500 ml/min.
-- 26 -
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L9
l The perfusion fluid was prepared by using the M.R.S. of
Preparation Example l as the basal salt solution and
adding thereto the PFC emulsion of Preparation Example
2 and a bovine serum albumin. The concentrations of the
PFC and albumin in the perfusion fluid were lO~W/V)
and 6~(W/V), respectively. After 12 hours or 24 hours
of preservation, the kidneys were autotransplanted.
The contralateral nephrectomy was carried out one week
after the autotransplantation and the symptomes were
observed. In Table 5 are shown the results of the above
experiments, together with the results of a control
experiment wherein no PFC was added and a comparative
experiment wherein M.C.S. was used as the basal salt
solution.
:, , , , - ~ . ............................................. ;
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Table 5
.
Ex- Hours of After contralateral
ample Perfusate perfu- ne~hrectomy
_ sionFunction Survival
1 M.R.S. No function Died
albumin 6%
2 (W/V) 12 "
3 ll ll
4 " "
.
No function Died
6 M.C.S. " ,.
7 albumin 6% 12 ,i ,.
PFC 10%(W/V)
, .
9 M.R.S. Early function Survived
albumin ,.
6%(W/V) 12
11 PFC 10%(W/V) .. ll
12 " "
. . . _ . . _
13 M.R.S. Early function Survived
14 6%(W/V) 24 No function Died
PFC 10%(W/V) Early function Survived
16 Late function Survived
2 days
....__
- 28 -
.
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