Note: Descriptions are shown in the official language in which they were submitted.
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S014-543.571
DR. KARL THOMAE GMBH Case 5/1141-Ro
D-88397 Biberach 0891
Methods, apparatus and perfusion-solutions for
preservation of explanted organs
The invention relates to the preservation of organs
removed for transplantation, particularly human livers,
and more especially to methods, apparatus and perfusion
solutions for preserving these organs.
The first human liver transplant was carried out in 1963
by Dr. Thomas Starzl (Starzl et al., Surg. Gynecol.
Obstet 17: 659-676 (1963)).
Thereafter the increased capabilities of technological
and medical treatment caused the worldwide field of
liver transplant medicine to progress in leaps and
bounds. A limiting factor was the availability of
transplantable vital organs, of which there is still a
considerable shortage.
One of the most important prerequisites for the success
of liver transplants is, as in all transplants, the
damage-free extracorporeal preservation of the donated
liver.
Up to 1989, donated livers were preserved, in the period
between removal and transplant, in solutions having a
high osmolality and a high potassium concentration. A
typical composition was the Euro-Collins solution
described hereinafter (see Starzl et al., Current
Problems in Surgery, Liver Transplantation: A 31 - Year
Perspective, Part 1, Year Book Medical Publishers, Inc.,
1990, p. 69):
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bicarbonate 10 mM/l; chloride 15 mM/l;
phosphate 57.5 mM/l; sodium 10 mM/l;
potassium 115 mM/l; glucose 194 g/l;
osmolality 375 mOsm/l and pH 7.4.
When using this solution the liver must be kept under
refrigeration, e.g. at 4C, after being removed. The
time limit for safe storage is about 8 hours.
With this system of preservation there are two problems:
firstly, hypothermia causes swelling of the cells with
the result that the sinusoid cell lining is exposed.
Secondly, the storage time of only 8 hours is very
short.
The conventional perfusion solution developed by Belzer
(University of Wisconsin) is an improved solution which
in some cases allows a storage time of up to 24 hours.
The solution developed at the University of Wisconsin
has the following composition (see Starzl et al.,
Current Problems in Surgery, Liver Transplantation: A 31
- Year Perspective, Part 1, Year Book Medical
Publishers, Inc., 1990, p. 69):
phosphate 25 mM/l; lactobionate 100 mM/l;
sodium 30 mM/l; potassium 120 mM/l;
magnesium 5 mM/l; hydroxyethyl starch 50 g/l;
raffinose 17.8 g/l; adenosine 1.34 g/l;
glutathione 0.922 g/l; insulin 100 units;
allopurinol 0.136 g/l; sulphamethoxazole 40 mg/l:
trimethoprim 8 mg/l; dexamethasone 8 mg/l;
and osmolality of 320 mOsm/l and a pH of 7.4.
Although, in experimental studies of specific clinical
applications, an isolated liver has successfully been
stored hypothermically at between 0C and 4C for up to
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48 hours using this solution and has then been
successfully transplanted orthotopically, the systematic
use of this system has nevertheless increasingly led to
reports of so-called "cold damage" which appears to be
responsible for primary post-transplant liver failure.
To avoid this damage, attempts have been made in some
research projects to preserve the isolated liver at
higher temperatures of 7C, 15C and 37C. However,
these attempts have not hitherto resulted in the
reported development of any practicable procedures,
either in discussions with recognised research groups or
in the literature (Starzl et al., Current Problems in
Surgery, Liver Transplantation: A 31 - Year Perspective,
Part 1, Year Book Medical Publishers, Inc., 1990, p.
49-116).
The limited storage time and the above-mentioned damage
which occurs during hypothermic preservation leads to
shortages of available vital organs.
The aim of the present invention is to store a donor
organ for a longer period than before and/or at higher
temperature, e.g. ambient temperature, for an adequate
length of time.
The invention relates to the use of an aqueous fatty
emulsion for preparing an aqueous, electrolyte-
containing perfusion solution for preserving a
surgically removed liver (a donated liver) in a viable
state.
The perfusion solution additionally contains as an
oxygen carrier, a perfluorocarbon emulsion (PFC
emulsion).
In practice, when a donated liver is preserved according
to the invention viability is maintained by the adequate
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supply of oxygen from the PFC emulsion and by the
provision of the fatty emulsion, which if sufficiently
oxygenated, provides a physiological substrate for the
liver.
Conveniently, during perfusion of the removed organ,
oxygen is introduced in the form of gas bubbles into the
perfusion solution through a suitable line and a filter.
The oxygen flow rate is, for example, 0.1 to 1 l/min,
preferably 0.3 to 0.7 l/min. A flow rate of 0.5 l/min
is particularly preferred.
The fatty emulsions used may comprise commercially
available components. Preferably they are taken from
clinical infusion therapy. Stable fatty emulsions based
on soya bean oils emulsified with egg lecithins in
aqueous electrolyte-containing isotonic solutions are
used, such as for example Abolipid~ 10%-20% (Abbott),
Intralipid~ 10%/Intralipid 20% (Pfrimmer Kabi),
LipofundinXMCT 10%-20% (Braun Melsungen), Lipofundin~S
10%-20% (Braun Melsungen), Lipoharm~ 10%, 20%
(Schiwa/Hormonchemie), Lipovenoes~ 10%, 20% (Fresenius)
(see Rote Liste, BPI e.V., 1992).
The fatty emulsion may also be prepared by known
methods, as described for example in Clinical Nutrition
11, 223-236 (1992).
The fat particles of the fatty emulsion may have, for
example, an average particle size of 200 to 2000 nm,
preferably 600 to 1000 nm.
The invention further relates to an aqueous electrolyte-
containing isotonic solution for the perfusion and
preservation of organs removed for transplant,
particularly a surgically removed liver, characterised
in that it contains a fatty emulsion and as an oxygen
carrier, a perfluorocarbon emulsion. The perfusion
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solution preferably contains 0.5 to 3% (v) of a 20%
(w/v) fatty emulsion per litre of the perfusion solution
or an equivalent quantity of a fatty emulsion having a
different concentration, so as to obtain a perfusion
solution with a fat content of 0.1 to 0.6% (w/v). The
fat content of the perfusion solution is preferably 0.2
to 0.5% (w/v); a fat content of 0.4% (w/v) is especially
preferred. The perfluorocarbon content of the perfusion
solution is 10 to 30% (w/v), preferably 20% (w/v). The
percentages specified refer to the volume (v) or the
weight/volume (w/v).
In the literature, a number of perfluorocarbon
substances are described as oxygen carriers, which may
be used in this invention, see for example EP-A 282948,
EP-A-231Q70, EP-A-91820, EP-A-190393, EP-A-220152 and
French Patent No. 850992.
Perfluorooctylbromide (PFOB) or a mixture of
perfluorodecalin (PFD) and perfluorotripropylamine (PFT)
are preferred. A commercial preparation of a mixture of
PFD and PFT known as Fluosol DA is made by the firm
Green Cross Corporation.
As described above, a perfluorocarbon emulsion is
already known and is commercially available. The PFC
emulsion may also be prepared using known methods, as
described in the above-mentioned Patent publications.
In order to prepare a PFC emulsion by conventional
means, an emulsifier (e.g. Serval, Pluronic or
Synperonic) is mixed with fresh electrolyte solution and
stirred vigorously. Some of the resulting mixture is
then placed in a high pressure homogeniser and
homogenisation is effected as the remainder of the
-mixture and PFC are slowly added. The resulting
emulsion is then cooled to 5C, for example, and
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homogenised once more.
Normally, the PFC emulsion used in this invention has an
average particle size of 100 to 400 nm, preferably 150
to 250 nm. An average particle size of 180 to 240 nm is
particularly preferred.
The electrolyte solution which constitutes the
continuous phase of the perfusion solution may, for
example, be any of the solutions hitherto used in liver
preservation (see, for example, Starzl et al., Current
Problems in Surgery, Liver Transplantation: A 31 - Year
Perspective, Part 1, Year Book Medical Publishers, Inc.,
1990, p. 49-116). Preferred solutions are the
Brettschneider solution (see below), the Euro-Collins
solution and the solution of the University of
Wisconsin.
Preferably, the continuous phase of the perfusion
solution is an aqueous electrolyte-containing isotonic
solution which contains 3.5 to 100 mMol/l of potassium
ions, 0.8 to 5 mMol/l of magnesium ions and 15 to
146 mMol/l of sodium ions.
The osmolality of the perfusion solution is preferably
350 to 400 mOsmol/kg.
The process for preserving a surgically removed liver
according to the invention is characterised in that the
liver is stored in an aqueous, electrolyte-containing,
isotonic solution, which contains a fatty emulsion and a
perfluorocarbon emulsion, and is perfused with such a
solution.
Preferably the liver is perfused with the solution
through the Vena portae.
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The process can be performed without cooling
(hypothermia) at temperatures between 15 and 30OC,
preferably at an ambient temperature of 19 to 22C. At
this temperature, which is substantially higher than in
conventional methods, the occurrence of the above-
mentioned cold damage is avoided.
An important advantage of the process according to the
invention is the preservation period of up to 48 hours,
which is longer than with conventional methods. A
preservation period of up to 24 hours at ambient
temperature is preferred.
An apparatus according to the invention for preserving
an organ removed for transplant is characterised in that
it consists of a container for a perfusion solution, in
which the organ can be held surrounded by solution,
provided with a feed line and a pump adapted to
circulate a perfusion solution between the container and
the organ, and with means for maintaining the oxygen
concentration of the perfusion solution contained in the
apparatus.
In the apparatus according to the invention, preferably
at least part of the feed line as well as the pump for
circulating the perfusion solution are located outside
the container.
If the organ is a liver, a feed line is adapted to
circulate the perfusion solution between the container
and the Vena portae and the Vena cava.
Preferably, the apparatus contains means for holding or
suspending the organ, the entire surface of the organ
being in contact with the perfusion solution. In
addition, the apparatus may be equipped with a device by
means of which the temperature of the perfusion solution
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and hence the perfused organ can be regulated.
Preferably this device allows a temperature of between
15 and 30C to be maintained for the perfusion solution
and hence for the organ lying in the solution. It is
particularly preferred for perfusion to be carried out
at ambient temperature, so that the perfusion solution
and the organ both have a temperature of 19 to 22C.
Urea synthesis is a process specific to the liver and
can therefore be used to monitor the viability of the
organ.
The present invention provides a method of testing the
viability of a liver which has been removed for
transplanting and has been perfused in an aqueous
electrolyte-containing solution. In this method, at
least one amino acid which can be metabolised by the
liver is added to the perfusion solution during the
preservation process according to the invention. At a
specified time interval after administration, the
concentration of ammonia and urea in the perfusion
solution is determined.
Preferably, a mixture of amino acids is added, e.g. 10
to 100 ml, preferably 40 to 60 ml of a 10 to 20% amino
acid solution may be used.
The amino acid is added to the perfusion solution from
time to time during the extracorporeal preservation of
the liver, e.g. when the solution is added to the
container or preferably through the feed line.
The preferred quantities of amino acid used are between
5 g and 10 g per test. Preferably, a mixture of amino
acids originating from infusion therapy is used (e.g.
one of the infusion solutions on sale under the name
Thomaeamin (see the Rote Liste, ibid)). The use of
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individual amino acids is theoretically possible but it
is not preferred, since it leads to imbalances and in
most cases the toxicities of the amino acids vary and
are not known precisely. If urea synthesis is
functional, there will be an increase in urea production
of, for example, 2.5 mMol in 4 hours after the addition
of 7.5 g of amino acid, administered by the method
according to the invention in the form of 50 ml of a 15%
amino acid solution. The ammonia concentration remains
below 50 ~Mol/l if the liver perfusion is adequate. It
increases dramatically if the energy supply to the liver
breaks down.
Liver-specific urea synthesis can thus be tested through
the administration of amino acids into the perfusate and
by analysis for urea and ammonia. If there is
perfusion, there is an increase in urea production of,
for example, more than 2.5 mMol in 4 hours. If the
energy supply is inadequate, no urea is synthesised and
there is a corresponding rise in the ammonia
concentration to above 100 ~Mol/l.
The use of fatty emulsions, to provide a substrate for
the organ, is monitored in the perfusate by analysis of
the triglyceride content and of the "free fatty acidsi'
to examine the utilisation of the fatty acids.
Analytical determination is carried out by methods known
from the literature, e.g. measurement of the fatty acid
methylesters by gas chromatography after esterification
of the free fatty acids with methyliodide over solid
potassium carbonate, analogously to the technique
described in Z. Klin. Chem. Klin. Biochem. 13: 407-412
(1975). If there is no utilisation, there is a
resultant rise in the concentration of free fatty acid,
since lipolysis occurs in the perfusate as a result of
the lipase situated in the vascular walls of the liver.
In the perfusion experiments described above, there was
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no increase in the free fatty acids and hence the fatty
acids were obviously being utilised by the liver.
The present method thus provides a method of testing the
viability of an organ removed for transplant,
particularly a liver, which is perfused in an aqueous
electrolyte-containing solution, characterised in that a
fatty emulsion is added to the solution and after a
certain length of time the concentration of the free
fatty acids is determined.
Example 1
The following three solutions are investigated:
A. Brettschneider(HTK) solution having the following
composition:
Sodium chloride 15 mM/l; potassium chloride 9 mM/l;
potassium hydrogen-2-oxoglutarate 1 mM/l;
magnesium chloride x 6H2O 4 mM/l;
histidine x HCl x H2O 18 mM/l;
histidine 180 mM/l; tryptophan 2 mM/l;
mannitol 30 mM/l;
in water for injections
osmolality: 310 mOsm/kg;
anion: Cl 50 mval;
H = histidine, T = tryptophan, K = potassium.
B. Brettschneider solution as in (A) together with a
perfluorocarbon (PFC) emulsion
C. Brettschneider solution and PFC emulsion as in (B)
together with fatty emulsion
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Preparation of solutions B and C
Under vigorous stirring, 8 1 of fresh Brettschneider-
electrolyte solution are combined, in batches, with
396 g of emulsifier (Serva, Heidelberg).
As soon as the emulsifier has fully dissolved, the
solution is topped up to 8.1 1 with electrolyte solution
and cooled to 5C.
A high pressure homogeniser (Lab 60 made by APV Gaulin)
is rinsed with about 600 ml of pure electrolyte
solution. Then some of the electrolyte solution is
poured into the storage container of the homogeniser.
The mixture is then homogenised under 500 bar pressure
of CO2, whilst the remainder of the electrolyte
emulsifier solution and 1000 ml of perfluorocarbon are
each added to the storage container, through separating
funnels, slowly enough so that the first homogenisation
run ends shortly after the addition of perfluorocarbon
is complete.
The emulsion is then cooled to 5C with ice water and
homogenised once more under 500 bar pressure of CO2.
This procedure is repeated 5 times.
The finished emulsion contains perfluorocarbon 20% w/v
and has an average particle size of 180-240 nm. It is
storable at about 5C for a short period before use.
Solution C is obtained from solution B, by mixing a 20%
(w/v) fatty emulsion, e.g. Intralipid-20, so as to
obtain a fat content of for example 0.2% (w/v) in the
finished perfusion solution C. Accordingly, 1 1 of
solution C contains for example 20 ml of the 20% (w/v)
fatty emulsion.
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Example 2
A number of domestic pigs weighing 20 kg are
anaesthetised intravenously, laparotomised,
thoracotomised and their livers are removed by the usual
surgical method with the intravenous administration of
125 units of heparin per kg of body weight.
The Vena Portae and Vena cava inferior are dissected out
and cannulated.
The residual blood remaining in the liver is flushed out
by the infusion of 300 ml of solution A, B or C (Example
1) through the cannulated Vena portae. During this
flushing the Ductus choledochus is cannulated.
The cannulated liver [11] is placed in container [1]
(see Figure 1) until it is totally submerged in the
perfusion solution contained therein. A solution C
prepared according to Example 1 with an initial fat
content of 0.1% (w/v) is used as the perfusion solution.
The perfusion circuit [2] connects the V.portae [13] and
V.cava [12] via the external feed line to the outer
container, whilst the perfusion solution is continuously
circulated between the liver and the container by means
of the pump [3], e.g. a peristaltic pump.
Oxygen gas bubbles are piped into the perfusion solution
from an oxygen gas bottle [4] via a line [5] through a
filter (not shown in Figure 1) on the inner edge of the
container [1] at a flow rate of about 0.5 l/min. In the
container is a net [6] of plastics or metal for securing
the liver in position whilst ensuring that no essential
part of the liver surface loses contact with the
perfusion solution.
Neither the liver nor the apparatus nor the perfusion
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solution need to be warmed or cooled.
By means of the cannulation [7] of the D.choledochus the
outflowing bile is collected in the external container.
Continuous perfusion begins 10 minutes after removal of
the organ, through the Vena portae.
The median flow rate of the perfusion solution is
adjusted to 0.3 ml/g of weight of liver/min.
The liver is kept at an ambient temperature of about
22C in the suitably buffered solution throughout the
entire extracorporeal preservation period.
Reference numeral [8] denotes a removal point for
samples of the perfusion solution for analytical
purposes.
Integrated in the feed line [2] is a pressure gauge [9]
and an input point [10] for fatty emulsions, consisting
for example of an injection syringe coupled with a valve
incorporated in the feed line.
In order to compensate for the consumption of fatty
emulsion, after each period of six hours 12.5 ml of
Intralipid-20 are fed directly into the perfusion
solution.
At intervals of four hours, biopsies are taken for
examination under the electron microscope and samples of
the perfusate are taken in order to analyse its
composition (electrolytes, pH, osmolarity and gas
analysis).
The perfusate is sampled at 2 hour intervals in order to
check on the function of the liver.
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Urea synthesis:
A urea production test is carried out by adding
50 ml of 15% amino acid solution (Thomaeamin N15 of
Dr. Karl Thomae GmbH) to the perfusate every four
hours and taking a sample for the measurement of
urea and ammonia at intervals of two hours. The
urea and ammonia are measured using methods known
from the literature. The urea measurement is
described for example in R. Spayd et al., Clin.
Chem. 24, 1343-1344 (1978), whilst the ammonia
level is determined as described by W.A. Bruce et
al., Clin. Chem. 24, 782-787 (1978).
Figure 2 shows, by a comparison of solutions A, B
and C (see Example 1), the increase in the
concentration of urea in the perfusate from a pig's
liver, in which perfusion has been carried out as
described above with the following modifications:
a) perfusion solution A but with no oxygen supply
b) perfusion solution A with an oxygen supply
c) perfusion solution B with an oxygen supply
d) perfusion solution C with an oxygen supply.
Figure 3 shows, analogously to Figure 2, the
development of the ammonia concentration in the
same perfusion solutions.
Figure 2(d) clearly shows that when perfusion
solution C according to the invention is used with
an oxygen in-flow, the concentration of urea
increases constantly over a perfusion period of 48
hours, and to a significantly greater extent than
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is the case when perfusion is carried out with the
comparison solutions A and B (cf. Figure 2(a)-(c)).
Figure 3(d) shows that, by contrast with solutions
A and B (Figures 3(a)-(c)) the ammonia
concentration in the perfusate C remains negligibly
small during this perfusion period.
This clearly demonstrates the superior maintenance
of function of the test organ using the perfusion
solution C according to the invention.
Electron microscope slides are prepared from the tissue
samples, and these slides are subjected to planimetry
using a computer system and thus make it possible to
obtain quantitative figures for the number of
mitochondria and the diameter and shape of the
mitochondria.
