CA2112952A1 - Methods, apparatus and perfusion-solutions for preservation of explanted organs - Google Patents
Methods, apparatus and perfusion-solutions for preservation of explanted organsInfo
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- CA2112952A1 CA2112952A1 CA002112952A CA2112952A CA2112952A1 CA 2112952 A1 CA2112952 A1 CA 2112952A1 CA 002112952 A CA002112952 A CA 002112952A CA 2112952 A CA2112952 A CA 2112952A CA 2112952 A1 CA2112952 A1 CA 2112952A1
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- perfusion
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The invention relates to the preservation of organs removed for transplant, particularly the human liver removed for transplant, and especially methods, apparatus and perfusion solutions for preserving these organs, the perfusion solutions containing an aqueous fatty emulsion and, as an oxygen source, a perfluorocarbon emulsion.
Description
21123~2 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):
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):
- 2 _ ~ 952 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 ~21129~2 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 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 21~29~2 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 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.
~1129~2 -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 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 ~112952 g 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 2112~52 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 2112~52 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.
- ~112g52 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 21~2952 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.
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 ` 2112952 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.
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 ~21129~2 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 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 21~29~2 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 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.
~1129~2 -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 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 ~112952 g 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 2112~52 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 2112~52 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.
- ~112g52 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 21~2952 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.
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 ` 2112952 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.
Claims (18)
1. An aqueous, electrolyte-containing, isotonic solution for the perfusion and preservation of a surgically removed liver, characterised in that it contains a fatty emulsion and, as an oxygen carrier, a perfluorocarbon emulsion, the fat content of the solution being 0.1 to 0.6% (w/v) and the perfluorocarbon content being 10 to 30% (w/v).
2. A solution according to claim 1, characterised in that the solution 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.
3. A solution according to claim 1, characterised in that the osmolality of the solution is 350 to 400 mOsmol/kg.
4. A solution according to claim 1, characterised in that the fat particles of the fatty emulsion consist of soya bean oils emulsified with egg lecithin.
5. A solution according to claim 1, characterised in that the fat particles of the fatty emulsion have an average particle size of 200 to 2000 nm.
6. The use of an aqueous fatty emulsion for preparing a solution according to at least one of claims 1 to 5 for the perfusion and preservation in a viable state of a surgically removed liver.
7. A process for extracorporeal conservation of a surgically removed liver, characterised in that the liver is stored in a solution according to at least one of claims 1 to 5 and is perfused with this solution.
8. A process according to claim 7, characterised in that the preservation is carried out at a temperature between 15 and 30°C.
9. A process according to claim 7 or 8, characterised in that the preservation lasts up to 48 hours.
10. A process according to at least one of claims 7 to 9, characterised in that oxygen is introduced into the perfusion solution during preservation.
11. An apparatus for preserving an organ removed for transplant, characterised in that it consists of a container for a perfusion solution, in which the organ may be contained surrounded by the solution, which is provided with a feed line and a pump adapted to circulate a perfusion solution between the container and the blood vessels of the organ, and with means for maintaining the oxygen concentration of the perfusion solution contained in the apparatus.
12. An apparatus according to claim 11, characterised in that the feed line and pump for circulating the perfusion solution are located outside the container.
13. An apparatus according to one of claims 11 or 12, characterised in that it contains means for holding or suspending an organ, the entire surface of the organ being in contact with the perfusion solution.
14. An apparatus according to at least one of claims 11 to 13, characterised in that it is provided with a device by means of which the temperature of the perfusion solution and hence of the perfused organ can be regulated.
15. A method of testing the viability of a liver removed for transplanting which is perfused in an aqueous electrolyte-containing solution, characterised in that at least one amino acid is added to the perfusion solution and after a certain length of time the concentration of the ammonia or urea in the perfusion solution is measured.
16. A method according to claim 15, characterised in that a mixture of amino acids is added.
17. A method according to claim 15 or 16, characterised in that 10 to 100 ml, preferably 40 to 60 ml of a 10 to 20% amino acid solution are used.
18. A method of testing the viability of a liver removed for transplanting which is perfused in an aqueous electrolyte-containing solution, characterised in that a fatty emulsion is added to the solution and after a certain time interval the concentration of the free fatty acids is measured.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4342728.6 | 1993-12-15 | ||
| DE4342728A DE4342728A1 (en) | 1993-12-15 | 1993-12-15 | Aq. isotonic soln. for perfusion and preservation of explanted liver |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2112952A1 true CA2112952A1 (en) | 1995-06-16 |
Family
ID=6505045
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002112952A Abandoned CA2112952A1 (en) | 1993-12-15 | 1994-01-06 | Methods, apparatus and perfusion-solutions for preservation of explanted organs |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPH07196401A (en) |
| CA (1) | CA2112952A1 (en) |
| DE (1) | DE4342728A1 (en) |
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-
1993
- 1993-12-15 DE DE4342728A patent/DE4342728A1/en not_active Withdrawn
-
1994
- 1994-01-06 JP JP6000222A patent/JPH07196401A/en active Pending
- 1994-01-06 CA CA002112952A patent/CA2112952A1/en not_active Abandoned
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Also Published As
| Publication number | Publication date |
|---|---|
| DE4342728A1 (en) | 1995-06-22 |
| JPH07196401A (en) | 1995-08-01 |
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