EA045517B1 - METHOD AND APPARATUS FOR ORGAN RESTORATION - Google Patents

Description

The invention relates to the removal of organs from a donor, as well as to the preservation and evaluation of organs.

BACKGROUND OF THE INVENTION

Currently, organ banking for transplantation is largely limited to obtaining organs from patients who, while brain dead, are still mechanically ventilated and whose hearts are still beating.

Organs from patients who die of cardiac arrest before or during transport to hospital are not usually used for transplantation. In rare cases, such organs have been used, especially if the time from cardiac arrest to organ removal is short, for example, less than 30-60 minutes. If the time from cardiac arrest to organ removal is more than 1 hour, such organs are usually not suitable for transplantation. If such a second organ bank could be used, the number of organs available for transplantation would increase by 10-100 times.

After cardiac arrest, organs are subjected to warm ischemia, which leads to the accumulation of toxic metabolic end products in the organs. This is caused by the cessation of circulation of oxygenated blood, which leads to the accumulation of metabolic end products.

Immediately after cardiac arrest, the blood coagulation system is activated, and fibrin blood clots form in the capillary vessels, which leads to thrombotic phenomena, the elimination of which will take hours or days if the patient, for example, is resuscitated and survives.

In the event of death, the microbial barrier in the intestine ceases, resulting in bacterial overgrowth with the release of endotoxins such as lipopolysaccharides and cytokines, which begins, in some cases, within 5 minutes. Therefore, the use of organs from donors who have died from circulatory arrest is considered marginal and in most cases is carried out in situations where circulatory arrest is controlled. Organs that have been subjected to warm ischemia for more than two hours are essentially considered unsuitable for transplantation.

If organs are cooled, metabolism slows down by about 6% per degree Celsius. At 28°C the metabolism slows down to approximately 50%, and at 22°C to approximately 25%.

Normal cooling of a dead body occurs at a rate of up to 2°C per hour. Therefore, after 5 hours the body and organs may have a temperature of approximately 27°C.

Thus, there is a need in the art for a method of recovering organs from a donor that allows for greater use of a second organ donor bank.

Patent document EP0631786A1 (abstract) discloses a method of treating ischemia and concomitant reperfusion injury, including the administration of plasmin and plasmin-forming proteins, including lys-plasminogen and the like. Lys-plasminogen, which can be produced by proteolytic cleavage of glu-plasminogen, has been found to have a protective effect on tissue affected by ischemia. Lys-plasminogen administration may be used to treat subjects during and after reperfusion. Lys-plasminogen can also be administered in conjunction with clot dissolving drugs, such as those that use tissue plasminogen activator or the like. It is indicated that ischemic conditions and subsequent reperfusion injury caused by surgery can be prevented or treated by proteins having the effect of lys-plasminogen or lys-plasminogen precursors. Such proteins may even have beneficial effects on already transplanted donor organs or tissues, as well as on the surrounding organs and tissues of the donor and recipient. Administration of proteins having the lys-plasminogen effect or lys-plasminogen precursors allows organs and tissues to withstand longer periods of ischemia as well as the physiological stress caused by reperfusion after transplantation. Damage to an organ or tissue can be reduced or completely prevented by administering proteins having the effect of lys-plasminogen or lysplasminogen precursors before surgery. In the case of transplantation, proteins can be administered to the donor before the organ or tissue is removed. The donor may receive treatment systemically or locally into the artery supplying an organ or tissue before the organ or tissue is removed. Likewise, an organ or tissue recipient may receive treatment prior to transplantation to protect the organs and tissues surrounding the transplant site as well as the organ or tissue being placed in the recipient's body. It is also possible to administer proteins having the effect of lys-plasminogen or lysplasminogen precursors during or after reperfusion. Thus, this patent document discloses the introduction of lys-plasminogen into the body of a donor or recipient in which blood flow is present, otherwise there will be no result.

The essence of the invention

Accordingly, it is an object of the present invention to reduce, partially or completely eliminate one or more of the above-mentioned disadvantages and defects, separately or together.

In one aspect of the invention, there is provided a method for recovering an organ obtained from a donor, for example from a donor who has died from circulatory arrest, comprising: obtaining the organ from the donor at least two hours after the donor's circulatory arrest; introducing lys-plasminogen into the organ after taking it, wherein lys-plasminogen is contained in the first hyperoncotic solution;

- 1 045517 administration of tissue plasminogen activator (tPA) simultaneously or after administration of lysplasminogen, wherein tissue plasminogen activator is contained in the second hyperoncotic solution; at the first stage of recovery, ensuring circulation through the organ of the third hyperoncotic fluid containing albumin and electrolytes at a low temperature from 5°C to 25°C; at the second stage of recovery, ensuring circulation through the organ of the fourth hyperoncotic fluid containing oxygenated red blood cells at a temperature of 28°C to 37°C; assessment of the organ according to the usual criteria.

In one embodiment, the first stage of recovery may include: allowing said third hyperoncotic fluid to circulate through the organ, wherein said third hyperoncotic fluid contains albumin at a concentration of from 50 g/L to 120 g/L, wherein the circulation pressure is increased, for example from about 20 mmHg Art. up to 90 mm Hg Art., for 30-75 minutes, for example, in increments of 5 mm Hg. Art. every 5 min. The second recovery step may include: circulating said second hyperoncotic fluid through the organ, wherein said fourth hyperoncotic fluid contains albumin at a concentration of from 50 g/L to 120 g/L, wherein the circulation pressure is increased, for example from about 20 mm Hg. Art. up to 90 mm Hg Art., for 30-75 minutes, for example, in increments of 5 mm Hg. Art. every 5 min.

In yet another embodiment, the method may further include: storing the organ at a low temperature from 4°C to 16°C, with circulation of a preservation liquid through the organ at a pressure below 30 mm Hg. Art. and for a period of time from one hour to 7 hours. The storage step may be performed after the second recovery step or between recovery steps.

In yet another embodiment, at least one of the first or second hyperoncotic fluids contains electrolytes at physiological concentrations and albumin, wherein the first hyperoncotic fluid and the second hyperoncotic fluid contain albumin at a concentration of from 50 g/L to 120 g/L.

In yet another embodiment, at least one of the first, second, third or fourth hyperoncotic fluids may further comprise at least one of the following: a coagulation inhibitor such as antithrombin III, a direct thrombin inhibitor such as argatroban, protein C , protein S, and a platelet inhibitor such as abciximab.

In yet another embodiment, at least one of the third or fourth hyperoncotic fluids circulates through the leukocyte filter. In addition, at least one of the third or fourth hyperoncotic fluids may be contacted with a cytokine scavenger, such as a cytosorbent, to absorb the cytokines. In addition, at least one of the third or fourth hyperoncotic fluids is contacted with an endotoxin scavenger, such as a lipopolysaccharide scavenger, to absorb the endotoxins.

In yet another embodiment, the method may further include: obtaining an organ from the donor after the donor's circulatory arrest has occurred for at least three hours, wherein said at least three hours include no more than two hours of local cooling with cold saline, ice, or ice. sludge placed into the donor's abdominal cavity.

In yet another embodiment, a method is provided for recovering an organ obtained from a donor, for example, from a donor who has died of cardiac arrest, comprising: obtaining the organ from the donor at least four hours after the donor's circulatory arrest; introducing lys-plasminogen into the organ after its collection, wherein lys-plasminogen is contained in a first hyperoncotic solution containing albumin at a concentration of 50 g/L to 70 g/L and a coagulation inhibitor such as antithrombin III; administering tissue plasminogen activator (tPA) into the organ simultaneously or after administration of lysplasminogen, wherein tissue plasminogen activator is contained in a second hyperoncotic solution containing albumin at a concentration of 50 g/L to 70 g/L and a coagulation inhibitor such as antithrombin III; in the first stage of recovery, circulation through the organ of a third hyperoncotic fluid containing albumin in a concentration of 50 g/l to 120 g/l and electrolytes and a coagulation inhibitor such as antithrombin III, at a low temperature of 5 ° C to 25 ° C, while pressure increases from 20 mm Hg. Art. up to 70-90 mm Hg. Art.; in the second stage of recovery, circulation through the organ of the fourth hyperoncotic fluid containing red blood cells, albumin in a concentration of 50 g/l to 120 g/l and electrolytes and a coagulation inhibitor such as antithrombin III, at a temperature of 30°C to 37°C; assessment of the organ according to the usual criteria.

According to another aspect, there is provided a device for regenerating an organ obtained from a donor, for example, from a donor who died of circulatory arrest, comprising: a container for housing the organ to be treated; connector for connecting to an artery of an organ with an open vein; a circulation pump connected between the container and said connector for circulating fluid present in the container through the organ; a drain connected to the container by a drain valve; at least one bag connected to the container by hydraulic valves for supplying liquids to the container; an oxygenator for oxygenating the liquid pumped by the pump; heater/cooler to regulate the temperature of the liquid pumped by the pump; leukocyte filter for removing leukocytes from the liquid pumped by the pump; endo absorber

- 2 045517 toxins, configured to remove endotoxins from the container liquid; a cytokine absorber configured to remove cytokines from the container liquid; and a leukocyte filter configured to remove leukocytes from the container liquid.

According to another aspect, a liquid is provided for performing any of the methods described above, containing: lys-plasminogen, tPA, electrolytes and albumin in a concentration of from 50 g/L to 120 g/L.

Brief description of drawings

Other objects, features and advantages of the invention will become more apparent from the following detailed description of embodiments of the invention with reference to the accompanying drawings.

Fig. Figure 1 schematically illustrates two kidneys removed together by cutting the aorta and vena cava.

Fig. 2 schematically illustrates the capillary systems of the kidney.

Fig. 3 schematically illustrates a block diagram of an embodiment of an apparatus for performing the method.

Fig. 4 schematically illustrates a block diagram of another embodiment of an apparatus for performing the method.

Fig. 5 schematically illustrates a block diagram of another embodiment of an apparatus for performing the method.

Fig. 5A schematically illustrates a block diagram of another embodiment of an apparatus for performing a method.

Fig. 5B schematically illustrates a block diagram similar to that shown in FIG. 5A for treating two buds separately.

Fig. 6 illustrates a diagram showing the change in arterial blood flow in Example 1.

Fig. 7 illustrates a diagram showing urine production in Example 1.

Fig. 8 illustrates a diagram showing arterial blood flow in the kidney in Example 2.

Fig. 9 illustrates a diagram showing arterial blood flow in the kidney in Example 3.

Fig. 10 illustrates a diagram showing arterial blood flow in the kidney after transplantation in Example 4.

Fig. 11 illustrates a diagram showing arterial blood flow in the kidney after transplantation in Example 6.

Fig. 12 illustrates a diagram showing arterial blood flow in the kidney after transplantation in Example 7.

Fig. 13 illustrates a diagram showing arterial blood flow in the kidney after transplantation in Example 9.

Fig. 14 is a photograph showing the kidney in Example 9.

Fig. 15A-15D are photographs showing a transplanted kidney according to Example 9.

Fig. 16A is a graph showing changes in IL-6 levels primarily from red blood cells.

Fig. 16B is a graph showing changes in IL-8 levels also primarily from red blood cells.

Fig. 16C is a graph showing changes in IL-1e levels also primarily from red blood cells.

Fig. 16D is a graph showing changes in TNF-α levels also primarily from red blood cells.

Fig. 17 is a graph showing the flow rate after perfusion with the modified solution using an osmolarity of approximately 300 mOsmoles according to Example 12.

Fig. 18 is a diagram showing pressure, flow rate and resistance according to Example 14.

Fig. 19-22 are graphs showing creatinine before and after transplantation according to Example 16.

Fig. 23 and 24 are photographs showing buds according to Example 16.

Fig. 25-28 are photographs showing the liver of Examples 17 and 18.

Fig. 29 is a graph showing the results for the liver in Examples 17 and 18.

Detailed description of embodiments

Several embodiments of the invention will be described below. These embodiments are described for illustrative purposes to enable one skilled in the art to practice the invention and to disclose the best embodiment. However, these embodiments do not limit the scope of the invention. In addition, certain combinations of features are shown and described. However, other combinations of various features are possible within the scope of the invention.

When the heart stops beating, blood circulation stops. This can lead to a cascade with

- 3 045517 events that occur in the body trying to maintain blood circulation, which in the case of brain death after brain herniation is called an autonomic catecholamine storm, in which the body releases large quantities of adrenaline and norepinephrine in an attempt to maintain cardiovascular stability. This eventually leads to brain death and cardiac arrest. When cardiac arrest occurs before brain herniation, another cascade of events will follow involving the coagulation system and the inflammatory system, without a catecholamine storm. Less is known about the events following death due to cardiac arrest and circulatory arrest.

After cardiac arrest and death, a process called cadaveric pallor occurs within 15-25 minutes, in which the skin turns pale. Corpse pallor is caused by the cessation of capillary blood supply in the body.

A process called cadaveric cooling then occurs, in which the body changes its temperature until it reaches the ambient temperature. After approximately 4 hours, body temperature is approximately 27-29°C or lower, depending on the ambient temperature.

A process called rigor mortis or rigor mortis then occurs, in which the muscles become rigid. After death, breathing stops, depleting the source of oxygen used in the production of adenosine triphosphate (ATP). ATP is required to promote actin-myosin cross-bridge separation during muscle relaxation. The body undergoes rigor mortis because it cannot break these bridges. In rigor mortis, the myosin heads continue to bind to the active sites of actin proteins via adenosine diphosphate (ADP), and the muscle cannot relax until other enzymatic activity destroys the complex.

Rigor mortis begins approximately 1-4 hours after cardiac arrest and reaches a maximum after approximately 12 hours. It affects all muscles of the body and all organs.

When a patient in cardiac arrest is far from the hospital, it is difficult to assess the duration of cardiac arrest and its impact on organs. Therefore, the bodies of patients who suffer cardiac arrest far from the hospital are usually not used for transplantation. The purpose of this invention is the restoration of such organs, as well as the regeneration, evaluation and storage of such organs prior to transplantation.

The sooner the organs are removed, the better the result of their use.

However, the recovery process according to embodiments of the present invention allows recovery of organs to rigor mortis and up to maximum rigor mortis, with slightly worse results when used for significant periods of time after death and cessation of circulation.

All of the above actions affect the organs in the body when the heart has stopped.

Cardiac arrest can activate the coagulation system, leading to a procoagulative state in which microthrombi eventually form in the capillary system. Little is known about how low body temperature affects blood coagulation processes, the process of activation and the process of deactivation of coagulation processes.

When the circulatory system of a living body is working, whether the blood will clot or not depends on the balance between two groups of substances, those substances that promote clotting are called procoagulants, and those that prevent clotting are called anticoagulants. Normal blood flow is dominated by anticoagulants so that the blood does not clot as it circulates through the blood vessels.

When blood circulation stops after cardiac arrest, little is known about what happens to the coagulation system over time. Without being bound by any theory, it is believed that when circulatory arrest occurs, the amount of anticoagulants is reduced and procoagulants are activated, also depending on the cause of circulatory arrest. This process can be slow and also depends on the temperature decreasing over time.

It is known that if blood is collected in a chemically clean glass tube, it usually clots within 6-10 minutes, this can be used to detect clotting disorders. However, if you replace the glass tube with a siliconized container, the blood may not clot for an hour or more because the platelets are not activated. Therefore, it can be assumed that the blood remaining in the donor organ may not clot for 30 minutes, one hour or more. Significant curling will occur after 2-4 hours.

When the pressure of the heart stops, some blood vessels, namely the capillaries, become narrower, which can contribute to the onset of cadaveric pallor. After some more time, the presence of albumin and other oncotic substances in the blood causes ultrafiltration of water from the surrounding tissue into the blood vessels, causing dilation of the blood vessels, namely arterioles and venules and larger vessels. Over time, the red blood cells separate and settle at the very bottom of the blood vessels in a sedimentation reaction. The buffy coat, containing white blood cells and platelets, forms above the red blood cells. The buffy coat has an increased concentration of platelets and other proteins. Vasodilation can open up the parts of the blood vessels that interact with platelets and activate platelets in about 2-4 hours. In addition,

- 4 045517 as there is no blood circulation, platelets can also interact with endothelial cells, especially if there is vessel damage, on the surface of blood vessels and attach to endothelial cells. This interaction can further damage the endothelial cells and can eventually also lead to the formation of blood clots. Such blood clots can form in any part of the vessels that do not have circulation. A decrease in temperature also affects the blood clotting system in various ways. Typically, lower temperatures will slow down chemical reactions. Thus, after some time, usually after a few hours, for example after 1-4 hours, the blood vessels may have many larger or smaller clots adhered to the walls of the blood vessels. These clots cannot be washed away by flushing the organs' blood vessels, which typically occurs after receiving organs from a donor. Of course, if you try to wash away blood clots using high pressure and high-speed flows, endothelial cells may be damaged at the sites where the blood clots break off. Instead, these clots remain during organ storage after organ retrieval and before transplantation. When the organs are finally transplanted into the recipient, the recipient's blood enters the blood vessels, which can lead to the formation of new blood clots in the damaged areas of the blood vessels. Thus, it is important to remove blood clots as quickly as possible, especially if the organ was received from the donor some time later, for example, 2, 3, 4 hours or more after circulatory arrest. Since such blood clots form some time after circulatory arrest, this problem is increased in organs donated after an extended period of time, such as 4 hours or longer. On the other hand, this process slows down at low temperatures, which means that if the donor's body is cooled more quickly before organ removal, it may reduce the process of blood clots.

When a blood clot forms, it traps large amounts of plasminogen, as well as other plasma proteins. Plasminogen will not become plasmin or cause clot dissolution until it is activated. In the living body, the affected tissues and vascular endothelium very slowly produce a powerful activator called tissue plasminogen activator (tPA), which later finally converts plasminogen into plasmin, which in turn removes the remaining blood clot. Many small blood vessels in which blood flow has been blocked by blood clots are known to reopen by this mechanism in the living body. Thus, a particularly important function of the plasmin system is to remove tiny clots from millions of tiny peripheral vessels that would be blocked if they were not cleared.

The plasminogen trapped in the thrombus is usually glu-plasminogen, which is slowly converted to plasmin upon interaction with tPA. This causes the system to start up slowly. However, after some time, glu-plasminogen is converted to lys-plasminogen, which is converted to plasmin much faster. So, there is a positive amplification system that increases the rate of clot dissolution after an initial period of time, which can be up to 48 hours.

After much experimentation, the author came to the conclusion that the formation of blood clots during the first few hours of ischemia can be detrimental to organs. It is believed that platelets are activated due to the lack of blood flow and the resulting clots can influence and damage endothelial cells near the clot. Since there is no blood circulation after death and cardiac arrest, blood clots will affect the endothelial cells for a long time, which will damage them.

The endogenous dissolution system can be used to dissolve microthrombi in the donor kidney. However, the dissolution of blood clots occurs slowly. Of course, adding large amounts of tPA will not increase the rate of activation of plasminogen to plasmin.

However, it was found that the addition of lys-plasminogen and the addition of tPA would enhance clot dissolution and speed up the process. You can also pay attention to the local environment after dissolution of the thrombus to prevent recurrent thrombus formation, since the local endothelium is more vulnerable after fibrinolytic treatment.

The present invention is useful in transplantation of any organ such as liver, kidney, pancreas, pancreatic islet, uterus, small intestine, multivisceral graft.

Further embodiments of the invention will be described using the example of kidney and liver transplantation. However, embodiments are useful in transplantation of other organs, as indicated above, and other tissues.

A feature of the kidney is that it contains two capillary systems connected in series, the glomerular capillaries and the peritubular capillaries, with efferent arterioles located between these two capillary systems, as shown in Fig. 2. So microthrombi can form in both capillary systems. In addition, larger clots can form in the efferent arterioles and be transferred between capillary systems. The blood clotting process will affect the kidneys and block the two capillary systems and the efferent arterioles between them. Thus, it is difficult to flush out blood clots from kidneys where microthrombi have formed.

To test the recovery process, pig kidneys were used for experimental purposes.

- 5 045517 women who were subjected to warm ischemia for 4 hours or more. There is evidence that kidneys exposed to ischemia for 6 hours, 8 hours, 10 hours, 12 hours or more can be restored. In addition, kidneys exposed to ischemia for one hour or less may also benefit from this process, including kidneys from brain-dead donors that are considered marginal either due to prolonged warm or cold ischemia or other associated factors , such as age, hypertension, diabetes, hypotension, intensive care unit (ICU) time, anuria, elevated laboratory values, poorly perfused kidneys on the instrument table, or any other reason for primary donor rejection from transplantation.

According to an embodiment of the present invention, kidneys that have been subjected to warm ischemia for a long and unknown time are restored using the basic method steps mentioned below. These steps do not need to be performed in exactly the same sequence as listed below, as will be explained below.

The first step may involve the administration of lys-plasminogen and tPA into the renal arteries immediately after removal and on the instrument table, sequentially or jointly, or administration may be possible using an extracorporeal, ex-vivo perfusion device sequentially or jointly. Lysplasminogen is contained in a carrier fluid, which is a physiological isotonic electrolyte fluid possibly containing a hyperoncotic agent. Approximately 5 to 20 mL of lys-plasminogen may be administered (using 5 to 100 units per kidney). Lys-plasminogen will adhere to any blood clots present in the blood vessel of the kidney. After approximately 15 minutes (or simultaneously), 5 to 20 mL of tPA (using 0.5 to 10 mg per kidney) can be injected into the arteries in a similar manner. TPA may be contained in a carrier fluid, which is a physiological isotonic electrolyte fluid, possibly containing a hyperoncotic agent. The first stage can be carried out at a temperature equal to or above room temperature, such as 20-37°C.

The kidneys may further be subjected to a circulation or perfusion step, in which the kidneys are connected to a perfusion device by placing connectors in the artery of the kidney and placing the kidney in a container to collect fluid exiting the veins. The container may contain 500-5000 ml of circulating fluid, which is a physiological isotonic electrolyte fluid and may also contain a hyperoncotic agent, for example albumin 57 g/l alone or in combination with additional hyperoncotic agents. The circulating fluid circulates through the kidney at 15-24°C (room temperature) for 35 minutes or longer, starting at a pressure of 20 mmHg. Art. and with a multiple increase in pressure by 5 mm Hg. Art. every 5 minutes to a maximum value of 70 mmHg. Art., followed by a 30-minute period at low pressure, for example, 30 mm Hg. Art. During this period of time, lys-plasminogen and tPA also circulate in the kidney and resistance progressively decreases. The color of the bud is checked and treatment at high pressure can be stopped to continue at low pressure when the bud turns pale.

It is possible to add one or more of the following substances:

a thrombin inhibitor such as antithrombin III (ATIII); argobatran or another direct thrombin inhibitor, such as inogatran, melagatran (and its prodrug ximelagatran), dabigatran or hirudin and their derivatives;

allosteric inhibitors;

platelet inhibitor such as glycoprotein IIb/IIIa receptor antagonists (abciximab, eptifibatide, tirofiban);

irreversible cyclooxygenase inhibitors (aspirin, triflusal);

adenosine diphosphate (ADP) receptor inhibitors (cangrelor, clopidogrel, prasugrel, ticagrelor, ticlopidine);

phosphodiesterase inhibitor (cilostazol);

protease-activated receptor-1 (PAR-1) antagonists (vorapaxar);

adenosine reuptake inhibitors (dipyridamole);

thromboxane inhibitors (thromboxane synthase inhibitors such as ifetroban, picotamide and thromboxane receptor antagonists such as terutroban);

or any other platelet inhibitor, alone or in combination, to prevent re-thrombosis of treated blood clots. These substances may be introduced during the first injection step or the first circulation step or any other step. The addition of heparin or low molecular weight heparin is optional.

The second stage of the process involves perfusion and restoration of the circulatory system by circulating hyperoncotic fluid through the renal vessels. Hyperoncotic is defined as pressure caused by plasma proteins, but can also be an artificial construct such as dextran or polyethylene glycol (PEG). A common property is that the substance must have a colloid-oncotic pressure greater than that of the surrounding kidney tissue through which it flows. In addition, the liquid can be perfused at a low temperature, for example, from 12°C to 24°C, and at a low pressure, for example, from 20 mm Hg. Art. up to 30 mm Hg Art. at one stage of the reduction stage, and at a higher temperature, for example from 18°C to 32°C at another stage of the recovery stage, at

- 6 045517 in which the perfusion pressure can also be higher, for example from 25 mm Hg. Art. up to 70 mm Hg Art. or 90 mm Hg. Art. One hyperoncotic fluid contains albumin in a high concentration of 40 g/L to 120 g/L, such as 50 g/L to 80 g/L, such as 57 g/L to 72 g/L, but may also contain others substances with similar properties or a combination thereof. Hyperoncotic fluid may contain large amounts of potassium compared to normal extracellular levels, approximately 10 mmol to 25 mmol. Hyperoncotic fluid may also contain the osmotic membrane impermeable agent gluconate and glucose, but may contain other agents with similar properties instead of or in combination with it (lactobionate, raffinose, mannitol). A liquid can be oxygenated by exposure to a gas containing oxygen (O ₂ 20%), carbon dioxide (CO ₂ 6.5%) and nitrogen (N2 73.5%) at normal atmospheric pressure, the amount of oxygen being varied according to metabolic needs after blood gas analysis. Hyperoncotic fluid removes water from the interstitial tissue of the kidney and restores the capillary system. In addition, toxic products of the faulty metabolic process are flushed out and the pH level is restored using a buffer system such as bicarbonate, but may additionally or alternatively contain other agents or substances with similar effects (phosphate, histidine/histidine HC).

Because the kidney was subjected to ischemia for a long period of time, glycogen stores were depleted, resulting in a lack of ATP. So, the cellular ion pumps do not work and the Na/K balance inside and outside the cells is disrupted. The pH level decreases, lactate and pyruvate are produced and accumulate in the tissue. Hyperoncotic fluid contains glucose and/or adenine as a metabolic substrate, but may additionally or alternatively contain other agents with similar effects (α-ketaglutorate, histidine, glutamic acid). However, the metabolic rate is very low at such low temperatures.

Circulation is performed at reduced pump pressure. During circulation, vascular resistance gradually decreases, and circulation continues until vascular resistance is sufficiently low, which may take several hours.

The third step of the process involves additional microenvironmental restoration and evaluation of the kidney. The temperature is increased to approximately 32°C and the pressure is increased to 30 mmHg. Art. or up to 70 mm Hg. Art. Washed red blood cells (RBC) are added to a hematocrit number of 3 to 20, such as from to 10, for example from 6 to 8. Oxygenation is carried out using an oxygenator with increased oxygen levels. Typically, the kidney begins to produce urine and the ability to store creatinine is measured as an indicator of its performance. A known amount of creatinine may be added to the solution as a marker of kidney filtration capacity. In addition, the kidney is assessed visually. If the kidney has (large) dark areas, this may indicate kidney failure. In addition, the vascular resistance of the kidney is assessed.

If the kidney is deemed suitable for transplantation, the kidney is transplanted immediately or cooled to a low temperature of 4 to 15°C and stored until transplantation.

A storage period is also possible between the second and third stage.

The various steps can be modified in various ways.

The first step may be modified by performing one or more of the first steps after the second step. For example, the hyperoncotic fluid can be perfused for one hour, after which lys-plasminogen and tPA and optionally ATIII are added and the perfusion can be continued for another hour, after which lys-plasminogen and tPA and optionally ATIII are added again, and so on.

The second stage can be modified by perfusing hyperoncotic fluid during the first period for about one hour, and then lowering the colloid oncotic pressure, for example, by reducing the albumin concentration from, for example, 72 g/L to, for example, 57 g/L and circulate the liquid for another one to three hours. The hyperoncotic fluid may additionally contain dextran 40 at a dose of 0.1 to 10%, alone or in combination with albumin or any other hyperoncotic agent.

The third step can be modified by adding a coagulation inhibitor and/or a platelet inhibitor to prevent re-thrombosis of the treated thrombi. The same substances can be used as described above for the first step. The substances can be added to the red blood cell suspension before it is added to the kidney in the third step, to the perfusion solution before adding the red blood cells, or to both. It is believed that blood clots formed in the microcirculation and blood vessels are sticky and adhere to endothelial cells. When thrombi dissolve, they leave damage to the endothelial cells and glycocalyx. This damage will activate possible platelets and clotting factors in the erythrocyte suspension, which remain despite the erythrocytes being washed away. This activation can lead to new blood clots forming in the same location, which should be avoided. Antithrombin III, or any other direct thrombin inhibitor, will react with thrombin and deactivate and remove thrombin. Abciximab, or any other platelet inhibitor, prevents platelets from sticking together

- 7 045517 together and their adhesion to damaged endothelial cells. The addition of heparin or low molecular weight heparin is optional.

The third stage can be modified by performing it at a temperature of 28 to 37°C and a pressure of 30 to 90 mm Hg. Art.

Washing steps can be performed between steps and during steps. The flushing fluid passes through the kidney and is then disposed of. Thus, flushing fluid does not circulate through the kidney.

The kidneys are removed as quickly as possible, and it is not known how long the kidneys were subject to ischemia due to cessation of circulation or other reasons, but the ischemia time is more than 2 hours, such as about 3 hours or 4 hours or more. The kidney is removed by freeing the kidneys, aorta and vena cava, cutting the aorta and vena cava above and below the renal arteries and renal veins, as shown in Fig. 1. The kit is placed on the instrument table and the remains of the aorta and vena cava are cleared of visible blood clots.

As quickly as possible, lysplasminogen (5 to 1000 units) is injected into the renal arteries using a needle and syringe, and the renal artery is compressed in front of the syringe to ensure that all lysplasminogen reaches the renal arteries and into the kidney. When fluid leaves the kidney veins, it indicates that the kidneys have been perfused with lys-plasminogen. Lys-plasminogen interacts with clots in blood vessels and binds to clots and fibrins in clots. Then, the veins of the kidney are clamped, as well as both arteries of the kidney, until it is time to administer tPA.

Next, tPA is administered in the same way as lys-plasminogen. However, the veins can be left closed so that a slight excess pressure builds up inside the kidney. The aorta is then cannulated at one end and clamped at the other end, preparing the system for ex-vivo machine perfusion. Both ureters are cannulated, allowing urine monitoring.

A thrombin inhibitor, such as antithrombin III (ATIII) or argobatran, can be added along with lys-plasminogen and/or along with tPA.

It should be noted that the volume of lys-plasminogen administered, approximately 10 ml, corresponds to approximately half the volume of blood normally available in the kidney, which is approximately 15 to 30 ml per kidney (if the kidney weighs 150 g) (10 to 20 ml per 100 g kidney). The volume of tPA is also approximately 10 ml and the dose ranges from 0.5 to 10 mg per kidney.

Finally, the kidneys are placed in a closed container, with the kidneys suspended from the remnant aorta and connected to the circulation system, as shown in FIG. 3. The lower end of the vena cava is open so that fluid can flow freely from the kidneys while fluid is supplied to the aorta by the container's circulatory system.

Sometimes the kidneys are treated separately, with the aorta divided longitudinally into two parts and catheters connected to the renal arteries that still extend from the divided portion of the aorta, as shown in FIG. 3, which allows multiple arteries to be treated as one and makes it possible to monitor each kidney individually. In humans, in more than 30% of all kidneys, more than one artery can be seen per kidney. With the proposed technology this will not cause any problems.

As shown in FIG. 3, the aortic remnant 33 of the kidney 35 is connected to a connector 32 provided in the container 31. The vena cava 34 is open to discharge fluid to the bottom 37 of the container 31, as shown by the dotted line 36. The fluid level 36 may be below the kidney or above the kidney, or in between. levels, the last option is shown in Fig. 3.

The bottom 37 of the container is connected to the drainage bag 41 by a valve 42. The bottom is also connected to the pump 43 by a first switching valve 44. The first switching valve 44 connects the inlet of the pump 43 to either the flushing fluid bag 45 or the bottom 37 of the container 31, the first of these provisions is shown in Fig. 3.

The output of the pump 43 is connected to a heater/cooler 48, which regulates the temperature of the fluid passing through the pump. The output of heater/cooler 48 passes through second switch valve 46 to oxygenator 47 and leukocyte filter 49, or directly to connector 32 and kidney, the first of these positions being shown in FIG. 3. In the position shown, the liquid is oxygenated only by interaction with the surrounding atmosphere (oxygen dissolved in the liquid).

Thus, the fluid present at the bottom of the container 37 circulates through the first switch 44 to the pump 43 and then through the heater/cooler 48 to the second switch 46 and to the connector 32. The fluid then passes from the connector 32 to the aorta and to the kidney and through the vessels of the kidney and further to the veins of the kidney and finally comes out to the bottom of the container.

The pump 43 is a pressure-controlled pump such that the pump pressure is adjusted to a desired value, for example, 20 mmHg. Art., and the flow rate depends on the resistance of the kidney.

The liquid at the bottom 37 of the container 31 is supplied through a number of bags 50, 51, 52, 53 and 54, as shown in FIG. 3. Each bag is connected to container 31 using valves 55, 56, 57, 58 and 59. By opening the valves, the contents of the bags are transported to the bottom of the container by gravity.

In one embodiment, the operation proceeds as follows.

After the kidneys were exposed to lys-plasminogen and tPA on instrumental

- 8 045517 table after the kidneys are removed, the kidneys are transferred to the container 31 and the remainder of the aorta is connected to the connector 32 so that the kidneys hang inside the container 31. The veins are opened and the liquid is allowed to drain straight down to the bottom 37 of the container. The kidneys can be located on a support (not shown), for example, on a mesh, and can have different angles relative to the horizontal axis. The temperature in the container is set to the desired initial value, for example, from 18°C to 28°C. A circulating fluid containing electrolytes and optionally albumin at a concentration of 57 g/l can be supplied to container 31 from a circulating fluid bag 50 by opening the appropriate valve 55. The kidneys are then perfused starting at a pressure of 20 mm Hg. Art. for 5 minutes, then increase the pressure by 5 mmHg. Art. every 5 minutes until the pressure reaches 50 mm Hg. Art. up to 90 mm Hg Art., which was selected in advance. Then the pressure can be lowered, for example, to 20-30 mm Hg. Art. and perfuse for an additional period of time selected in advance. The resistance of the kidneys has decreased and now the kidneys are of a normal pale color, there are no dark zones at all or there are only small ones. This suggests that the blood clots in the blood vessels of the kidneys have dissolved and disappeared. The liquid inside the container now flows to drain 41 by opening drain valve 42.

Without being bound by any theory, it is believed that dark areas in the kidney indicate areas of lack of circulation, which may be caused by blood clots or other causes. If circulation is later achieved in these dark areas, the kidneys can restore these areas to functional tissue.

During the perfusion step, drain valve 42 is closed and 1000 ml of perfusion liquid 51 flows into container 31 by gravity by opening valve 56. The liquid level in the container will rise above line 36 when 1000 ml of liquid has accumulated at the bottom of the container, after which valve 56 is closed. The first switch valve 44 is in the first position shown in FIG. 3, the pump starts and pumps with a pressure of 20 mmHg. Art. The heater/cooler 48 regulates the temperature from 12 to 18°C. The liquid at the bottom of the container circulates at a pressure of 20 to 30 mmHg. Art. for several hours, for example one to four hours. During the perfusion phase, the chemistry of the kidney is restored and toxic products are removed. The pump is now stopped and flushed. The perfusion step can be repeated with a different perfusion solution composition after the wash steps, during the hypothermic perfusion period, or the perfusion can also be continued at a higher temperature, with or without red blood cells.

In the evaluation step, the same solution from the perfusion step can be used after opening the drain valve 42 to drain the contents of the container onto the valve 41, or the drain valve 42 is closed after the two washing steps described above, and 500 ml of the assessment liquid 52 flows into the container 31 by gravity. by opening valve 57. After 500 ml of liquid has entered, valve 57 is closed. The first switching valve 44 is in the first position shown in FIG. 3, and the pump starts and pumps at a pressure of 20 mmHg. Art. Heater/cooler 48 increases the temperature to 28-32°C. After 5 to 30 minutes, during which the pH and environment are checked, red blood cell suspension from bag 53 is added by opening valve 58 until a hematocrit value of 5 to 10 is reached, after which valve 58 is closed. The second switching valve 46 is moved to a second position containing an oxygenator 47 in the circuit for oxygenating fluid and red blood cells. Circulation continues for 1-4 hours with a pressure of 30 mm Hg. Art. until the kidney is deemed suitable for transplantation. The kidneys are now assessed at a temperature of 32-37°C and a pressure of 70-90 mmHg. Art. for 15 minutes, noting vascular resistance, flow rate, and appearance to determine how well the kidneys are perfused. The surgeon decides whether the kidneys were well, moderately or poorly perfused, with average meaning that there are a few blue/black spots and poor meaning that there are several large dark blue or black areas that are not perfused. Urine output is measured and flow is recorded in units of ml/min per 100 g of kidney tissue. The pump is now stopped and drain valve 42 is opened to drain all the liquid at the bottom of the container into drain 41.

Any flushing step can be accomplished by moving the first switching valve 44 to a second position connecting the pump 43 to the flushing fluid bag 45. The pump is activated and circulates approximately 200 ml to the kidneys. The liquid leaving the kidneys and passing to the bottom of the container passes further to drain 41 through the open valve 42. Thus, all red blood cells are washed out of the kidney and into the drain. Such washing steps can be performed several times and between other steps.

At the conservation stage, the drain valve 42 is closed and 500 ml of the preservation liquid 54 passes into the container 31 under the influence of gravity and by opening the valve 59. When 500 ml of liquid has entered, the valve 59 is closed. The first switch valve 44 is in the first position shown in FIG. 3, the pump starts and pumps at a pressure of 20 to 30 mmHg. Art. The heater/cooler 48 regulates the temperature to 6-18°C, for example 12-15°C. The liquid at the bottom of the container circulates at a pressure of 20-30 mmHg. Art. for several hours, up to 48 hours or more, until a recipient is found and the kidneys are prepared for transplantation into the recipient. The pump is stopped and drain valve 42 is opened to drain all liquid at the bottom of the container into drain 41. The kidney is removed from connector 32.

The fluids used for the embodiment shown in FIG. 3, can represent with

- 9 045517 fighting liquids indicated in the table. A.

During the procedure on the instrument table, lys-plasminogen is added to a carrier liquid, which may be identical to the base solution in the table. A.

In an embodiment, the carrier fluid, rinsing fluid, perfusion fluid, and preservation fluid may have the same basic components. These components contain sodium from 113 to 129 mmol, potassium from 5 to 25 mmol, magnesium from 2.5 to 5 mmol and calcium from 1 to 5 mmol. In addition to the above electrolytes, one or more of the following may be present: albumin at a concentration of 10 to 120 g/L, such as 40 to 75 g/L, such as 57 to 72 g/L; optionally dextran 40 from 0 to 100 g/l, hydroxyethyl starch (HES) from 0 to 70 g/l, polyethylene glycol (PEG 20) from 0 to 25 g/l or PEG 35 from 0 to 3 g/l, alone or in combinations. Dextrose 5 mmol and bicarbonate from 10 to 35 mmol are also optional. Additionally, amino acids such as L-arginine (a precursor to nitric oxide (NO), regulating blood pressure during physiological stress), L-leucine (protein synthesis), L-glutamine (synthesizes L-arginine, essential for amino acid production), or others, alone or in combination, may be added in normal concentrations (see Table A). Other substances that can be added are: l-inositol (membrane potential stabilizer), adenine and ribose (produces adenosine - part of ATP). You can also add microelements (cofactors and vitamins), such as those listed in the table. A. Naturally occurring hormones such as novorapid, T3, T4, progesterone and estrogen may be added at physiological concentrations (Table A). Antibiotics such as thienam may be added. This base liquid according to table. A may be used as at least one of the following: a carrier fluid, a flushing fluid, a circulation fluid, a perfusion fluid, and a preservation fluid.

- 10 045517

TABLE A Vitamins mmol COMPONENT L-ascorbic acid Na 0-2 Inorganic salts mmol D-biotin 0.005 CaC1 ₂ 2H ₂ O 0-5 Choline chloride 0-0.05 MgSO ₄ (anhydrous) 0-10 Folic acid 0-0.01 KN ₂ RO ₄ 0-60 Myo-inositol 0-0.1 KS1 0-25 Nicotinamide 0-0.1 _NaHCO3 0-80 D-pantothenic acid ACa 0-0.01 NaCl 0-140 Pyridoxine hydrochloride 0-0.01 Na ₂ HPO ₄ (anhydrous) 0-5 Riboflavin 0-0.001 Gluconate Na 0-110 Thiamine HC1 0-0.01 Gluconate K 0-25 Vitamin B12 0-0.01 Gluconate Mg 0-10 Hormones Lactobionate Na 0-110 TK 0-0.0000030 Lactobionate K 0-25 T4 0-0.0000030 Lactobionate Ca 0-10 Cortisol 0-0.0000166 Amino acids Insulin novorapid (U) 0-40 L-alanine 0-1 Progesterone 0-0.0100 L-arginine HC1 0-1 Estrogen 0-0.1004 L-asparagine H ₂ 0 0-1 Other L-aspartic acid 0-1 Adenosine 0-5 L-cysteine HC1 H ₂ 0 0-0.1 Cytidine 0-0.1 L-cystine 2HC1 0.5 2'-deoxyadenosine 0-0.1 L-glutamic acid/ketoglutarate 0-5 2'-deoxycytidine HC1 0-0.1 L-glutamine 0-15 2'-deoxyguanosine 0-0.1 Glutathione 0-5 Guanosine 0-0.1 Glycine 0-5 Pyruvic acid 0-5 L-histidine HC1 H ₂ 0 0-210 Thioctic acid 0-0.01 L-isoleucine 0-1 Thymidine 0-0.1 L-leucine 0-1 Uridine 0-0.1 L-lysine HC1 0-1 Allopurinol 0-5 L-methionine 0-1 Dextrose 0-200 L-phenylalanine 0-1 D-ribose 0-10 L-proline 0-1 Raffinose 0-60 L-serine 0-0.02 Mannitol 0-120 L-threonine 0-2 Hyperoncotic fluid L-tryptophan 0-4 HES 0-5 L-tyrosine 2Na 2H ₂ 0 0-1 PEG 35 0-0.5 L-valine 0-2 Albumin (g/l) 0-120 Medicines Dextran 40/70 (%) 0-15 Verapamil Heparin 0-10000 units Minirin 0-000000009

The perfusion fluid may have a high oncotic pressure, which is achieved by adding a hyperoncotic solution such as albumin at a concentration of 70 to 120 g/L, for example 72 g/L, and optionally dextran 40 (or dextran 70) at a concentration of 0 to 150 g/l, or any other known hyperoncotic fluid.

The potassium level can be maintained higher than in normal plasma, in the range of 13 to 25 mmol, for example 17 to 22 mmol, for example 18 mmol. Alternatively, the potassium concentration may be low, for example 1-13 mmol. Potassium and sodium levels will change during the red blood cell stage due to normalization of the physiological environment and pH.

The assessment fluid may also contain a coagulation inhibitor such as argobatran (or another direct thrombin inhibitor, such as inogatran, melagatran (and its prodrug ximelagatran), dabigatran or hirudin and their

- 11 045517 derivatives, or allosteric inhibitors; and platelet inhibitors such as glycoprotein Ilb/IIIa receptor antagonists (abciximab, eptifibatide, tirofiban);

irreversible cyclooxygenase inhibitors (aspirin, triflusal);

adenosine diphosphate (ADP) receptor inhibitors (cangrelor, clopidogrel, prasugrel, ticagrelor, ticlopidine);

phosphodiesterase inhibitor (cilostazol);

protease-activated receptor-1 (PAR-1) antagonists (vorapaxar);

adenosine reuptake inhibitors (dipyridamole);

thromboxane inhibitors (thromboxane synthase inhibitors such as ifetroban, picotamide and thromboxane receptor antagonists such as terutroban);

or any other platelet inhibitor, alone or in combination, to prevent re-thrombosis of treated blood clots.

The addition of heparin, protein C and protein S is optional.

Verapamil may also be added.

In fig. 4 shows another embodiment. This embodiment may be used in a local hospital that has facilities for kidney donation. The kidneys are pre-processed at this local hospital and then transported to the central hospital responsible for the transplantation to the recipient.

The cardiac arrest patient, the donor, is admitted to a local hospital after an unknown amount of time has passed since death, but less than 12 hours, 10 hours, 8 hours, 6 hours, or 4 hours. Once the donor arrives, the body can be cooled to slow down the body. harmful processes, namely metabolism and blood clotting. Such cooling may be external, in which the body is placed in a cooled room or space, for example, having an air temperature of approximately 0°C. Another method of cooling may be placing ice slush around the body or in the abdominal cavity, or injecting cold liquid into the abdominal and/or chest cavity.

If your local hospital has an organ harvesting team, the removal can be done as soon as possible, with or without refrigeration.

During kidney removal, routine procedures are performed and the kidneys are placed on an instrument table, either together or each kidney individually. The first stage of introducing lys-plasminogen into the arteries of the kidneys is performed on the instrument table. Lys-plasminogen is left in the blood vessels of the kidney for approximately 15 minutes or more, during which time the kidney is further prepared by attaching a connector to the arteries of the kidney or to the aorta.

After approximately 15 minutes, or as quickly as possible, the buds are transferred to the container device 60 shown in FIG. 4. Connector 63 of kidney 61 is connected to a corresponding connector 34 at the top of container 62, with kidney 61 suspended within container 61 as shown in FIG. 4. The kidney vein is opened directly into the container 61.

With connector 64 still open, syringe 75 is connected and approximately 10 mL of tPA (alteplase) is injected into the renal arteries through connector 64. It should be noted that administration of lys-plasminogen can alternatively be performed using a syringe connected to connector 64 prior to administration of tPA and instead of insertion on the instrument table. Again, alternatively, lys-plasminogen and tPA can be administered simultaneously using a syringe through connector 64 or administered simultaneously on the instrument table.

Other tPAs such as streptokinase, urokinase, reteplase and tenecteplase can be used instead of alteplase.

Tube 65 is then attached to connector 64. Tube 65 is installed in a spiral configuration and connects vacuum bag 66 to connector 64. Vacuum bag 66 is supported by stand 67 at a predetermined height, for example, 27 cm above container 64 (corresponding to a pressure of 20 mm Hg .). The height position of the vacuum bag 66 is adjusted along the rack 67 using a motor 68 with a worm gear. The spiral configuration of tube 65 allows tube 65 to be positioned at a desired height position. Initially the bag is empty.

The circulating fluid bag 76 contains circulating fluid containing electrolytes and a hyperoncotic agent such as albumin (Table A). The circulating fluid in bag 76 is introduced into container 62 as indicated by arrow 77. Approximately 1 L of circulating fluid is used and the volume of container 62 is approximately 1.3 L, which means that container 62 is almost completely filled with circulating fluid. The circulating liquid is oxygenated using an oxygenator using a mixture of gases O ₂ , CO2 and N2 at a speed of 1 l/min. The operation is performed at room temperature. The circulating fluid circulates in the system along with lys-plasminogen and tPA.

The amount of circulating fluid can be less than 1 L, but must be greater than the volume of tubes and pumps (excluding the container) and kidneys. In addition, it is necessary that a small volume be contained in the container. This volume may be 10 ml, 20 ml, 50 ml or more. Thus, from 150 to 1000 ml of circulating fluid can be used.

- 12 045517

Pump 69 is connected to container 62 via tube 70 and pumps circulating fluid from container 62 into vacuum bag 66. Level sensor 71 starts pump 69 when the liquid level in container 62 reaches a predetermined value. The pump may be a peristaltic pump having a fluid flow rate corresponding to the rotational speed of the pump. There is no need to adjust the pump pressure.

The pump 69 may operate at a rate of, for example, 75 ml/min for one minute, with 75 ml of fluid being transferred from the container 62 to the vacuum bag 66. The pump flow rate is selected in accordance with the desired flow rate through the kidney, which indicates that The kidney resistance is quite low. A flow rate of 50 ml/min per 100 g of kidney is generally considered sufficient. A typical adult male kidney weighs approximately 150 g.

Since the vacuum bag is located at a height of approximately 27 cm above the container 62, the flow of circulating fluid passes through the kidney at a specified pressure of 27 cm of water, which corresponds to 20 mm Hg. Art. If the kidney resistance is high, this pressure of 27 cmH2O may result in a kidney flow rate of, for example, 7.5 ml/min, which means that the fluid level in container 62 returns to the level sensor 71 value in 10 minutes. Then the pump 69 is started by the level sensor 71 and the procedure is repeated.

The vacuum bag can be moved to a higher position using a 68 worm gear motor. In an embodiment, the worm gear motor 68 increases the height of the vacuum bag by 1 cm per minute. When the pressure increases, for example after 10 minutes to 35 cm H2O, the flow through the kidneys increases, for example, to 15 ml/min. This means that pump 69 is switched on again after 5 minutes. When the pressure has increased so that the flow rate through the kidney is 75 ml/min, the pump 69 will operate continuously. This indicates that the kidney resistance is quite low. The increase in height of the vacuum bag 66 will now be stopped.

The maximum height of the vacuum bag can be, for example, 95 cm, which corresponds to a pressure of 70 mmHg. Art. If the desired flow rate (75 ml/min for a 150 g kidney) has been achieved before placing the vacuum bag in the highest position (after 70 min maximum), the procedure is interrupted. If the desired flow rate has not been achieved, the operation is continued for another 30 minutes at a pressure of 70 mmHg. Art. (95 cm water column).

Until now, the operation was carried out at room temperature from 18 to 28°C.

Container 62 has a jacket 72 covering a large bottom portion of the container. Slush ice can now be placed in the jacket to cool the container 62 to a temperature of about 5-18°C, for example 12-15°C. The vacuum chamber is lowered to 25 cm and a pump is turned on to circulate circulating fluid through the kidney. The jacketed container and vacuum bag may be placed in an isolation housing (not shown) and the entire device transported to the main hospital responsible for long-term storage. The kidney can be stored in this state for a long time, up to 48 hours or longer.

At the main hospital where the transplant can be performed, the kidney is processed further. The bud can be left in the same container 62 or moved to a different container 82 as shown in FIG. 5. Container 82 has a connector 84 to which the kidney is connected. Pump 89 pumps fluid from container 82 through tube 87 into the inlet artery of the kidney and fluid exits container 82 through a vein as previously described. The pump 89 is a pressure controlled pump and maintains the pressure at a desired level, for example, 20-30 mmHg. Art. A heater/cooler (not shown) maintains the temperature at the desired level, for example, 12-32°C, during perfusion and preservation. The drain bag 85 is connected to the bottom of the container 82 by means of a valve 86.

The device according to Fig. 5 also contains a bag 91 for red blood cell suspension. The red blood cell suspension bag is connected to a circulation system containing three pumps 92, 93, 94 connected in parallel. A cytokine filter 95 is mounted in series with pump 92, an endotoxin filter 96 is mounted in series with pump 93, and an oxygenator 97 and leukocyte filter 98 are mounted in series with pump 94. When the suspension valve 99 located downstream of the red blood cell suspension bag 81 is opened, the red blood cell suspension is circulated by the pumps. 92, 93, 94 through appropriate filters 95, 96, 98 and through oxygenator 97. Circulation can be carried out for approximately 30 minutes at room temperature. Thus, the erythrocyte suspension is processed to remove endotoxins, cytokines and leukocytes. In addition, the erythrocyte suspension is oxygenated.

The evaluation solution is contained in a bag 101 and is connected to container 82 by valve 102. When the valve is opened, the evaluation solution flows by gravity into container 82, which was previously emptied through drain 85 by opening valve 86. The evaluation solution is heated to a temperature of approximately 32°C. . The evaluation solution is circulated through the kidney using pump 89, and the kidney assumes a temperature of 32°C. The red blood cell suspension is then introduced into the container by opening the valve 99 and the other two valves 103 and 104, as shown in FIG. 5. After the suspension has flowed by gravity into the container 82, the red blood cell suspension valve 99 is closed.

Now the pumps 92, 93, 94 pump the liquid present in the container 82 through the open valves 104 and 103 and through the filters 95, 96, 98 and through the oxygenator 97. Thus, ensuring the absence of

- 13 045517 assessment fluid of any endotoxins, cytokines and leukocytes. In addition, red blood cells are oxygenated. The kidney can now be assessed, for example, by measuring blood parameters such as partial pressure of oxygen, partial pressure of carbon dioxide, HCO ₃ , oxygen saturation, hemoglobin, hematocrit, lactate, glucose, pH and others. In addition, the kidney can be examined visually. The resistance can be calculated from the pump data. Urine production, including creatinine concentration, can be tested if creatinine is added to the assessment fluid. In this way, the suitability of the kidney for transplantation is assessed.

The contents of container 82 are then drained to drain 85 by opening valve 86. Preservation liquid 105 is introduced into container 82 by opening valve 106. Preservation liquid is circulated by pump 89 at a pressure of 20 to 30 mmHg. Art. and low temperature 12-15°C to remove all red blood cells. In addition, it happens that the kidney increases its weight, and the preservation fluid contains albumin to remove excess water accumulated in the kidney. After a storage period of up to 7 days (or longer), the kidney is transplanted.

Another embodiment is shown in FIG. 5A. The apparatus includes a device 110 for washing the red blood cell suspension (not shown in detail). The washed red blood cells in the bag 111 enter the conditioning container 114 through a tube 112 and through a valve 113. The container 114 has a conditioning circuit 115 containing a first pump 116, an oxygenator 117, a cytokine absorber 118, a leukocyte filter 118, a first valve 120, and a second valve 121. In addition In addition, the first valve connector 122 is connected to the first valve 120, and the second valve connector 123 is connected to the second valve 121.

When the first and second valves 120, 121 are open and the valves in the connectors 122, 123 are closed, the conditioning circuit operates to condition the fluid in the container 114 by circulating the fluid within the container 114 using the pump 116 through the oxygenator 117, the leukocyte filter 119 and the cytokine absorber 118 to remove leukocytes and cytokines in the red blood cell suspension present within the container 114. When circulation has already taken place for at least 30 minutes, for example 60 minutes or more, the red blood cell suspension is ready.

The processing system 130 is shown on the right in FIG. 5A. The treatment system includes a treatment container 131 having a third valve connector 132 that can be connected to the first valve connector 122, and a fourth valve connector 133 that can be connected to the second valve connector 123.

The treatment container 131 has three liquid bags 134, 135, 136, which are connected to the container 131 via tubes provided in the valves. By opening such valves, liquid from the corresponding liquid bag can be poured into the processing container. The waste bag 137 is connected to the bottom of the treatment container via a tube provided in the valve. By opening the valve, the liquid in the container 131 can be drained into the waste bag 137.

The harvested kidney(s) 140 comprise a kidney connector 141 connected to the remnant aorta or to the renal artery(s). The kidney connector 141 may be connected to a connector 142 provided in the container. The container connector 142 is connected to a renal circulation system containing a circulation pump 143 and an oxygenator 144. The pump 143 circulates fluid in the treatment container from the bottom thereof through the pump and oxygenator to the connector 142 and to the renal artery 140. Fluid exiting the renal vein , remains inside the container for further circulation.

The conditioning system includes a second pump 145 connected to the bottom of the treatment container 131 to pump the liquid in the treatment container 131 through the endotoxin absorber 146 and the leukocyte filter 147 and back to the treatment container.

Two medical infusion pumps 148, 149 are available to infuse medications into the renal artery stream. Infusion pumps may be syringe pumps. Medications to be infused may be lys-plasminogen, tPA, ATIII, platelet inhibitor, thrombin inhibitor, coagulation inhibitor and others. Medical infusion pumps 148, 149 may be replaceable so that additional syringe pumps can be attached.

Heater/cooler 150 is installed to heat/cool liquid exiting the treatment container.

The system can work as follows.

The red blood cell suspension is washed in the washing system 110 according to known methods. When the washing is completed, the red blood cell suspension is transferred by gravity to the conditioning container 114 by opening the valve 113.

The liquid in conditioning container 114 is circulated by pump 116 by opening valves 120 and 121 and operating pump 116 to pump liquid through leukocyte filter 119 and cytokine absorber 118, thereby removing remaining leukocytes and cytokines.

Meanwhile, the kidney 140 is taken from the donor and placed in the processing container 131 and connected to the container connector 142 using the kidney connector 141. This operation can be performed away from the left side of the system shown in FIG. 5A, for example, in a remote hospital.

- 14 045517

The treatment container 131 holds treatment fluid from bags 134, 135, 136 as needed. The used liquid is discharged into the waste bag 137 by opening the drain valve.

A second circulation pump 145 is included to pump the liquid in the treatment container through the leukocyte filter 147 and endotoxin absorber 146 to continuously remove leukocytes and endotoxins.

The circulation pump 143 is activated to pump fluid from the container through the oxygenator 144 and into the kidney artery and then through the kidney vein back to the kidney treatment container as described above.

Medical pumps 148, 149 can be used to administer medical supplies.

Next, the treatment system 130 is connected to the air conditioning system 115 by connecting the valve connector 122 to the valve connector 132 and connecting the valve connector 123 to the valve connector 133 and by opening valves other than valve 123. The first valve 120 is closed and the second valve 121 is opened. , while the pump 116 operates and pumps the liquid in the conditioning container 114 through the connectors 122, 132 into the treatment container. Next, the second valve 121 is closed and the valve 123 is opened, with the pump 116 pumping the treatment fluid in the treatment container 131 through the oxygenator 117, the leukocyte filter 119 and the cytokine absorber 118.

Processing can be performed according to any of the methods described above.

As shown in FIG. 5B, the processing container 151 may be modified to process two buds separately, although the buds may also be arranged as a block. The two kidneys have two kidney connectors 161, 171 connected to container connectors 162, 172, which are connected to circulation pumps 163, 173 as shown. The oxygenator 164 is installed in a common line of the two circulation systems as shown. With such a system, each individual kidney can be supplied with individual medical supplies via medical infusion pumps 168, 169 and 178, 179 for various treatments.

The entire ex-vivo perfusion device can be adapted to different organs. For example, the liver is larger than the kidneys and may require more fluid.

Example 1.

Procedure before kidney removal: 30 pigs were anesthetized and ventilated, after which the ventilator was turned off. Heart failure and circulatory arrest appeared after 15 minutes. After two hours at room temperature, cold saline was placed into the abdominal and chest cavities, after which no further action was taken for one hour.

Kidney removal: three hours after the cessation of blood circulation, the operation to remove the kidneys began, which takes an average of 45 minutes.

Instrument table procedure: On the instrument table, the kidneys were flushed through the renal artery with SOLTRAN (renal perfusion fluid, Baxter Healthcare) after administering approximately 500 units of heparin in 10 ml lidocaine 0.5-1% diluted to 20 ml using 0 .9% saline, per kidney.

Treatment procedure: The kidneys were divided into two groups: group A of 11 pigs, group B of 13 pigs, group C of one kidney from each of 6 pigs and group D of the remaining one kidney from the same 6 pigs.

Group A underwent cold storage for 2 hours, after which transplantation was performed.

- 15 045517

TABLE B COMPONENT g/l mmol Vitamins g/l mmol Inorganic salts L-ascorbic acid Na 0.05 0.284 CaC1 ₂ 2H ₂ O 0.263 2.3698 D-biotin 0.0001 0.00041 MgSO ₄ (anhydrous) 0.09767 0.81144 Choline chloride 0.001 0.00716 KS1 0.4 5.36543 Folic acid 0.001 0.00227 _NaHCO3 4.72 56.18606 Myo-inositol 0.002 0.0111 NaCl 6.8 116.35269 Nicotinamide 0.001 0.00819 Na ₂ HPO ₄ (anhydrous) 0.122 0.8594 D-pantothenic acid ’LSa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-alanine 0.025 0.2806 Riboflavin 0.0001 0.000266 L-arginine HC1 0.126 0.72329 Thiamine HC1 0.001 0.00296 L-asparagine H ₂ 0 0.05 0.37845 Vitamin B12 0.00136 0.00136 L-aspartic acid 0.03 0.22539 Other L-cysteine HC1 H ₂ 0 OD 0.00186 Adenosine 0.01 0.03742 L-cystine 2HC1 0.0313 0.0999 Cytidine 0.01 0.04112 L-glutamic acid 0.075 0.50975 2'-deoxyadenosine 0.01 0.0698 L-glutamine 0.292 2.00 2'-deoxycytidine HC1 0.011 0.04841 Glycine 0.05 0.66607 2'-deoxyguanosine 0.01 0.03742 L-histidine HC1 H ₂ 0 0.042 0.200 Guanosine 0.11 0.03531 L-isoleucine 0.052 0.396 Pyruvic acid 0.11 1,249 L-leucine 0.052 0.396 Thioctic acid 0.0002 0.00097 L-lysine HC1 0.0725 0.397 Thymidine 0.01 0.04111 L-methionine 0.015 0.101 Uridine 0.01 0.04095 L-phenylalanine 0.032 0.194 Hormones L-proline 0.04 0.34743 technical specifications 0.000000001953 0.000000030 L-serine 0.025 0.0002356 T4 0.00000000233 0.000000030 L-threonine 0.048 0.403 Cortisol 0.000006 0.0000166 L-tryptophan 0.01 0.0490 Insulin novorapid 5 units L-tyrosine 2Na 2H ₂ 0 0.0519 0.199 Minirin 0.00000001 0.000000009 L-valine 0.046 0.393 Medicines Verapamil 0.005 Tienam 0.050 Geparin 500 units

Group B was cold stored for 2 hours, followed by 90 minutes of reconstitution at 37°C with a solution of the composition shown in Table 1. B, and mixed with washed red blood cells until the hematocrit reached approximately 15, after which transplantation was performed.

Group C was treated with hypothermic perfusion for 4 hours immediately after kidney retrieval using the Lifeport kidney transfer device and renal perfusion solution (RPS) according to the manufacturer's instructions (manufacturer Organ Recovery Systems), followed by transplantation.

Group D contained kidneys from the same donors as group C but were processed by cold storage for 2 hours before transplantation.

Fig. Figure 6 illustrates the change in arterial blood flow (ml/min) after reperfusion and when observed for 90 minutes after transplantation.

Fig. 7 illustrates the average urine output in ml/min 90 minutes after transplantation. Using the Mann-Whitney U test, rescue group B had better flow in perfusion (p<0.05) (Figure 6) and rescue group B also had better urine flow (p<0.05) at 90 min after transplantation (Fig. 7).

Example 2.

The pigs were given anesthesia and provided with normal ventilation, after which the ventilator was turned off. Heart failure appeared 15 minutes later. After two hours at room temperature, a cold preservation solution (saline solution) was placed into the abdominal and chest cavities.

Kidney removal began 4 hours after death.

The kidneys were flushed on the instrument table with cold Ringer's solution after administering approximately 500 units of heparin in 10 mL of lidocaine 0.5-1% diluted to 20 mL with 0.9% saline, per kidney, through the renal artery.

The kidneys were connected to an ex-vivo perfusion device similar to that shown in FIG. 3. 0.4 mg tPA (alteplase) and 150 units of apyrase (purinergic drug CD39/CD73, manufactured by Sigma Aldrich) were injected through the renal artery. The composition of the solution is shown in table. WITH.

The solution was perfused for 30 min at a temperature of 15°C and a pressure of 20 mmHg. Art. no ok

- 16 045517 sigenation. The temperature was increased to 32°C, the pressure to 30 mm Hg. Art., and perfusion was continued for another 90 minutes during the recovery phase. Washed red blood cells with white blood cells removed were then injected and perfusion was continued for another 90 min during assessment.

Then a kidney transplant was performed.

Fig. 8 illustrates renal blood flow after reperfusion and 90 minutes later. The microcirculation in the kidneys has not been completely cleared and perfusion defects are visible on the surface of the kidneys. Fibrinolytic drugs as well as purinergic drugs (CD39/CD73 apyrase) added to the ex-vivo system did not significantly improve vascular resistance and flow.

TABLE C COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ 0 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 21.80 100.00 D-pantothenic acid ’ACa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-glutamine 0.292 2.00 Riboflavin 0.0001 0.000266 L-arginine HC1 0.105 0.603 Thiamine HC1 0.001 0.00296 L-cystine 2HC1 0.0313 0.0999 Other L-histidine HC1 H ₂ 0 0.042 0.200 Adenine 0.68 5.00 L-isoleucine 0.052 0.396 Dextrose 1.00 5.55 L-leucine 0.052 0.396 D-ribose 0.75 5.00 L-lysine HC1 0.0725 0.397 Albumen 72 L-methionine 0.015 0.101 Minirin 0.00000001 L-phenylalanine 0.032 0.194 Verapamil 0.005 L-proline 0.04115 0.100 Hyenas 0.050 L-threonine 0.048 0.403 Geparin 500 units L-tryptophan 0.01 0.0490 Apyrase 150 units L-tyrosine 2Na 2H ₂ 0 0.0519 0.199 Alteplase 0.4 mg L-valine 0.046 0.393 Hormones technical specifications 0.000000001953 0.000000030 T4 0.00000000233 0.000000030 Sterile water Cortisol 0.000006 0.0000166 Insulin novorapid 5 units Progesterone 0.00315 0.001002 Estrogen 0.00001 0.00004

Example 3.

In another experiment, the protocol of example 2 was repeated, but apyrase was not introduced. Instead, lidocaine was added to the perfusion solution. The idea was to seek a stabilizing effect during the recovery phase, as a result of the proven effect of lidocaine on the sodium-potassium pump. The composition of the solution is shown in table. D.

TABLE D COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Mio-ino ZITO l 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 21.80 100.00 D-pantothenic acid ’ACa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-glutamine 0.292 2.00 Riboflavin 0.0001 0.000266 L-arginine HC1 0.105 0.603 Thiamine HC1 0.001 0.00296

- 17 045517

L-cystine 2HC1 0.0313 0.0999 Other L-histidine HC1 H ₂ 0 0.042 0.200 Adenine 0.68 5.00 L-isoleucine 0.052 0.396 Dextrose 1.00 5.55 L-leucine 0.052 0.396 D-ribose 0.75 5.00 L-lysine HC1 0.0725 0.397 Albumen 72 L-methionine 0.015 0.101 Minirin 0.00000001 L-phenylalanine 0.032 0.194 Verapamil 0.005 L-proline 0.04115 0.100 Tienam 0.050 L-threonine 0.048 0.403 Heparin 500 units L-tryptophan 0.01 0.0490 Lidocaine 0.060 0.256 L-tyrosine 2Na 2H ₂ 0 0.0519 0.199 Hormones L-valine 0.046 0.393 technical specifications 0.000000001953 0.000000030 T4 0.00000000233 0.000000030 Cortisol 0.000006 0.0000166 Sterile water Insulin novorapid 5 units Progesterone 0.00315 0.001002 Estrogen 0.00001 0.00004

Fig. 9 illustrates the flow unchanged for the first 90 minutes, with the flow typically decreasing. In addition, the change in weight was minimal between the different stages of recovery, despite the fact that the osmolarity was 330 mOsmol and the potassium level was approximately 5 mmol/L. In example 2, the kidneys gained significantly more weight from the beginning of perfusion until the end after 90 minutes of reperfusion.

Example 4.

The pigs were given anesthesia and provided with normal ventilation, after which the ventilator was turned off. Heart failure appeared 15 minutes later. After two hours at room temperature, cold saline was placed into the abdominal and chest cavities.

Removal of both kidneys from each pig began 4 h after death.

The kidneys were flushed on the instrument table with cold Ringer's solution after administration of approximately 500 units of heparin in 10 ml of lidocaine 0.5-1% diluted to 20 ml using 0.9% saline, per kidney, through the renal artery as described in example 2. The composition of the solution is shown in table. E.

TABLE E COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1DZ 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 21.80 100.00 D-pantothenic acid ¹ HPa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-glutamine 0.292 2.00 Riboflavin 0.0001 0.000266 L-arginine HC1 0.105 0.603 Thiamine HC1 0.001 0.00296 L-cystine 2HC1 0.0313 0.0999 Other L-histidine HC1 H ₂ 0 0.042 0.200 Adenine 0.68 5.00 L-isoleucine 0.052 0.396 Dextrose 1.00 5.55 L-leucine 0.052 0.396 D-ribose 0.75 5.00 L-lysine HC1 0.0725 0.397 Albumen 72 L-methionine 0.015 0.101 Minirin 0.00000001 L-phenylalanine 0.032 0.194 Verapamil 0.005 L-proline 0.04115 0.100 Tienam 0.050 L-threonine 0.048 0.403 Geparin 500 units L-tryptophan 0.01 0.0490 Hormones L-tyrosine 2Na 2H ₂ 0 0.0519 0.199 technical specifications 0.000000001953 0.000000030 L-valine 0.046 0.393 T4 0.00000000233 0.000000030 Cortisol 0.000006 0.0000166 Insulin novorapid 5 units Sterile water Progesterone 0.00315 0.001002 Estrogen 0.00001 0.00004

The kidneys were perfused ex-vivo for 30 min at a temperature of 15°C and a pressure of 20 mmHg. Art. The perfusion solution was then oxygenated first using a gas mixture of _O2 (20%), CO2 (5.6%) and N2 (74.4%). After one hour, the solution was replaced with a new solution containing

- 18 045517 becoming according to table. E, the temperature was raised to 32°C and the pressure was set to 30 mmHg. Art. Washed red blood cells with white blood cells removed were then injected and a hematocrit of approximately 10-15 was achieved, after which perfusion was continued for another 5 hours. One kidney was removed from the perfusion system, transplanted (n=7) into a recipient pig and observed for a period of 90 minutes up to 8 hours. The remaining kidneys in the ex-vivo perfusion system were perfused for another 90 minutes for comparison.

Fig. 10 illustrates the arterial blood flow of the kidney after transplantation. The kidneys continued to function and produce urine for more than ten hours (n=3). Only a few animals were observed for up to ten hours, which explains the increased variation in fluxes. At the end of the test, the pigs were still under anesthesia.

Example 5.

In another trial, treatment was carried out according to the same protocol as example 4 (n=5), but ex-vivo perfusion continued for 11 hours instead of 6 hours. Flow rates were 104 ml/min during reperfusion and 109 ml/min. min 14.5 hours after reperfusion. Thus, even increasing the duration of perfusion can result in a significant flow rate, which provides additional time for transporting kidneys and waiting for recipients to arrive.

TABLE F COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 21.80 100.00 D-pantothenic acid ’LSa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-glutamine 0.292 2.00 Riboflavin 0.0001 0.000266 L-arginine HC1 0.105 0.603 Thiamine HC1 0.001 0.00296 L-cystine 2HC1 0.0313 0.0999 Other L-histidine HC1 H ₂ 0 0.042 0.200 Adenine 0.68 5.00 L-isoleucine 0.052 0.396 Dextrose 1.00 5.55 L-leucine 0.052 0.396 D-ribose 0.75 5.00 L-lysine HC1 0.0725 0.397 Albumen 80 L-methionine 0.015 0.101 Minirin 0.00000001 L-phenylalanine 0.032 0.194 Verapamil 0.005 L-iroline 0.04115 0.100 Tienam 0.050 L-threonine 0.048 0.403 Geparin 500 units L-tryptophan 0.01 0.0490 Hormones L-tyrosine 2Na 2H ₂ 0 0.0519 0.199 technical specifications 0.000000001953 0.000000030 L-valine 0.046 0.393 T4 0.00000000233 0.000000030 Cortisol 0.000006 0.0000166 Insulin novorapid 5 units Progesterone 0.00315 0.001002 Sterile water Estrogen 0.00001 0.00004

Example 6.

In another experiment, 7 pigs were killed according to the same protocol as in example 4. Warm ischemia time (WTI) ranged from 4 to 6 hours.

Removal of both kidneys from each pig began 4 h after death.

Ex-vivo perfusion was started at 15°C. During the first hour, 72 g of albumin per liter was used (solution according to Table E) and the pressure during perfusion was 20 mmHg. Art. Oxygenation began after 30 minutes. After 1 hour, the solution was drained and a new solution was used with 80 g of albumin per 1 liter (solution in accordance with Table F) for 2 hours after increasing the temperature to 32 ° C, the pressure to 30 mm Hg. Art. and adding red blood cells.

Fig. 11 illustrates the arterial blood flow of the kidney after transplantation. All seven kidneys showed good flow rates at 4 hours (118.7 ± 15.0 ml/min) and 8 hours (85.0 ± 10.5 ml/min) after kidney transplantation and maintained urine production. The test was completed after 8 hours on pigs that were still under anesthesia. Although the flow rate deteriorated during the follow-up time, the kidneys still had a good flow rate at the end of the follow-up. For ethical reasons, the pigs were not awakened.

- 19 045517

TABLE G COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1DZ 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 21.80 100.00 D-pantothenic acid ’LSa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-glutamine 0.292 2.00 Riboflavin 0.0001 0.000266 L-arginine HC1 0.105 0.603 Thiamine HC1 0.001 0.00296 L-cystine 2HC1 0.0313 0.0999 Other L-histidine HC1 H ₂ 0 0.042 0.200 Adenine 0.68 5.00 L-isoleucine 0.052 0.396 Dextrose 1.00 5.55 L-leucine 0.052 0.396 D-ribose 0.75 5.00 L-lysine HC1 0.0725 0.397 Albumen 57 L-methionine 0.015 0.101 Minirin 0.00000001 L-phenylalanine 0.032 0.194 Verapamil 0.005 L-proline 0.04115 0.100 Hyenas 0.050 L-threonine 0.048 0.403 Geparin 500 units L-tryptophan 0.01 0.0490 Hormones L-tyrosine 2Na 2H ₂ 0 0.0519 0.199 technical specifications 0.000000001953 0.000000030 L-valine 0.046 0.393 T4 0.00000000233 0.000000030 Cortisol 0.000006 0.0000166 Insulin novorapid 5 units Sterile water Progesterone 0.00315 0.001002 Estrogen 0.00001 0.00004

Example 7.

In another experiment, 4 pigs were killed according to the same protocol as in example 4. Local cooling with ice in the abdominal cavity was carried out after 2 hours, resulting in a change in body temperature from 37°C to approximately 20°C, compared to 25-29°C using liquid as a coolant.

The kidneys were removed, and the ITI was from 4 to 5 hours.

The kidneys were washed with a solution having the composition indicated in the table. G with 57 g albumin per liter as a hyperoncotic agent. The kidneys were flushed in an ex-vivo device at pressures ranging from 20 to 70 mmHg. Art., with an increase in pressure by 5 mm Hg. Art. every 5 minutes, and at a temperature of 18°C until the buds turn white. To obtain well-perfused kidneys, on average, 2 to 3 liters of solution and up to 50 minutes of time are required.

Using the same solution, the temperature was reduced to 12°C and the kidneys were perfused for 1 hour at a pressure of 20 mmHg. Art. The solution was then replaced with a solution containing 80 g of albumin per liter, as shown in the table. F, at a temperature of 28°C and a pressure of 30 mm Hg. Art. Red blood cells were added to give a final hematocrit of approximately 10-15. The kidneys were perfused with this solution for approximately 2 hours. The temperature was then raised to 32°C and the kidneys were perfused for an additional hour before the system was emptied and the solution containing 57 g albumin per liter used initially was added again. With this solution, perfusion was continued for 30 minutes at 15°C and a pressure of 20 mmHg. Art. before transplantation.

Fig. 12 illustrates the arterial blood flow of the kidney after transplantation. High flow rates after reperfusion can be achieved by thoroughly flushing the kidneys after removal. Urine flow can be maintained in recipients for a long time, but decreases after a long period of observation, suggesting that flushing of several liters may negatively affect the final result due to endothelial activation. As a result, after 4 hours (145.7 ± 29.3 ml/min) and after 8 hours (96.73 ± 12.8 ml/min) the flow was better than with local cooling with a cold preservation solution placed in the abdominal and chest cavities, as in previous examples 1-5, where a temperature of 25-29°C was achieved. For ethical reasons, the pigs were still under anesthesia at the end of the trial.

Example 8.

In another experiment, 8 pigs were killed according to the same protocol as in example 4, but local cooling was not performed.

The kidneys were removed 4 hours after death, and the kidneys were perfused on an instrument table using 200 to 500 ml of the drug Perfadex. All kidneys showed poor or moderately poor perfusion and poor clearance.

- 20 045517

The kidneys were then immediately transplanted into nephrectomized pigs. The average flow rate at reperfusion was 14.15 ml/min and after 90 minutes 36.8 ml/min. All buds looked blue/black after 90 minutes and there was no urine production as a result. This experiment was a control experiment showing that without any cooling or other treatment procedure, for example, according to embodiments of the present invention, a TTI of 4 ppm results in poor graft survival consistent with previous experience.

Example 9.

In another experiment, 6 pigs were killed according to the described protocol. Local cooling with ice slush in the abdominal cavity was carried out after 2 hours, resulting in a change in body temperature from 37°C to approximately 10-12°C, compared with 25-29°C using liquid as a coolant and from 20 °C when using ice.

The kidneys were removed, and the ITI was from 4 to 5 hours.

On the instrument table, after removal, 20 ml of a solution containing 72 g/l albumin solution (Table I) mixed with 10 units of lys-plasminogen and 200 units of antithrombin III (ATIII) was injected into each kidney through the renal artery after clamping the veins , and then the arteries after the solution has been injected.

The kidneys were placed in an ex-vivo device. After 15 minutes, each kidney was injected with 1 mg tPA (alteplase) and 100 units of ATIII, divided into 4 portions of 5 ml after dilution to 20 ml of a solution containing 72 g/l albumin solution (Table I). TPA was injected into the renal artery at 5-min intervals at 20°C, starting at a pressure of 20 mmHg. Art. and increasing each time by 5 mm Hg. Art.

TABLE AND COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 NaHCO: 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 17,451 80.00 D-pantothenic acid %Ca 0.001 0.00210 Potassium gluconate 4.685 20.00 Pyridoxine hydrochloride 0.001 0.00486 Amino acids Riboflavin 0.0001 0.000266 L-glutamine 0.292 2.00 Thiamine HC1 0.001 0.00296 L-arginine HC1 0.105 0.603 Other Hormones Adenine 0.68 5.00 technical specifications 0.000000001953 0.000000030 Dextrose 1.00 5.55 T4 0.00000000233 0.000000030 D-ribose 0.75 5.00 Insulin novorapid 5 units Albumen 57 Progesterone 0.00315 0.001002 Hyenas 0.050 Estrogen 0.00001 0.00004 Sterile water

At the end of the tPA infusion, the kidneys were perfused with a solution containing 72 g of albumin per 1 liter and the pressure was increased every 5 minutes by 5 mmHg. Art. until reaching 50-70 mmHg. Art. or until the kidneys are cleared, whichever comes first. The temperature was then reduced to 12°C and perfusion was continued for another 1 hour at a pressure of 20 mmHg. Art.

After washing with a solution containing 57 g/l albumin (Table H), a second dose of lysplasminogen was administered, followed by tPA and ATIII diluted with a solution of 57 g albumin per liter and sequentially fed through the artery as described above.

Then the perfusion was continued for 30 minutes at a pressure of 20 mmHg. Art. and temperature 12°C. The temperature was increased to 28°C and the pressure to 30 mm Hg. Art., then added red blood cells to obtain a hematocrit of 5 to 10. Before adding to the solution, red blood cells were pretreated for 2 hours using an external pump and were passed through a leukocyte filter. This was done to ensure that the white blood cell count was reduced before contact with the kidneys. Perfusion with a combined solution of albumin (57 g albumin per 1 liter, Table H) and red blood cells lasted 1 hour. The temperature was then increased to 37°C and the pressure to 70-90 mm Hg. Art. for one hour during the assessment. After flushing the kidneys from the solution containing red blood cells, filling them with a new solution of albumin 57 g/l and lowering the temperature to 15°C, transplantation was performed.

Transplantation was performed in two pigs in which their own kidneys were nephrectomy immediately prior to kidney transplantation. Three animals were awakened from anesthesia (ten days of observation were provided for ethical reasons). On the third day, one animal had its liver checked and a new blood test taken. Immunosuppression consisted only of steroids during reperfusion. The kidneys looked good, blood creatinine was 615 µmol/l. The other pig was checked on day 4 and the kidneys also looked good and the blood creatinine was 884 µmol/L. The third pig was bled on day 5 and then continued for a total of 8 days. All three pigs showed signs of delayed

- 21 045517 translantate function (OFT), which in one case led to uremia and death after 8 days, in two other cases the animals were sacrificed on days 3 and 4 with elevated creatinine levels in the blood and high creatinine in the urine, which indicated ability to concentrate urine.

Fig. 13 illustrates arterial flow for each of six pigs after reperfusion and 90 minutes later. The addition of Lys-plasminogen and tPA cleansed the kidneys better than ever before, resulting in decreased resistance and improved microcirculation.

Fig. 14 illustrates that some kidneys had spots on the surface after reperfusion, which sometimes proliferated to produce blue/black poorly perfused kidneys several hours after transplantation, suggesting either re-thrombus formation due to the coagulation system or due to platelet-induced action /erythrocytes on the endothelium.

Example 10.

In three more pigs, the protocol according to example 9 was repeated with some differences.

After the kidneys were removed and on the instrument table, lys-plasminogen was administered as described above. After 15 min, the kidneys were flushed with 2 mg tPA (alteplase) in a 500 ml solution containing 57 g albumin per liter (Table H), after which the kidneys were placed in an ex-vivo device.

In the ex-vivo device, the kidneys were perfused with a solution containing 72 g/L albumin (Table I) at 24°C and increasing the pressure from 20 to 50-70 mm Hg. Art. Using the same solution, perfusion was continued for 2 hours at 12°C and a pressure of 20 mmHg. Art.

After emptying and washing, a solution containing 57 g of albumin per 1 liter (Table H) was introduced along with washed red blood cells to achieve a hematocrit of 5 to 10, the temperature was changed to 28 ° C, the pressure to 30 mm Hg. Art. A new dose of lys-plasminogen, 2 mg tPA and 200 ATIII units was administered, the temperature was increased to 32°C, after which perfusion was carried out for 15 minutes at a pressure of 70 mmHg. Art. for rate. After washing and rinsing, a solution containing 57 g/l albumin was added and the temperature was reduced to 15°C. All three pigs were recovered from anesthesia and observed. Two animals lived for 10 days, with blood creatinine levels in one being 166 µmol/L and in the other 174 µmol/L; at the end of the experiment the kidneys looked normal. The third pig was sacrificed on day 6 with delayed transplantation function and a blood creatinine level of 1231 μmol/L and a urine creatinine level of more than 2000, indicating the ability to concentrate urine. All recipients received steroids only as the sole immunosuppressive agent. Samples were taken for histological study of allo-rejection.

Fig. Figure 15a illustrates a normal-appearing kidney graft 10 days after transplantation. There are no macroscopic signs of rejection.

Fig. 15B illustrates the cut surface of the transplanted liver shown in FIG. 15a, with a normal macroscopic structure.

Fig. 15C illustrates the surface of a second kidney that has been in use for 10 days and has areas of black spots indicating microcirculation impairment.

Fig. 15D illustrates the cut surface of the transplanted liver shown in FIG. 15C, which has dark zones with impaired circulation in the lower pole. Data suggest that treatment can restore both function and structure, although the risk of local microcirculation disruption remains.

Example 11.

In another experiment with 19 pigs, cytokine levels were measured in three groups. Group A (n=5), containing kidneys from living donors perfused with a 72 g/L albumin solution (Table I) without any cytokine scavenger, was sampled at the start of perfusion, after 3 hours and at the end at 37 °C. Group B (n=5), containing living donor kidneys perfused with a 57 g/L albumin solution (Table H) without any cytokine scavenger, was sampled at the start of perfusion, after 3 hours and at the end at 37 °C. In group C (n=9), containing kidneys from donors who died of cardiac arrest, perfused using a 72 g/L albumin solution for 90 minutes and a 57 g/L albumin solution for 90 minutes before adding red blood cells and perfusion at 37 °C, and during all this a cytokine absorber (cytosorb, cytosorbents) was used. An enzyme-linked immunosorbent assay kit (Quantikin enzyme-linked immunosorbent assay kit, manufactured by R&D Biosystems) was used for the analysis. Values below detection limits were set to zero, and averages of the measured values were calculated for each group.

Fig. 16A illustrates a graph showing changes in IL-6 levels primarily due to red blood cells. The scavenger (group C) effectively removed all traces of IL-6.

Fig. 16B illustrates a graph showing changes in IL-8 levels, also primarily due to red blood cells and to a lesser extent due to a 57 g/L instead of 72 g/L albumin solution. The scavenger removed IL-8 (group C).

Fig. 16C illustrates a graph showing changes in IL-1e levels also primarily due to red blood cells. The absorber again removed all evidence of cytokines derived either from the kidney itself or from the injected red blood cells.

Fig. 16D illustrates a graph showing changes in TNF-α levels, also generally

- 22 045517 due to red blood cells. The absorber again removed all evidence of cytokines derived either from the kidney itself or from the injected red blood cells. Data not shown include analysis of IL-10 levels, where it was not possible to detect any levels in either group.

Example 12.

In another experiment, pigs were killed according to the above protocol, local cooling with slush ice was used after 2 hours, and kidney removal began after 4 hours.

The kidneys were obtained with a VTI of 4.5 to 5 hours.

TABLE I COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 21.80 100.00 D-pantothenic acid ’LSa 0.001 0.00210 Amino acids Pyridoxine hydrochloride 0.001 0.00486 L-glutamine 0.292 2.00 Riboflavin 0.0001 0.000266 L-arginine HC1 0.105 0.603 Thiamine HC1 0.001 0.00296 Hormones Other technical specifications 0.000000001953 0.000000030 Adenine 0.68 5.00 T4 0.00000000233 0.000000030 Dextrose 1.00 5.55 Insulin novorapid 5 units D-ribose 0.75 5.00 Progesterone 0.00315 0.001002 Albumen 72 Estrogen 0.00001 0.00004 Tienam 0.050 Sterile water

On the instrument table, 10 units of lys-plasminogen and 2 mg of tPA were injected into the kidneys, each in a solution of 15 ml.

The kidneys were placed in an ex-vivo device and perfused. The perfusion solution contained the ingredients shown in the table. J with albumin at a concentration of 57 g/l and with an increase in pressure from 20 to 70 mm Hg. Art., by 5 mm Hg. Art. every 5 min. During the perfusion phase (20°C), 600 units of ATIII were used along with 2 mg of abciximab (platelet inhibitor).

TABLE J COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 17,451 80.00 D-pantothenic acid ’LSa 0.001 0.00210 Potassium gluconate 3,982 17.00 Pyridoxine hydrochloride 0.001 0.00486 Amino acids Riboflavin 0.0001 0.000266 L-glutamine 0.292 2.00 Thiamine HC1 0.001 0.00296 L-arginine HC1 0.105 0.603 Other Hormones Adenine 0.68 5.00 Insulin novorapid 5 units Dextrose 1.00 5.55 D-ribose 0.75 5.00 Albumen 57 g/l Tienam 0.050 Sterile water

After emptying, rinse twice using 250 ml of solution according to table. J with 57 g of albumin per 1 liter, perfusion was continued with a solution in accordance with table. J with 57 g of albumin per 1 liter at 15°C for 2 hours.

Then the pressure was increased to 30 mmHg. Art. and temperature up to 28°C, after which perfusion was carried out for 30 minutes, while red blood cells were added. Red blood cells were pretreated with 800 units of ATIII plus 2 mg abciximab and perfused through a leukocyte filter before mixing with the solution. Thanks to this treatment, the kidneys were completely cleaned without stains or circulation problems.

The kidneys were transplanted and two pigs survived for 7 days before being sacrificed. One pig had an infection. Both pigs had elevated creatinine on day 7, blood creatinine was 1115 µmol/L, 1305 µmol/L, urine creatinine 1790 µmol/L, 4530 µmol/L - which can be interpreted as signs of delayed but improved transplant function functions

- 23 045517 urine concentration.

Fig. 14 illustrates a graph showing flow rates after perfusion with a solution having an osmolarity of approximately 300 mOsmoles with the addition of ATIII and platelet inhibitor. The diagram shows higher initial flow rates during reperfusion than in previous experiments.

Example 13.

Another example used a protocol similar to Example 12.

On the instrument table, 15 units of lys-plasminogen and 3 mg of tPA were injected into the kidneys, each in a solution of 15 ml.

The kidneys were placed in an ex-vivo device and perfused using a solution containing the ingredients shown in Table 1. J with albumin at a concentration of 57 g/l. During the perfusion phase (20°C), 600 units of ATIII were used along with 3 mg abciximab. The pressure was increased from 20 to 70 mm Hg. Art., by 5 mm Hg. Art. every 5 min.

After emptying, rinse twice using 250 ml of solution according to table. J with 57 g of albumin per 1 liter, perfusion was continued with a solution in accordance with table. J with 57 g albumin per liter at 15°C for 2 hours. During this phase, an additional dose of 600 ATIII units was administered along with 3 mg abciximab.

Then the pressure was increased to 30 mmHg. Art. and temperature to 28°C, after which perfusion was carried out for 30 minutes, while red blood cells were added. Red blood cells were pretreated with 800 ATIII units and 3 mg abciximab and perfused through a leukocyte filter before mixing with the solution.

After perfusion for 2.5 hours, the flow rate at a pressure of 30 mmHg. Art. decreased from 147 ml/min to 138 ml/min and the resistance increased. 800 units of ATIII and 3 mg of abciximab were once again added to the solution. The flow rate and resistance remained constant for 30 min, after which the flow rate increased and the resistance decreased.

Example 14

In another experiment using 6 pigs, the following protocol was used.

First, the living donor's right kidney was removed and discarded, while the donor's left kidney was left to function.

The recipient's left kidney was then removed and directly transplanted into a living donor in place of the removed right kidney.

The recipient's abdomen was sutured and the recipient was left to sleep awaiting a subsequent transplant, with the recipient's right kidney still functioning.

In a living donor setting, the recipient's left kidney, transplanted into the donor, was reperfused with the donor's blood and observed until it began producing urine. The donor's abdominal cavity was sutured.

The donor's heart was then induced to stop as in the previous examples.

The recipient's left kidney, which was transplanted into the donor, was removed after a VTI of 4.5 to 5 hours.

On the instrument table, 20 units of lys-plasminogen and 600 units of ATIII were injected into the kidneys, and both arteries and veins were clamped. After 15 min, 4 mg tPA was administered along with another dose of 600 units of ATIII. After 15 minutes, the kidneys were connected to the ex-vivo perfusion device.

TABLE K COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Sodium gluconate 17,451 80.00 D-pantothenic acid ’LSa 0.001 0.00210 Potassium gluconate 3,982 17.00 Pyridoxine hydrochloride 0.001 0.00486 Amino acids Riboflavin 0.0001 0.000266 L-glutamine 0.292 2.00 Thiamine HC1 0.001 0.00296 L-arginine HC1 0.105 0.603 Other Hormones Adenine 0.68 5.00 Insulin novorapid 40 units Dextrose 1.00 5.55 D-ribose 0.75 5.00 Albumen 57 g/l Hyenas 0.050 Sterile water

In an ex-vivo perfusion device, the kidneys were perfused using a solution in accordance with Table. K with 57 g albumin per 1 liter. After perfusion for 5 minutes at a pressure of 70 mmHg. Art., perfusion pressure was reduced to 30 mm Hg. Art. and perfused the kidneys for 30 minutes. Then the pressure was reduced to 20 mmHg. Art., and the temperature was up to 15°C, and the kidneys were perfused for 2 hours.

In parallel, washed erythrocytes were circulated through an external leukocyte filter for 1 hour at room temperature. 1000 ATIII units, 3 mg abciximab, and 4 mg argatroban (a direct antithrombin inhibitor) were added to circulating red blood cells.

The Cytosorb cytokine-absorbing filter and the Alteco endotoxin-absorbing filter were washed with NaCl and a solution in accordance with Table. K, and then connected to the perfusion device.

The temperature of the perfusion solution was increased to 28°C, and the pressure was increased to 30 mmHg. Art. for 30 min. The above red blood cells were added to the solution and filters were connected, for example, as shown in FIG. 5. After stabilizing the medium at 28°C, the temperature was increased to 32°C. The kidneys were perfused at this temperature for 3 hours.

1000 ATIII units, 3 mg abciximab, and 8 mg argatroban were then added to the solution. After perfusion for 1.5 hours at 32°C, 1.5 mg abciximab, 4 mg argatroban, and 1000 units of ATIII were added to the solution. The temperature was then reduced to 15°C, the pressure to 20 mm Hg. Art. and the kidneys were perfused for 30 min.

The kidneys were then removed and the recipient's kidney was transplanted back into the recipient, and the recipient's remaining own kidney was removed. The flow velocity and resistance values can be seen in Fig. 18.

Example 15.

In another experiment using 9 pigs, the same protocol as Example 14 was used.

The recipient's left kidney, which was transplanted into the donor, was removed after a VTI of 4.5 to 5 hours.

On the instrument table, 30 units of lys-plasminogen and 600 units of ATIII were injected into the kidneys, and both arteries and veins were clamped. After 15 min, 6 mg tPA was administered along with another dose of 600 units of ATIII. After 15 minutes, the kidneys were connected to the ex-vivo perfusion device.

In an ex-vivo perfusion device, the kidneys were perfused using a solution according to Table 1. K with 57 g albumin per 1 liter. After perfusion for 5 minutes at a pressure of 75 mm Hg. Art., perfusion pressure was reduced to 25 mm Hg. Art. and perfused the kidneys for 30 minutes. Then the pressure was reduced to 20 mmHg. Art., and the temperature was up to 15°C, and the kidneys were perfused for 2 hours.

In parallel, washed erythrocytes were circulated through an external leukocyte filter for 1 hour at room temperature. Circulating red blood cells were supplemented with 1000 ATIII units, 3 mg abciximab, and 4 mg argatroban.

The Cytosorb cytokine-absorbing filter and the Alteco endotoxin-absorbing filter were washed with NaCl and a solution in accordance with Table. K, and then connected to the perfusion device.

The temperature of the perfusion solution was increased to 28°C, and the pressure was increased to 30 mmHg. Art. for 30 min. The above red blood cells were added to the solution and the filters were connected. After stabilizing the medium at 28°C, the temperature was increased to 32°C. The kidneys were perfused at this temperature for 3 hours.

The kidneys were then infused with 1000 units of ATIII, 3 mg abciximab, and 8 mg argatroban. After perfusion for 1.5 hours at 32°C, the kidneys were infused with 1.5 mg abciximab, 4 mg argatroban, and 1000 units of ATIII.

Example 16.

In another example using 8 pigs, similar to Experiment 15, the pigs underwent the same protocol as in previous Examples 14 and 15. After transplantation, the pigs were awakened from anesthesia and monitored for up to three months. Plasma creatinine levels after 10 days and three months, as well as urine creatinine levels at the same time points, were consistent with the data shown in FIG. 19-22. Creatinine normalized within the first week and remained normal for three months without the need for dialysis. Fig. Figure 23 illustrates a typical kidney used 3 months after repair and transplantation, well perfused and without signs of fibrosis or atrophy. In fig. Figure 24 illustrates a kidney that has been removed and contoured to show normal renal parenchyma.

Example 17.

One pig was given anesthesia and provided with normal ventilation, after which the ventilator was turned off. Heart failure appeared approximately 15 minutes later. After two hours at room temperature, crushed ice was placed into the abdominal cavity.

Removal of one liver began 4 hours after death.

On the instrument table, the liver was flushed using 500 ml of cold Storeprotect solution through the portal vein. 60 units of lys-plasminogen and 1200 units of ATIII were injected into each of the vessels, the hepatic artery and the portal vein, after which the artery, portal vein and vena cava were clamped for 15 minutes.

The liver was connected to an ex-vivo perfusion device and perfused using

- 25 045517 solution according to table. L with 72 g albumin per 1 liter. 12 mg tPA and 1200 units of ATIII were then injected into the hepatic artery and portal vein, after which the liver was connected to the perfusion device and perfusion was started.

TABLE L COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 4.15 49.40 Nicotinamide 0.001 0.00819 Potassium gluconate 3.98 17.00 D-pantothenic acid ’LSa 0.001 0.00210 Sodium lactobionate 30.46 100.00 Pyridoxine hydrochloride 0.001 0.00486 Amino acids Riboflavin 0.0001 0.000266 L-glutamine 0.292 2.00 Thiamine HC1 0.001 0.00296 L-arginine HC1 0.105 0.603 Other Hormones Adenine 0.68 5.00 Insulin novorapid Yu unit Dextrose 1.00 5.55 D-ribose 0.75 5.00 Albumen 72 g/l Hyenas 0.050 Sterile water

Perfusion was started into the portal vein at a rate of 0.75 ml/min per gram of liver weight and into the artery at 0.25 ml/min/g, at a temperature of 24°C. The portal vein was first flushed for 10 min before arterial flow was turned on, and the solution was completely oxygenated.

12 mg of platelet inhibitor containing abciximab and 16 mg of direct thrombin inhibitor containing argatroban were injected into the artery and portal vein. The pressure was increased from 20 mm Hg. Art. up to 90 mm Hg Art., by 5 mm Hg. Art. every 5 min. After the liver was cleansed, the pressure was reduced to 30 mmHg. Art. and the liver was perfused for another 30 min at a temperature of 22°C-24°C. The temperature was then reduced to 15°C, maintaining a flow rate of 0.75 ml/min/g in the portal vein and 0.25 ml/min/g in the artery for 2 hours. The temperature was increased to 33°C, red blood cells and pressure were added increased to 75 mm Hg. Art. Perfusion was carried out for 3 hours with the addition of 24 mg of abciximab and 32 mg of argatroban every hour into both the artery and portal vein. At the end of the experiment, the liver had perfusion defects, as shown in Fig. 25 and 26.

Example 18.

Three pigs were given anesthesia and ventilated, after which the ventilator was turned off. Heart failure appeared approximately 15 minutes later. After two hours at room temperature, crushed ice was placed into the abdominal cavity.

Liver removal began 4 hours after death.

On the instrument table, the liver was flushed using 500 ml of cold Storeprotect solution through the portal vein. 60 units of lys-plasminogen and 1200 units of ATIII were injected into each of the vessels, the hepatic artery and the portal vein, after which the artery, portal vein and vena cava were clamped for 15 minutes. 12 mg tPA and 1200 units of ATIII were then injected into the hepatic artery and portal vein. After waiting 15 minutes, the liver was connected to the perfusion device.

- 26 045517

TABLE M COMPONENT g/l mmol g/l mmol Inorganic salts Vitamins CaC1 ₂ 2H ₂ O 0.6732 4.95 Choline chloride 0.001 0.00716 Magnesium Gluconate (Anhydrous) 1.13 5.00 Folic acid 0.001 0.00227 Potassium phosphate (monosubstituted) 0.68 5.00 Myo-inositol 0.002 0.0111 _NaHCO3 1.26 15.00 Nicotinamide 0.001 0.00819 Sodium gluconate 10.90 50.00 D-pantothenic acid ’LSa 0.001 0.00210 Sodium lactobionate 24.37 80.00 Pyridoxine hydrochloride 0.001 0.00486 Amino acids Riboflavin 0.0001 0.000266 L-glutamine 0.292 2.00 Thiamine HC1 0.001 0.00296 L-arginine HC1 0.105 0.603 Other Hormones Adenine 0.68 5.00 Insulin novorapid 40 units Dextrose 1.00 5.55 D-ribose 0.75 5.00 Albumen 72 g/l Tienam 0.050 Sterile water

Perfusion was carried out with a solution according to table. M. Perfusion was started into the portal vein at a rate of 0.75 ml/min/g and into the artery at 0.25 ml/min/g, at a temperature of 24°C. The portal vein was first flushed for 10 min before arterial flow was turned on, and the solution was completely oxygenated. 12 mg of abciximab and 16 mg of argatroban were injected into the artery and portal vein. The pressure was increased from 20 mm Hg. Art. up to 90 mm Hg Art., by 5 mm Hg. Art. every 5 min. After the liver was cleansed, the pressure was reduced to 30 mmHg. Art. and the liver was perfused for another 30 min at a temperature of 22°C-24°C. The temperature was then reduced to 15°C, maintaining a flow rate of 0.75 ml/min/g in the portal vein and 0.25 ml/min/g in the artery for 2 hours. The temperature was increased to 33°C, red blood cells and pressure were added increased to 75 mm Hg. Art. Perfusion was carried out for 3 hours with the addition of 24 mg of abciximab and 32 mg of argatroban every hour into both the artery and portal vein.

Fig. 29 illustrates the flow rates in the artery during the experiment. The liver was cleared of stains on the parenchyma.

Discussion.

Example 1 examined the basic principles of renal recovery from a donor subjected to warm ischemia for more than 4 hours after circulatory arrest using a solution containing minimal nutritional medium (MNM) and albumin as a hyperoncotic agent. Normometric ex-vivo perfusion resulted in significantly better blood flow and urine production compared with LifePort (LP) or cold storage (XX) preservation agents.

In Example 2, long-term flushing of kidneys obtained from a cardiac arrest donor following the use of heparin, apyrase—a purinergic inhibitor that removes ATP from the tissue—and tPA (alteplase) injected into both the artery and the perfusion solution slightly improved vascular resistance and blood flow. and did not completely cleanse the kidneys using a perfusion solution of similar composition. Apyrase can be administered into the kidneys after clearing the microcirculation of fibrin clots.

In Example 3, lidocaine was added which improved blood flow at 90 min, indicating stabilized membrane function, possibly due to suppression of the sodium-potassium pump.

In Example 4 it was also noted that prolonged perfusion times using red blood cells were possible at 32°C, some kidneys were observed for 8 hours after transplantation into the recipient pig, and in the following Example 5 the perfusion time was increased to 11 hours to ensure that the transplant was functioning. kidneys

In Example 6, the agent with a higher colloid oncotic pressure, albumin 80 g/L, was found to provide good renal flow rates above 115 ml/min with an IUT of 4 to 6 hours. Recipients were observed for 8 hours after transplantation.

In Example 7, local cooling was changed to ice instead of cold Ringer's solution 2 hours after death. This allowed a core body temperature of 20°C to be achieved, compared to 25-29°C when using cold Ringer's solution. Blood flow improved significantly at both 4 and 8 hours after transplantation.

Pigs that did not receive local cooling, see example 8, and only received perfusion on the instrument table did not show good flow rates at reperfusion (14.15 ml/min) or at 90 min (36.8 ml/min).

In examples 9 and 10, the temperature and colloid oncotic pressure were changed, and slush ice was used instead of ordinary ice. Now before extraction you can achieve a body temperature of 10 to 12°C. By treating the kidneys with lys-plasminogen in combination with tPA, the kidneys were much better cleaned and vascular resistance was significantly improved. We used alte

- 27 045517 plaza, but any fibrinolytic agent can be used, for example streptokinase, urokinase, reteplase and tenecteplase. This modification cleansed the kidneys better than in previous examples. Some of these kidneys continued to function for 10 days with functioning kidneys following nephrectomy of the donor's own kidneys performed at the time of transplantation, demonstrating the feasibility of using donor kidneys after cardiac arrest 4 hours after death. It has been found that treatment of red blood cells during the evaluation period can help release cytokines such as IL-6, IL-8, IL-1e and TNF-α, which are all involved in inflammatory processes and ischemia reperfusion.

By using a special absorber (cytosorb), these cytokines can be completely removed, see example 11.

In subsequent Examples 12 and 13, we observed that a platelet inhibitor such as abciximab added to the preservation solution and to the red blood cells before mixing with the perfusion solution provided very good flow rates. In addition, signs of increased vascular resistance during prolonged perfusion with red blood cells may be improved by adding ATIII and abciximab to the solution, suggesting that it is desirable to prevent recurrent thrombosis caused by both the coagulation system and platelet adherence upon completion of fibrinolysis. The dose of ATIII was increased in these examples compared to previous examples.

In Examples 14 and 15, the results were further improved by the addition of a direct thrombin inhibitor such as argatroban, which eliminated the risk of decreased flow and increased vascular resistance during the red blood cell step.

In Example 16, we successfully transplanted kidneys reconstituted according to Examples 14 and 15 and monitored them for 3 months, proving that the reconstituted kidneys were also functional under appropriate clinical conditions. The new transplant models used in Examples 14, 15 and 16 mean that allo-rejection mechanisms are eliminated due to the fact that the kidneys originally transplanted were taken from the recipient and transplanted into the donor before cardiac arrest was induced. The pigs survived without the need for dialysis, despite the fact that the regenerated kidney was the only one that worked in the recipient pig.

Example 17 examined the basic principles of liver regeneration, confirming our experiments on kidneys obtained from a donor subjected to warm ischemia for more than 4 hours after death due to cessation of blood flow. The solution had a composition similar to that shown to be effective in the kidney, with albumin as the hyperoncotic agent, but using the sodium salt of lactobionic acid instead of the gluconate. Initial irrigation and perfusion at 24°C cleared the liver parenchyma to a large extent, but the liver had perfusion defects at the end of the evaluation after RBC administration.

In Example 18, tPA and ATIII were administered on the instrument table, similar to the renal protocol. Ex-vivo normothermic perfusion resulted in clearing of the parenchyma, and better flow rates and lower resistance compared to control liver samples obtained from donors who died from cessation of blood supply.

We observed bile production and lower lactate levels compared to control animals after 6.5 hours of perfusion, and the liver appeared well perfused without defects.

In the claims, the terms contains and/or containing do not exclude the presence of other elements or steps. In addition, although described separately, the plurality of means, elements, or method steps may be implemented as, for example, a single unit. Also, although individual features may be included in different claims or embodiments, they may be advantageously combined, and reference to different claims does not imply that the combination of features cannot be implemented and/or is not advantageous. Moreover, the singular does not exclude the plural. Terms characterizing the singular number, first, second and others do not exclude the possibility of the plural. Position numbers are included in the claims for clarity only and are not intended to limit the scope of the claims in any way.

Although the present invention has been described above with reference to specific embodiments and experiments, it is not limited to the above specific forms. On the contrary, the invention is limited only by the appended claims and other embodiments other than those described above are equally possible within the scope of these appended claims.

Claims (8)

1. A method for restoring an organ obtained from a donor, including taking the organ from the donor at least 2 hours after the donor’s circulatory arrest, introducing lys-plasminogen into the organ after its removal, wherein lys-plasminogen is contained in the first hyperoncotic solution, introducing a tissue activator plasminogen (TAP) simultaneously with or after the administration of lys-plasminogen, with tissue plasminogen activator contained in the second hyperoncotic solution, at the first stage of recovery, ensuring circulation through the organ of the third hyperoncotic fluid containing albumin and electrolytes at a low temperature from 5 to 25 ° C, at the second stage of recovery, ensuring circulation through the organ of the fourth hyperoncotic fluid containing oxygenated red blood cells at a temperature of 28 to 37 ° C, assessing the organ for suitability for transplantation. 2. The method according to claim 1, where the donor is a donor who died from cessation of blood supply. 3. The method according to claim 1 or 2, in which, at the said first stage of recovery, said third hyperoncotic fluid is circulated through the organ, wherein said third hyperoncotic fluid contains albumin in a concentration of 50 to 120 g/l, and the circulation pressure is increased during 30-75 min. 4. The method according to any one of claims 1-3, in which, at the specified second stage of recovery, the specified fourth hyperoncotic fluid is circulated through the organ, and the specified fourth hyperoncotic fluid contains albumin in a concentration of 50 to 120 g/l, while the circulation pressure is increased for 30-75 minutes. 5. The method according to any one of claims 1 to 4, in which at least one of the hyperoncotic fluids, the first, second, third or fourth, contains at least one of the following substances: a coagulation inhibitor; direct thrombin inhibitor; protein C; protein S and platelet inhibitor. 6. The method according to any one of claims 1 to 5, wherein at least one of the third and fourth hyperoncotic fluids is circulated through a leukocyte filter. 7. The method according to any one of claims 1 to 6, wherein at least one of the third and fourth hyperoncotic fluids is contacted with a cytokine scavenger to absorb the cytokines. 8. The method according to any one of claims 1 to 7, wherein at least one of the third and fourth hyperoncotic fluids is contacted with an endotoxin scavenger to absorb endotoxins.