RU2847325C1 - Method for maintaining the viability of a transplantable organ in a preservative gas mixture and kit for its implementation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the invention relates to medicine, namely to pathophysiology and experimental transplantology. A method for hypothermic preservation of an isolated mammalian organ involves placing the organ in a chamber with a gas environment, where the gas environment comprises a mixture of dinitrogen oxide and oxygen "N₂ O+O₂ ", which constitutes from 60 to 100% of the volume of the gas environment for preservation. The types of gas mixtures for forming the gas environment in the chamber are selected from a list of mixtures belonging to a group consisting of: "N(₂₎ O+O(₂₎ "; "N(₂₎ O+O(₂₎ +Ar"; "N(₂₎ O+O(₂₎ +CO+Ar", where the percentage ratio of gases in the mixture "N(₂₎ O+O(₂₎ " is selected from 71/29 to 29/71, preferably 50/50, where the percentage ratio of gases in the mixture "N₂ O+O₂ +Ar" is 36/36/28, where the percentage ratio of gases in the mixture "N₂ O+O₂ +CO+Ar" is 60/17/14/9. To form a gas environment in the chamber, in the first stage, the chamber volume is purged using the first mini-cylinder with a gas environment for purging, and in the second stage, the chamber volume is filled with the selected gas mixture from the second mini-cylinder, while setting the pressure in the chamber preferably to 7 atm and performing hypothermic preservation at a temperature from +2° C to +12° C, preferably around +4° C, for 12 to 24 hours, preferably 24 hours. A kit for hypothermic preservation of an isolated mammalian organ includes a device in the form of a sterile chamber for placing the isolated organ, two mini-cylinders with gas media, where the first cylinder contains a gas medium for displacing air and purging the working volume of the chamber, and the second cylinder contains a gas mixture for displacing the purging medium and creating a high-pressure gas medium for prolonged preservation of the isolated organ, a set of liquid media and clamps for performing organ preservation and deconservation after the end of its transportation and storage.

EFFECT: ensuring long-term preservation of the viability of the organ for transplantation through the use of non-toxic preserving gas media, safety of storage and transportation.

4 cl, 2 dwg, 1 ex

Description

The invention relates to the field of transplantology and can be used to store a transplanted organ in safe conditions and maintain its viability for the purpose of subsequent transplantation.

In today's world, organ transplantation remains the only viable option for many people to prolong life and improve its quality. Advances in surgical technologies and the ability to perform complex procedures have led to a global transplant crisis—an acute shortage of donor organs. According to the World Health Organization (WHO), only 10% of patients receive a new organ. The heart, in contrast, has the shortest storage time (4-6 hours) when using standard static cold preservation methods, compared to the liver (12 hours) and kidneys (24 hours). In Russia, the average wait time for a donor heart is one and a half years.

Methods and devices for preserving biomaterials in an aerosol atmosphere of medical gas and preservative liquid are known [1]. Other technical solutions are based on the continuous perfusion of a preservative gas medium consisting of a mixture of CO and O ₂ gases through the transplant during storage [2]. Russian patent No. RU 2741219 describes a device for preserving donor organs of experimental animals through continuous oxygenation of the transplant with humidified oxygen under a constant pressure of 35 mm Hg with a volume flow rate of 3 ml/min [3].

One promising approach to organ preservation may be hypothermic storage of organs in a mixture of biologically active gases under pressure of up to 7 atmospheres. One method of organ preservation involves using a chamber filled with a mixture of toxic carbon monoxide and oxygen under elevated pressure [4]. A disadvantage of this technical solution is that the chamber is not protected from the external environment, and toxic gas may leak into the environment. An invention described in Russian Patent No. 2707532, November 27, 2019, addresses this disadvantage and allows for the safe transportation of organs using toxic gas under high pressure [5].

A disadvantage of the known method and the design of the device based on it is the need to use complex technical solutions in the implementation of the method and in the design of the device, caused by the need to increase safety when preserving transplants in a toxic gas mixture of carbon monoxide with oxygen at high pressure.

The present invention is aimed at expanding the range of non-toxic or low-toxic gas environments used in the method of hypothermic preservation and expanding the range of devices for implementing the method of maintaining the long-term viability of a transplanted organ under safe storage or transportation conditions.

The technical result consists of improving preservation technology and ensuring safe organ preservation by placing the transplanted organ in new non-toxic or low-toxic gas environments, improving the safety of personnel preparing the organ for preservation and de-preservation. The effectiveness of organ preservation is further enhanced by the use of a kit containing a device in the form of a sterile chamber for placing the transplanted organ, two disposable mini-cylinders pre-filled with the selected gas mixture at a specific pressure, and a set of liquid media and clamps for performing organ preservation and de-preservation after transportation and storage.

According to a first aspect, the present invention relates to a method for hypothermic preservation of an isolated mammalian organ for transplantation, taking into account its safe storage and transportation, according to which the organ is placed in a chamber with a gas medium, where the gas mixture includes a mixture of dinitrogen oxide and oxygen "N ₂ O + O ₂ ", which makes up from 60 to 100% of the volume of the gas medium for preservation. The gas mixture for forming the gas medium is selected from the list of mixtures included in the group consisting of: "N ₂ O + O ₂ ";"N ₂ O + O ₂ + Ar"; «N ₂ O + О ₂ + СО+Ar», where the ratio in percentage of gas volumes in the «N ₂ O+O ₂ » mixture is selected from 71/29 to 29/71, preferably 50/50, where the ratio in percentage of gas volumes in the «N ₂ O+O ₂ +Ar» mixture is 36/36/28, where the ratio in percentage of gas volumes in the «N ₂ O+O ₂ + СО+Ar» mixture is 60/17/14/9. In this case, to form a gas environment in the chamber, at the first stage the chamber volume is purged using the first mini-cylinder with purging gas under a pressure of up to 3 atm, and at the second stage the chamber volume is filled with the selected gas mixture from the second mini-cylinder, which is pre-filled with the selected gas mixture under a pressure of up to 16 atm. The operating pressure in the chamber is set to preferably 7 atm and hypothermic preservation is carried out at a temperature of +2 to +12°C, preferably around +4°C, for 12 to 24 hours, preferably 24 hours. The preferred organ for transplantation is the heart.

Another aspect of the invention is a kit for the hypothermic preservation of an isolated mammalian organ, comprising: a device in the form of a sterile chamber for accommodating the transplanted organ, two mini-cylinder gaseous media, wherein the first cylinder contains a gaseous medium for displacing air and purging the working volume of the chamber, and the second cylinder contains a gaseous medium for displacing the purging medium and creating a high-pressure gaseous medium for prolonged preservation of the transplant. The kit also includes an organ attachment unit and a liquid container, as well as a set of liquid media and clamps for performing organ preservation and depreservation after its transportation and storage.

Another aspect of the invention relates to the use of a kit for hypothermic preservation of an isolated mammalian organ, described in paragraph 3, for carrying out the method for preserving an isolated mammalian organ for transplantation given in paragraphs 1, 2.

Fig. 1. A diagram of the chamber for heart preservation is shown.

Fig. 2. The results of the analysis of the preservation of the isolated rat heart after preservation at 4°C are shown. Data are presented as mean ± standard deviation.

Where: A - cardiac activity in the Custodiol group; B - restoration of cardiac activity in the CO+ _O2 group in the percentage ratio of gases of 50/50; C - restoration of cardiac activity in the _N2O + _O2 group in the percentage ratio of gases of 71/29; D - restoration of cardiac activity in the _N2O + _O2 group in the percentage ratio of gas volumes of 50/50; E - restoration of cardiac activity in the _N2O + _O2 group in the percentage ratio of gas volumes of 29/71; F - restoration of cardiac activity in the _O2 +CO+ _N2O +Ar group in the percentage ratio of gas volumes of 60/17/14/9; G - restoration of cardiac activity in the _O2 + _N2O +Ar group in the percentage ratio of gas volumes of 36/36/28

During research into the complete or partial replacement of toxic carbon monoxide in multicomponent preservation gas mixtures, it was discovered that a gas mixture of oxygen and dinitrogen oxide as a gas medium for storing transplanted organs at high pressure improves safety and enhances the efficiency of organ storage and transportation. Dinitrogen oxide, a registered gas included in the "Vital and Essential Drugs" list, is known to have a positive effect on the preservation of the heart under hypoxic conditions; however, its use is known only at atmospheric pressure. It was also found that the inclusion of argon, an inert gas, in preservation gas mixtures, along with a gas mixture of oxygen and dinitrogen oxide, reduces ischemia-reperfusion injury to transplanted organs, including the heart, and improves the reliability of organ preservation for subsequent transplantation.

To improve the efficiency of gas mixtures during preservation, a quantitative ratio in percentage volume was discovered for several gas mixtures containing a mixture of dinitrogen oxide and oxygen "N ₂ O + O ₂ ", which constitutes from 60 to 100% of the total volume of the gas environment for preservation. The gas mixture for forming the gas environment is selected from a list of mixtures included in the group consisting of: "N ₂ O + O ₂ ";"N ₂ O + O ₂ + Ar";"N ₂ O + O ₂ + CO + Ar". The ratio in percent of the volumes of gases in the mixture “N ₂ O+O ₂ ” is selected from 71/29 to 29/71, preferably 50/50, where the ratio in percent of the volumes of gases in the mixture “N ₂ O+O ₂ +Ar” is 36/36/28, where the ratio in percent of the volumes of gases in the mixture “N ₂ O+O ₂ +CO+Ar” is 60/17/14/9.

The method for preserving a transplanted organ involves several stages. The first stage involves selecting the gas medium used to fill the chamber containing the organ, preparing the organ, and placing it in the chamber. Next, the chamber is purged using a first mini-cylinder of purging gas at a pressure of up to 3 atm, which is included in the organ preservation kit. The next stage involves filling the chamber with the selected gas mixture from a second mini-cylinder, which is pre-filled with the selected gas mixture at a pressure of up to 16 atm. The operating pressure in the chamber is set to a preferred value of 7 atm, and hypothermic preservation is performed at a temperature of +2 to +12°C, preferably around +4°C, for 12 to 24 hours, preferably 24 hours.

The structural diagram of the chamber for placing the transplanted organ is shown in Fig. 1.

To improve reproducibility, the chamber design must ensure reliability and consistent conditions during preservation. In known devices [1], the experimenter creates a gas mixture by connecting cylinders equipped with pressure gauges to the chamber inlets. However, manual pressure control makes it difficult to create consistent conditions in different experiments, and time is wasted setting the required parameters during preservation, leading to decreased reproducibility of the results.

According to the invention, the device comprises a chamber 1 containing a working volume 2, within which a transplantable organ 3 is placed, which is located on the surface of a moisture-permeable porous substrate 4 and fixed with tabs 5 to prevent the organ from moving during transportation. The moisture-permeable porous substrate 4 and tabs 5 are secured to a frame 6 fixed above a container 7, in which a liquid 8 is placed to create humidity and a possible additional cold gas zone with a reduced temperature at the initial stage of preservation. The chamber is equipped with a lid 9 on which a gas pressure gauge 10 and an electronic unit 11 are located, the inputs of which are connected to sensors: measuring temperature 12, humidity 13, pressure 14 in chamber 1, as well as a board for wirelessly transmitting data on temperature, humidity and pressure and transmitting data on the location of chamber 1 with the transplantable organ 3 (GPS module).

Chamber 1 additionally contains two inputs equipped with gas valves. The first valve 15 is intended for the alternate introduction into chamber 1, first of a gas mixture from the first 16 mini-cylinder under a pressure of up to 3 atm for purging the chamber volume, and at the second stage of introducing into chamber 1 a gas mixture for preservation from the second mini-cylinder under a pressure of up to 16 atm 17. To ensure the possibility of quickly connecting the first and second gas cylinders, and introducing gases into the working volume 2 of the chamber, a quick-release gas connection 18 is connected to the first valve 15. The second valve 19 ensures the possibility of smoothly reducing the pressure of the gas mixture in volume 2 of chamber 1. To eliminate gas leaks during preservation from the volume of chamber 1, a removable plug 20 is installed on the second valve. The designs of the chamber and valves must ensure operation with pressures of at least 10 atm. As an example, not limiting other options, chamber 1 can be made on the basis of chemical reactors capable of operating under high pressure.

In order to simplify the work on organ preservation, with the possibility of increasing the reproducibility of the preservation results, it was decided to prepare a set for carrying out preservation, which includes: chamber 1, container 7 with a frame 6 fixed to it for fastening a substrate made in the form of a water-permeable porous substrate 4 and paws 5 for fastening an organ 3, a set of liquid media, clamps, fittings, the first 16 and second 17 mini-cylinders - included in the set for hypothermic preservation of an isolated mammalian organ.

The device for placing and storing a transplanted organ, based on a sterile chamber 1, operates as follows. The open chamber, which is part of the kit, along with the container 7 and the organ fixation unit located thereon, containing the frame 6 with the porous support 4 secured to it and the attached tabs 5, is pre-disinfected using quartz treatment, hermetically sealed (until the organ is harvested), and cooled to +4°C.

Before organ collection, the operating room chamber is opened and a preservative solution 8 (example: Custodiol) is poured into container 7 to maintain humidity and provide additional local cooling, up to a level of 1 cm, or a 0.9% NaCl solution with the addition of 0.1 mg/ml ceftriaxone (when preserving an organ filled with blood).

The organ is placed in the chamber on the frame 6, on which a soft, medium-porous base 4 is fixed, and the organ 3 is fixed with special spring-loaded tabs 5 made of a neutral material (which do not exert pressure on the organ).

After the lid 9 is completely closed, the chamber is purged with a gas mixture. For the first stage of the purging procedure and the second stage of organ saturation with a pressurized gas environment, the first 16 and second 17 disposable gas cylinders with quick-release couplings are used. These cylinders create the purging gas environment and the positive pressure gas environment necessary for organ preservation.

A gas bag of the required volume is connected to pressure relief valve 19. With both valves 15 and 19 open, the first gas cylinder 16 is connected to quick-release coupling 18. The internal volume 2 of chamber 1 is purged with a gas mixture. Next, pressure relief valve 19 is closed, the gas bag is disconnected, and a special plug 20 is installed on valve 19. A second gas mini-cylinder 17 is connected to pressure increase valve 15 of storage chamber 1. When the pressure gauge needle 10 of the chamber registers a pressure of 7 atm, pressure increase valve 15 is closed and the second gas cylinder 17 is disconnected. The chamber is then transferred to an ice-filled organ transport container. The chamber is equipped with unit 11 for monitoring, recording, and transmitting temperature, humidity, and pressure readings in the chamber to a specialized application, as well as organ 3 location data via a GPS module and wireless communication.

After organ transport, remove the plug from pressure relief valve 19 and connect a device to depressurize the organ to atmospheric pressure (this device consists of a gas bag with an adapter that provides a degassing rate of 0.5 atm/min). After depressurizing the organ to atmospheric pressure, chamber 1 is opened and organ 3 is removed for transplantation.

The following examples include, but do not limit, the scope of the invention.

Example 1. Testing the efficiency of storing the heart in new gas environments.

The effectiveness of storing the heart in new gas environments for preservation is tested in gas environments containing a mixture of "N ₂ O + O ₂ ", which makes up 60 to 100% of the volume of the gas environment, compared to a two-component environment of CO + O ₂ containing toxic carbon monoxide.

The effectiveness of storing the heart in new gas environments was tested on 42 male Wistar rats weighing 250 ± 20 g.

After anesthetizing the rats with a mixture consisting of 80 μl of Zoletil 100 (Virbac, France) and 20 μl of Xyla (Interchemie, Netherlands), the animals were secured on a table and a longitudinal midline laparotomy was performed. Next, 1 ml of heparin solution (Moscow Endocrine Plant, Russia) was injected into the inferior vena cava at a dose of 100 IU/ml. After 5 min, a longitudinal median sternotomy was performed, and the heart was isolated along with a portion of the aorta at least 1 cm long. A cannula was inserted into the cardiac aorta and secured with a ligature. The organ, not washed free of blood, was then placed in a storage chamber pre-cooled to 4°C. To maintain humidity in chamber 1, before placing the heart in the chamber, a container is installed and secured. It is filled with a liquid solution selected from the following group: distilled water, Custodiol solution, 0.9% NaCl solution with 0.1 mg/ml ceftriaxone added, or a liquid solution with ice. An organ attachment unit is placed above the container. This assembly consists of a frame holding a support and tabs for securing the organ to the support.

After the lid is completely closed, the chamber is purged with a gas mixture. The gas mixture for purging is prepared before hypothermic preservation by mixing the gases in the first mini-cylinder in specified ratios under a pressure of up to 3 atm. The gas mixture for preservation is also prepared before hypothermic preservation by mixing the gases in the second mini-cylinder in the required ratios at a pressure of up to 16 atm. After purging, the chamber is pressurized with a gas mixture at 7 atm. The chamber is stored in a refrigerator at +4°C for 24 hours.

After storage, the chamber is removed from the refrigerator and degassed for 13 minutes, gradually manually releasing the pressure to atmospheric pressure, with a degassing rate of 0.5 bar/min.

After preservation, the rat heart was connected to the isolatedHEART-SR stand (Hugo Sachs Elektronik Harvard Apparatus GmbH, Germany). Perfusion was performed with oxygenated (aeration - 95% O ₂ /5% CO ₂ ) Tyrode's solution (in mM): NaCl, 135; KCl, 4; MgCl ₂ , 1; CaCl ₂ , 1.8: NaHCO ₃ , 13.2; NaH ₂ PO ₄ , 1.8; glucose, 11; pH 7.4, at a temperature of 37°C and a pressure of 80 mmHg. A latex balloon with a volume of 0.5 ml was introduced into the left ventricle, connected to a sensor for recording the developed pressure and heart rate. After a 20-minute stabilization period, experimental data were recorded using the isolated HEART software. The studied parameters were recorded in the interval from the twentieth to the sixtieth minute of the heart’s work on the stand.

To test the effectiveness of different gas mixtures, two control groups of 6 male rats and five experimental groups of 6 male Wistar rats each weighing 250 ± 20 g were used.

In the first control group, hearts were used that were washed from blood with Custodiol solution (Dr. F. KOHLER CHEMIE GmbH, Germany) and preserved at a temperature of 4°C for 24 hours.

In the second control group, hearts were used that were not washed from blood and preserved in a gas mixture of CO+ _O2 with a gas ratio (in percent) of 50/50 under a pressure of 7 atmospheres at a temperature of 4°C for 24 hours.

In the experimental groups, two-component gas mixtures consisting of _O2 and _N2O in various volume ratios were used for organ preservation, as well as multicomponent mixtures containing, in addition to the _O2 and _N2O mixture (which constitutes 60 to 100% of the gas medium volume), the inert gas Ar or Ar and CO. The experiments were conducted under a pressure of 7 atmospheres at a temperature of 4°C for 24 hours. Unwashed hearts were used for storage.

In the first experimental group, a gas mixture of “N ₂ O+O ₂ ” was used in a percentage ratio of gas volumes of 71/29.

In the second experimental group, a gas mixture of “N ₂ O + O ₂ ” was used in a percentage ratio of gas volumes of 50/50.

In the third experimental group, a gas mixture of “N ₂ O+O ₂ ” was used in a percentage ratio of gas volumes of 29/71.

In the fourth experimental group, a gas mixture of “ _O2 +CO+ _N2O +Ar” was used in a percentage ratio of gas volumes of 60/17/14/9.

In the fifth experimental group, a gas mixture of “ _O2 + _N2O +Ar” was used in a percentage ratio of gas volumes of 36/36/28.

Data analysis was performed using SigmaPlot 12.5 software (Systat Software Inc, USA); data were expressed as mean ± standard deviation. The significance of differences was determined using the Mann-Whitney U test. Values with p < 0.05 were considered statistically significant.

During the experiment, contractile activity was restored in all experimental groups and the CO2+ _O2 control group within 1-2 minutes of the onset of reperfusion. Hearts preserved in Custodiol cardioplegic solution did not recover their contractile activity. Within the first 5 minutes of reperfusion, the hearts of all experimental groups returned to normal function, with rare periods of arrhythmia observed in all groups.

The number of surviving hearts in the experimental groups “CO+ _O2 ” in relation to _{“ N2OO2} ” was 4/6

The heart rate indicators in the “CO+ _O2 ” and “ _N2O + _O2 ” groups did not differ significantly and were 262 ± 21 beats/min and 256 ± 23 beats/min.

The pressure developed by the left ventricle (PDLV) in the control group "CO + O ₂ " (50/50%) was 31 ± 19 mm Hg. In the experimental groups "N ₂ O + O ₂ " (71/29%) - 25 ± 5 mm Hg, in the group "N ₂ O + O ₂ " (50/50%) - 49 ± 10 mm Hg, in the group "N ₂ O + O ₂ " (29/71%) - 37 ± 6 mm Hg.

In the experimental group "O ₂ + CO + N ₂ O + Ar" (60/17/14/9%), the daily lipid level was 30 ± 9 mm Hg; in the group "O ₂ + N ₂ O + Ar" (36/36/28%), it was 34 ± 11 mm Hg. Fig. 2 shows graphs explaining the efficiency of the developed types of gas mixtures. These results confirm the efficiency of gas mixtures based on O ₂ + N2O.

Nitrous oxide, when used in combination with oxygen, is a key component of the gas compositions described in the invention. It exhibits a pronounced protective effect during hypothermic preservation, similar in organ preservation to that achieved in a gas mixture of oxygen and carbon monoxide. In the future, replacing carbon monoxide with nitrous oxide in oxygen-based protective gas compositions will improve the safety of gas preservation. The heart is the preferred organ for transplantation.

The proposed new method demonstrates the feasibility of using multicomponent gas mixtures based on oxygen and other biologically active gases with reduced carbon monoxide content for cardiac preservation. The feasibility of replacing carbon monoxide with dinitrogen oxide and argon is demonstrated, which will improve the safety of gas preservation technology. The feasibility of cardiac preservation using a gas mixture without prior cardiac arrest with cardioplegic solutions is demonstrated, which may be useful in emergency preservation of traumatically amputated limbs. In some amputation cases, it is not always possible to promptly locate the main artery for the administration of tissue-preserving solutions for subsequent replantation.

The use of disposable mini-cylinders, pre-filled with the selected type of gas mixture at a certain pressure, increases the reproducibility of the results obtained and further simplifies the preservation process.

Sources of information

1. HATAYAMA N. et al. PRESERVATION METHOD OF BIOMATERIAL, PRODUCTION PROCEDURE OF BIOMATERIAL, BIOMATERIAL, TRANSPLANT MATERIAL, TRANSPLANT METHOD AND PRESERVATION APPARATUS OF BIOMATERIAL, Japanese Patent JP 6242001 (2017-12-06).

2. LUKE PATRICK SUBNORMOTHERMIC MACHINE PERFUSION OF ORGANS WITH CARBON MONOXIDE AND OXYGEN. US Application US 2023073834 (A1) (2023-03-09).

3. ERMOLAEV P.A. et al. DEVICE FOR PRESERVATION OF DONOR ORGANS Russian Federation Patent RU 2741219 (01.22.2021).

4. NAOYUKI HATAYAMA ET AL. DIFFERENT EFFECTS OF PARTIAL PRESSURE IN A HIGH-PRESSURE GASEOUS MIXTURE OF CARBON MONOXIDE AND OXYGEN FOR RAT HEART PRESERVATION. Sci Rep. 9: 7480 (2019).

5. FESENKO E.E. ET AL. METHOD FOR INCREASING THE SAFETY AND EFFICIENCY OF STORAGE AND TRANSPORTATION OF A TRANSPLANT ORGAN UNDER THE PRESSURE OF A PRESERVATIVE GAS MIXTURE AND A DEVICE BASED THEREON Russian Federation Patent No. 2707532 (11/27/2019).

Claims (10)

1. A method for hypothermic preservation of an isolated mammalian organ for transplantation, characterized in that the organ is placed in a chamber with a gas medium, where the gas medium contains a gas mixture of dinitrogen oxide and oxygen "N ₂ O + O ₂ ", which makes up from 60 to 100% of the volume of the gas medium for preservation, where the gas mixture for forming the gas medium is selected from a list of gas mixtures included in the group consisting of: "N ₂ O + O ₂ ";"N ₂ O + O ₂ + Ar"; «N ₂ O+О ₂ +СО+Ar», where the ratio in percent of the volumes of gases in the gas mixture «N ₂ O+O ₂ » is selected from 71/29 to 29/71, preferably 50/50, where the ratio in percent of the volumes of gases in the mixture «N ₂ O+O ₂ +Ar» is 36/36/28, where the ratio in percent of the volumes of gases in the mixture «N ₂ O+О ₂ +СО+Ar» is 60/17/14/9, while in order to form a gas environment in the chamber, at the first stage, the chamber volume is purged using the first mini-cylinder with a gas mixture for purging under a pressure of up to 3 atm, and at the second stage, the chamber volume is filled with a gas mixture from the second mini-cylinder, which is pre-filled with a gas mixture under a pressure of up to 16 atm, while the working pressure in the chamber is set preferably 7 atm and hypothermic preservation is carried out at a temperature of from +2°C to +12°C, preferably about +4°C, for 12 to 24 hours, preferably 24 hours. 2. The method according to claim 1, characterized in that the organ for transplantation is preferably the heart. 3. A kit for hypothermic preservation of an isolated organ of a mammal for carrying out the method according to paragraph 1 or 2, characterized in that it contains: a) a device in the form of a sterile chamber, in the working volume of which the following are installed: - an organ attachment unit, which includes a flexible support on which the organ is placed, and side tabs are attached that hold the organ during transportation, - a container for liquid to form a humidity zone and additional cooling in the chamber; b) two mini-cylinders with gaseous media, where the first cylinder contains a gas mixture under a pressure of up to 3 atm for displacing air and purging the working volume of the chamber, and the second cylinder contains a gas mixture under a pressure of up to 16 atm for forming a gaseous medium in the working area of the chamber, while the gas mixture is selected when forming a set from a list of gas mixtures included in a group consisting of: "N ₂ O + O ₂ ";"N ₂ O + O ₂ + Ar";"N ₂ O+O ₂ +CO+Ar", where the ratio in percent of the volumes of gases in the gas mixture "N ₂ O+O ₂ " is selected from 71/29 to 29/71, preferably 50/50, where the ratio in percent of the volumes of gases in the mixture "N ₂ O+O ₂ +Ar" is 36/36/28, where the ratio in percent of the volumes of gases in the gas mixture "N ₂ O+O ₂ +CO+Ar" is 60/17/14/9, and the gas mixture is used to displace air, purge and create a high-pressure gas environment for prolonged preservation of an isolated mammalian organ for transplantation; c) a set of liquid media, d) a set of clamps for carrying out work on preserving and depreserving an organ after its transportation and storage. 4. Use of the kit according to item 3 for hypothermic preservation of an isolated organ of a mammal for carrying out the method according to item 1 or 2.