Perfusion fluid, methods of producing perfusion fluid, and its uses

Abstract

The subject matter of the present invention is a perfusion fluid in the form of an oil-in-water PFC nanoemulsion, methods of producing this perfusion fluid, and its use in particular for organ perfusion and as a blood substitute preparation.

Description

[0001] Perfusion fluid, methods of producing perfusion fluid, and its uses Description

[0002] Field of invention

[0003] The subject of the present invention is a perfusion fluid in the form of an oil-in-water PFC nanoemulsion, methods of production of this perfusion fluid, and its applications, in particularly for organ perfusion and as a blood substitute preparation.

[0004] Background of the invention

[0005] In Poland in 2023, 977 kidney transplants, 523 liver transplants and 178 heart transplants were performed, and the waiting list for a vascularized organ transplant includes an averages 1,903 patients annually in Poland (Statistics 2023. https: / / www.poltransplant.org.pl / statystyka 2023.html) . It is estimated that up to 25 per cent of patients die each year while waiting for an organ transplant, and an equal number are removed from the list due to a disqualifying health condition (Committee on a Fairer and More Equitable, Cost-Effective, and Transparent System of Donor Organ Procurement, Allocation, and Distribution. Realizing the Promise of Equity in the Organ Transplantation System. 26364 (National Academies Press, Washington, D.C., 2022). doi: 10.17226 / 26364). On the other hand, patients receiving dialysis awaiting kidney transplantation account for up to 7% of the total cost of the entire health care budget (Himmelfarb, J., Vanholder, R., Mehrotra, R. & Tonelli, M. The current and future landscape of dialysis. Nat. Rev. Nephrol. 16, 573-585 (2020)).

[0006] The growing problem of too few donors can be addressed by allowing the transplantation of organs from donors currently often excluded after irreversible cardiac arrest, i.e. donation after circulatory death (DCD) and donors qualified as expanded criteria donors (ECD). The ECD category includes donors aged 60 years and older and those aged 50-59 years with at least two of the following risk factors: hypertension, death due to cerebrovascular event, blood creatinine level at the moment of death above 1.5 mg / dL.

[0007] The risk of rejection of a kidney transplant taken from an ECD-qualified donor is 70% higher than that from SCD (ang. Standard Criteria Donor), not belonging to the ECD and DCD group (Rao, P. S. & Ojo, A. The Alphabet Soup of Kidney Transplantation: SCD, DCD, ECD-Fundamentals for the Practicing Nephrologist. Clin. J. Am. Soc. Nephrol. 4, 1827 (2009)). DCD kidney recipients due to ischaemic damage require dialysis much more frequently and for a longer time after kidney transplantation due to delayed graft function and have a markedly reduced long-term graft survival.

[0008] A solution to increase the availability of organs from ECD and DCD donors is to employ methods that enable organ procurement and storage [pl. przechowywanie], thereby extending their viability, allowing reliable assessment of future function, and ideally initiating ex vivo regeneration. Storage is a critical period, the only one in which interventions are possible to reduce the effect of ischaemic damage that has already been initiated.

[0009] The basic method of organ preservation is simple hypothermia (pl. prosta hipotermia, ang Static Cold Storage, SCS,). In this method, a cooled preservation solution is used to fill the organ’s vascular bed. The organ is then packed under sterile conditions and stored on ice. The first attempts at storage using simple hypothermia (ang. the static cold storage, SCS) method, using a dedicated fluid, a Collins formulation based on electrolytes with a high potassium and low sodium content, took place in 1969 (Wesson, L. G., Colberg, J. E., deGuzman, A., Elsasser, W. & Dunn, S. Extracellular fluid of the kidney preserved by the Collins technique. Transplantation 27, 380-383 (1979)). Hypothermia, by reducing the rate of chemical reactions, only slows the process of organ deterioration. This method is insufficient to maintain the organ’s full functional and metabolic capacity.

[0010] The solution to this problem is to maintain fluid circulation to provide nutrients and protect the organ from further degradation.

[0011] Further improvement in organ condition for transplantation as a result of the activation of repair processes requires raising the temperature of the organ with the simultaneous supply of oxygen and other substrates for active metabolism. Normothermic perfusion with blood at 37°C has been shown to improve transplantation outcomes compared to simple hypothermia in a large animal model (pig). It was already observed that when kidneys were stored at 20°C (subnormothermia) after reperfusion with blood, improvements in flow, urine excretion and creatinine clearance were observed.

[0012] In view of these premises, there is a clear opportunity to increase the pool of available high-quality organs qualifying for transplantation by using mechanical kidney perfusion under subnormothermic and normothermic conditions, which could realistically increase the number of transplants performed, shorten the time of dialysis use and reduce the number of deaths among patients awaiting transplantation.

[0013] A solution to this problem may be to conduct the perfusion process using an acellular fluid in the form of a perfluorocarbon nanoemulsion, acting as a synthetic nanocarrier for respiratory gases. This fluid should provide adequate oxygenation and essential nutrients to the organ awaiting transplantation. Perfusion fluids based on perfluorocarbons [pl. perfluorozwiqzkow] will also have significant advantages over products containing morphotic elements of blood, as they prevent phenomena such as leukotriene release and other inflammatory and vasoconstrictive substances.

[0014] Perfluorinated compounds (PFCs, pl. zwiqzki perfluorowane) are substances in which the hydrogen atoms are completely substituted with fluorine atoms and sometimes with other additional halogens (e.g. bromine). This substitution radically changes the physical properties of these organic compounds. The carbon-fluorine bond is very strong (-484 kJ / mol) and highly polar (almost ionic), which gives these compounds very high thermal and chemical stability. However, this does not result in water solubility, as the internal symmetry removes the polarity of each C - F bond and the entire PFC molecule becomes non-polar. These compounds also have twice the density and approximately 50 times the gas solubility compared to water. For example: perfluorooctyl bromide (PFOB) can dissolve 527 mLCh / L PFOB at a pressure of 1 atm. Carbon dioxide, on the other hand, can dissolve up to 4 times more than oxygen. For comparison, the solubility of O2 in water is about 9 - 10 mLCF / L water, and in blood about 200 mLCF / L blood. The presence of fluorine atoms affects the shape and size of the PFC molecule, which needs more space between the H2O molecules, increasing the hydration energy. This effect reduces the solubility of the PFC in water compared to the corresponding hydrocarbon derivative. On the other hand, this extreme polarity inhibits the formation of induced dipoles, which would lead to the van der Waals forces necessary to obtain solubility in lipids. Therefore, PFCs are both hydrophobic and lipophobic compounds.

[0015] Due to their unique physico-chemical properties, perfluorinated compounds and their derivatives have a wide range of industrial applications. In medicine and pharmaceutics, research is being conducted into their use mainly as PFC-based oxygen carriers (PFOCs) in preparations intended to replace blood and in organ perfusion fluids, contrast agents and as excipients in anticancer therapies. The extreme hydrophobicity of PFCs makes it necessary to use emulsifiers such as lipids, partially fluorinated compounds, proteins or other surface active agents to combine with aqueous media, e.g., blood or organ perfusion fluids. These preparations are usually in the form of an emulsion, i.e., a mixture of two immiscible phases (in this case water and perfluorinated compounds). The formation of such kinetically stable systems is made possible by the use of surface tension-reducing compounds and high-pressure emulsification methods. Oxygenation of PFC preparations results in the dissolution of large amounts of O2 in the perfluorinated core of the emulsion particles. The emulsifier present in the envelope [pl. otoczka] does not interfere with this process. If such a formulation, is then exposed to an environment with lower oxygen content (e.g., body tissues), oxygen diffuses out of the mixture. WO2024046999A1 discloses compositions for the production of stable synthetic oxygen carriers based on perfluorocarbons, methods for their manufacture and use as synthetic blood-oxygen carriers and as volume expanders. The manufactureing of perfluorocarbon-based synthetic oxygen carriers (PFOCs) disclosed herein comprises the following steps: (a) preparation of a suitable aqueous albumin solution and mixing it with at least one suitable perfluorocarbon-based artificial oxygen carrier, (b) suitable addition of lecithin, (c) preemulsification by rapid mixing with suitable cooling, (d) emulsification of the pre-emulsion from step (c) under pressure using a high-pressure homogenizer with suitable cooling, and storage for at least 30 minutes, also with suitable cooling. Albumin used was from among the mammalian albumin group, human serum albumin (HSA) and bovine serum albumin (BSA), or suitable derivatives such as pegylated albumin. Albumin was prepared in a suitable aqueous buffer, water or balanced electrolyte solution for infusion therapy, such as Sterofundin® ISO, Ringerfundin®, Ringer solution, STEEN solution, OCS solution and hydroxyethyl starch solution. The addition of lecithin as a stabiliser was used in the preparation of emulsions. WO200877641 Al discloses nanoemulsions comprising at least one aqueous component and a carrier phase, the carrier comprising at least one lipophilic component, at least one surface active agent and at least one alcohol. The method of preparing an emulsion comprises the steps of (a) preparing an aqueous component, (b) preparing a carrier comprising at least one lipophilic component, at least one surface active agent and at least one alcohol, the at least one alcohol having at least three carbon atoms, and (c) mixing the aqueous component of step (a) with the carrier of step (b). The document also discloses a composition comprising the nanoemulsion and an active substance. In particular, the composition is in the form of a gel and the active substance is 5-aminolevulinic acid (ALA), a derivative, precursor and / or metabolite thereof. The invention also relates to the preparation of the said nanoemulsion and / or composition and to their use in the treatment of dermatological diseases, viral diseases, and diseases associated with abnormal cell proliferation, in particular cancer and / or psoriasis, and to the use of the said nanoemulsion in cosmetics.

[0016] Document US 11406722 B2 discloses a method for producing a composition of liquid perfluorocarbon (PFC) nanodroplets, the method comprising the following steps: combining the PFC liquid with a surfactant and a co-surfactant to produce a liquid composition; and emulsifying the said PFC liquid with the surface active agent and the said co-surfactant within the said liquid composition by direct microfluidization at a pressure of at least 3,000 psi to reduce the size of the PFC droplets within the said liquid composition, thereby forming a nanodroplet composition with an average particle diameter of less than 300 nm, the PFC having a boiling point of below approximately 0°C. The said surface active agent comprises an amphiphilic phospholipid-based compound, wherein the said co-surfactant comprises a semifluorinated alkane and wherein emulsification is carried out at a temperature below 0°C. The document discloses compositions having an average particle diameter of less than about 250 nm, for example less than about 200 nm or less than about 150 nm. The composition disclosed in document US 11406722 B2 shows less than 10 % change in average particle diameter over a period of 1 week at 4°C, or less than 10 % change in average particle diameter over a period of 1 hour at 37°C. The composition has applications in the field of medical diagnostics and therapeutics, where the stabilised nanodroplets are used in ultrasound imaging and potentially in therapy, e.g. by forming microbubbles under ultrasound.

[0017] Document KR20230124148, discloses a composition for inhibiting fat accumulation using a nanoemulsion containing a perfluorocarbon, a surface active agent and an auxiliary surface active agent dispersed in a continuous phase. The document discloses the effective delivery of perfluorocarbon to cells, tissues or body tissue causing an inhibitory effect on fat accumulation. The document describes studies on nanoemulsion particle size under different conditions. Compositions containing EggPC or Span80 with Tween80 as an auxiliary surfactant were found to form stable emulsions with small particles (100 - 200 nm). However, the addition of Pol oxamer 188 as an auxiliary surfactant negatively affected the stability of the emulsions, causing an increase in particle size above 1000 nm and instability with changes in temperature and time.

[0018] In W02020209752 Al, discloses an emulsion comprising the perfluorocarbon compound perfluorodecalin, a perfluorocarbon additive and phospholipids in the form of liposomes (vesicles) prepared by pressure homogenization in an aqueous saline medium. The perfluorocarbon additive is a mixture of perfluorinated products: perfluorooctyl bromide and perfluorotripropylamine, and the liposomes (vesicles) contain natural oils as an excipient. The method for producing the emulsion comprises producing the liposomes (vesicles) by homogenization at a pressure of 60 - 120 atm in an aqueous saline medium, followed by hot sterilization, homogenizing the said perfluorocarbon compounds under pressure into a liposomal form (vesicles) of phospholipids and performing hot sterilization of the prepared emulsion. The emulsion is intended to improve blood oxygen supply during the treatment of hypoxic conditions and to conserve isolated perfused organs and tissues. The stability of the emulsion is increased while the particle structure is maintained, thus improving the quality of the emulsion and maintaining biocompatibility with the biological medium (blood, plasma, serum, blood substitutes). The shelf life of the emulsion after thawing at +4°C is a at least 23 months, while maintaining the emulsion structure.

[0019] Document WO201356246 Al, discloses an oxygen therapeutic composition comprising a perfluorocarbon material, a viscosity modifier, a buffer, the buffer stabilizing the pH of the composition at from about 6.5 to about 7.5, the composition having a viscosity of from about 2.0 to about 3.5 mPa-s and the perfluorocarbon having a boiling point of from about 4°C to about 60°C. The document indicates that DDFPe absorbs more oxygen at room temperature than other agents including perfluorodecalin and perfluorooctyl bromide and is even more effective at physiological temperature (above the boiling point of DDFP). The document discloses that DDFPe forms an emulsion in water consisting of submicron-sized droplets at room temperature and converts to a gas at 29°C. In order to maintain the stability of the DDFPe emulsion, a viscosity modifying material was used which prevents particle settling and agglomeration. The document indicates that sucrose is the preferred viscosity modifying material.

[0020] Document CN106176601A discloses a perfluorocarbon emulsion and a method for producing and using it, in particular for improving oxygen content in tumours. The perfluorocarbon emulsion is prepared from perfluorocarbons and proteins preferably bovine serum albumin or human albumin. Such an emulsion has a particle size of 180 - 200 nm and has a high oxygen carrying capacity and can rapidly release the contained oxygen under ultrasound. In the method for producing the emulsion, the protein is dissolved in phosphate buffer, then perfluorocarbon liquid is added, and the resulting solution is sonicated by ultrasound, the bottom layer obtained is centrifuged and subsequently dissolved in phosphate buffer. The experiment showed that the oxygen carried by the perfluorocarbon emulsion could be rapidly released under the mechanical action of ultrasound, and the oxygen content at the tumour site increased steadily. With this method, it is possible to effectively alleviate oxygen deficiency at the tumour site, which results in improved therapeutic outcomes with photodynamic therapy and radiotherapy.

[0021] Document W02010 / 121082 discloses an emulsion comprising a perfluorocarbon dispersed as particles in a liquid phase, the dispersed particles having a monomodal particle size distribution. According to the disclosure, the perfluorocarbon may be perfluoro (tert-butylhexane), perfluorodecalin, perfluoroisopropyldecalin, perfluorotripropylamine, perfluorotributylamine, perfluoromethylcyclohexylpiperidine, perfluorooctyl bromide, perfluorodecyl bromide, perfluorodichlorohexane, perfluorohexane, dodecafluoropentane or mixtures thereof. The emulsion additionally contains an emulsifier, which is a phospholipid from egg yolk. In addition, the emulsion contains an aqueous carrier buffered to pH 6.8 - 7.4. The method for producing the emulsion comprises the following steps: mixing the emulsifying agent with the aqueous medium (step a), adding a perfluorocarbon to this mixture (step b), mixing this mixture to form a coarse emulsion (step c), obtaining a sample of the emulsion and determining the particle size distribution (step d), if the particle size distribution is monomodal, homogenization of the emulsion is carried out (step e), obtaining the final emulsion (step f). The document indicates the usefulness of the emulsion in the treatment of sickle cell anaemia, decompression sickness, air embolism or carbon monoxide poisoning in a patient by administering the described emulsion in an amount effective to treat these conditions. The document also discloses the use of the emulsion in a method of conserving an organ prior to transplantation, which involves contacting the organ with the described emulsion to extend organ viability. In one variant, the organ is also perfused with the emulsion.

[0022] Document WO2015 / 147705 discloses a method for producing a stable, sterile nanoemulsion of perfluoroorganic compounds (PFOC) with an average particle size of not more than 150 nm, preferably 30 - 100 nm, and most preferably 30 to 80 nm. The method for producing the nanoemulsion comprises filling the circuit of the installation for producing the PFOC nanoemulsion with an aqueous solution of a stabilizing additive, preferably a poloxamer, adding the PFOC mixture to the aqueous solution of the stabilizing additive and then homogenizing the resulting PFOC mixture in the aqueous solution of the stabilizing additive to obtain the PFOC pre-emulsion of the required particle size, under thermostatic control of the homogenizer working chamber. The PFOC pre-emulsion is then added to an aqueous salt solution to produce a PFOC nanoemulsion with the required concentration of PFOC, stabilizing additive and salt. The PFOC nanoemulsions are stored at 2 - 10°C, with optional pre-packaging of the finished product into sterile consumer packaging and freezing of the finished product. According to the disclosure in the document, two types of perfluoroorganic compounds are typically used for PFOC emulsions. One is selected from the C8 - CIO group, comprising, for example, perfluorodecalin (PFD) or perfluorooctyl bromide (PFOB), while the other is selected from the Cll - C12 group, comprising, for example, perfluorotripropylamine (PFTPA), perfluoromethylcyclohexylpiperidine (PFMCP) and perfluorotributylamine (PFTBA). Compounds of the first type are rapidly (within a month) removed from the body, but do not provide sufficient emulsion stability. Compounds of the second type, on the other hand, have a high emulsion stability that allows them to be stored without freezing, but can persist in the body for long periods of time (8 months to 2 years). It is also possible to use mixtures of two PFOC compounds, such as FPD / PFTBA, PFD / PFMCP, PFOB / PFTBA, PFOB / PFMCP, or three compounds, for example PFOB / PFD / PFMCP, PFOB / PFD / PFTBA, or even four, such as FPD / PFOB / PFMCP / PFTBA. Special stabilisers are added to the emulsion to reduce the average particle diameter of the emulsion, increase dispersion and stability during long-term storage. Non-toxic high-molecular-weight non-ionic surface active agents (non-ionic surfactants), in particular poloxamers (proxanols, Pluronics, Kolliphor), are preferred as stabilisers. Their amount in PFOC emulsions is kept to a minimum, resulting only from the need to ensure satisfactory emulsification and homogenization of PFOC. Minimising the amount of surfactants is important because they affect the toxicity and reactivity of PFOC emulsions.

[0023] Document WO2007 / 139827 A2 discloses a storage-stable perfluorocarbon emulsion for in vivo oxygen delivery in patients, comprising a continuous aqueous phase and a discontinuous fluorocarbon phase, wherein the discontinuous fluorocarbon phase comprises perfluorooctyl bromide and perfluorodecyl bromide, wherein perfluorooctyl bromide is present in the fluorocarbon emulsion in an amount of about 57 - 60 % w / v of the total emulsion and perfluorodecyl bromide is present in the emulsion in an amount of about 2 - 3% w / v of the total emulsion. The emulsion additionally contains an emulsifying agent comprising egg yolk phospholipid in an amount of about 3 - 4 % w / v. The method for producing comprises (a) preparing an aqueous solution of sodium salts in hot water (preferably at a temperature of 65°C to 80°C) in the first tank and saturating with nitrogen; (b) adding of perfluorooctyl bromide and perfluorodecyl bromide to the second tank and saturation with nitrogen; (c) adding the emulsifying agent to the third tank and adding the contents of the first tank to the third tank; (d) adding perfluorooctyl bromide and perfluorodecyl bromide to the third tank and mixing the contents to emulsify the perfluorooctylbromide and perfluorodecyl bromide; (e) diverting the contents of the third tank into one or more homogenizing tanks. Although not limited to the following, some other therapeutic applications of the emulsion include its use in improving post-operative organ function (bowel, liver, heart, brain, kidney), treating sickle cell anaemia, organ conservation, organ transplantation or enhancing the effectiveness of chemotherapy. Document W02007 / 105978, discloses a composition formed from a mixture of fast (C8 - CIO) and / or slow (Cll - C12) excreted types of perfluorocarbons, such as perfluorodecalin (PFD) / perfluoromethylcyclohexylpyridine (PFDCP), or PFD / perfluorotributylamine (PFTBA), or perfluorooctylbromide (PFOB) / PFMCP, or PFOB / PFTBA in a ratio of 1 / 1 to 10 / 10, respectively; or mixtures of three types of perfluorocarbons: two rapidly excreted (C8 -CIO) and one slowly excreted (Cll - C12), or one rapidly excreted (C8 - CIO) and two slowly excreted (Cll - C12) perfluorocarbon types such as PFOB / PFD / PFDCP, or PFOB / PFD / PFTBA, or PFOB / PFDCP / PFTBA, or PFD / PFDCP / PFTBA, in a ratio of 1 / 1 / 1 to 10 / 10 / 10, respectively; or a mixture of four perfluorocarbon types: two rapidly excreted (C8 -CIO) and two slowly excreted (Cll - Cl 2) types of perfluorocarbons, such as PFOB / PFD / PFDCP / PFTBA in a ratio of l / l / l / l to 10 / 10 / 10 / 10, respectively. A mixture of perfluorocarbons in an emulsion of 1% (0.5% by volume) to 100% (50% by volume) and an average particle size of 30 - 80 nm is emulsified with proxanol-268 in an amount of 0.2% to 20% with a molar mass of 6 - 12 Da and contains an acceptable electrolyte solution. The method of producing the perfluorocarbon emulsion comprises: producing the emulsion by mixing the total perfluorocarbons with an emulsifier, repeated homogenization of the resulting mixture in a high-pressure homogenizer, the entire emulsion preparation process being carried out in at least two high-pressure extrusion units by sequentially passing the emulsion through a primary and secondary extrusion unit (extruders), passing the emulsion through a buffer volume located between the two extruders to equalise pressure between the devices, passing the emulsion through the primary extruder, increasing the homogenization pressure by 2 - 3 times compared to the pressure in the secondary extruder, passing the emulsion through the buffer volume and introducing (supplying) carbon dioxide gas. The disclosed emulsion can be used as a synthetic perfluorocarbon blood substitute for the treatment of various diseases or as a synthetic perfusion agent. Document CN1286081 A, discloses a high concentration, ultra-fine particle perfluorocarbon emulsion for injection which contains a perfluorocarbon, an emulsifier, an isotonic buffer solution, an additive and water. The emulsion is prepared by sterilizing all equipment and containers and producing the emulsion under aseptic conditions. Its advantages are that the product does not need to be sterilized at high temperatures, has a long storage time and has a long duration of action in the circulatory system. In the disclosed method for producing, prior to emulsification, all parts in direct contact with the materials, the product packaging containers and all materials are sterilized and pyrogens are removed prior to emulsification. The material is then weighed in a clean environment, all ingredients except perfluorocarbon are added to water to form an aqueous solution and then sterilized using high-pressure steam. The next step is to heat the aqueous solution to a temperature 2 - 10°C higher than the turbidity point of the emulsifier, stir until the aqueous solution becomes transparent, then add the perfluorocarbon, stir evenly, and pass through the homogenizer circuit; the first pass through the homogenizer has a pressure of 100 - 200 kg / cm2, the second pass has a low pressure of 100 - 200 kg / cm2and a high pressure of 500 - 700 kg / cm2. The circulation pump pumps the emulsion 5 - 30 times to achieve an emulsion particle size of 40 - 80 nm. The disclosed perfluorocarbon emulsion, with high concentration and ultrafine particles for injection, contains 20.0 - 115.0 g of perfluorocarbon per 100 ml of emulsion; 0.5 - 10.0 g of emulsifier; 0.3 - 1.0 g of isotonic buffer, 0.5 - 10.0 g of additive and the remaining amount of water, with an average particle size in the emulsion in the range of 40 - 80 nm. The perfluorocarbon is selected from the group including perfluorooctyl bromide, bis(perfluorobutyl)ethylene, perfluoroalkyl ethylene, perfluorocarbon ether or perfluorodecalin. The emulsifier is selected from the group comprising egg yolk lecithin, partially hydrogenated egg yolk lecithin or polyether F-68.

[0024] Document US5684050 discloses stable emulsions of highly fluorinated organic compounds for use as oxygen transport agents, "artificial blood" or red blood cell substitutes and as contrast agents for biological imaging. These emulsions contain a highly fluorinated organic compound, an oil that is neither significantly surface active nor significantly water-soluble, a surfactant and water. The highly fluorinated organic compound is selected from the group comprising perfluorodecalin, perfluorodimethyladamantanes, perfluorooctyl bromide, perfluoro-4-methyl-octa-hydroquinolidine, perfluoro-N-methyl-decahydroquinoline, F-methyl-1 -oxa-decalin, perfluoro-bicyclo[5.3.0]decane, perfluoro-octa-hydroquinolidine, perfluoro-5,6-dihydro-5-decene and perfluoro-4,5-dihydro-4-octene. The oil is selected from the group comprising liquid fatty oils, C12 - C18 fatty acid diglycerides with one unsaturation, silicone oils and mineral oil, in addition the emulsion may further comprise at least one compound from the group comprising isotonic agents, osmotic pressure controlling agents and antioxidants. The disclosed emulsion is prepared by mixing the respective components in a Fisher brand blender and then passing them through a microfluidizer for 30 minutes at 60 psi.

[0025] Document US4987154 A, discloses a perfluorocarbon emulsion with a concentration of up to 125% for use in animals and their organs, which maintains emulsion stability during standard sterilization procedures due to selective osmotic and buffering agents, maintains the emulsion at a specified osmolality level and, when desired, is free from excessive calcium precipitation, reduces damage to red blood cells in vivo and in vitro, reduces the adverse effects of anaemia, reduces viscosity and slows the rate of oxidation, and seeks to equilibrate its distribution in major body organs, thereby reducing toxicity. Osmotic agents can act as buffers and provide nutrients in the form of sugars. Osmotic and buffering agents can selectively include hexahydric alcohols such as mannitol and sorbitol; certain sugars such as glucose, mannose and fructose; and buffering agents that will affect osmolality, including imidazole, tris(hydroxymethyl)aminomethane, sodium chloride, sodium bicarbonate, monobasic potassium phosphate, dibasic potassium phosphate, calcium chloride, magnesium sulphate, monobasic sodium phosphate, dibasic sodium phosphate or combinations thereof. The emulsion may contain tocopherol. The method for emulsifying a perfluorocarbon comprises forced flows under pressure after mixing the perfluorocarbon in the dispersed phase. The perfluorocarbon emulsion can be used to deliver drugs and therapeutic substances that are soluble in the emulsion or transported through the emulsion.

[0026] Document JP94062402 B2, discloses a sterilizable brominated perfluorocarbon emulsion suitable for intravenous injection, excellent for transporting oxygen to cellular tissue in the animal body and for maintaining the emulsion, comprising a brominated perfluorocarbon, cholesterol, etc., and a specific compound. The emulsion disclosed comprises an aqueous phase, a small amount of emulsifying agent (e.g. phospholipid or anionic surface active agent) and a compound selected from the group comprising of brominated perfluorocarbon, cholesterol, steroid hormone, tocopherol and combinations thereof in an amount to ensure the presence of the compound in the body. Steroid hormone, cholesterol, tocopherol and a mixture thereof are used as the compound. The method of producing a perfluorocarbon emulsion, comprises the steps of: (a) adding a compound selected from the group comprising of steroid hormones, cholesterol, tocopherol and combinations thereof in an in vivo compatible amount to a buffered aqueous solution of glycerol; (b) dispersing an emulsifying agent comprising a phospholipid, brominated perfluorocarbon at a concentration of 50% to 125% by weight / volume in the liquid prepared in step (a); and (c) dividing the resulting mixture into multiple streams and diverting the divided streams under a pressure of at least 281.2 kg / cm2(a method of emulsion production involving a step of diverting the divided streams at a pressure of 4000 psi).

[0027] Document Johannes Jagers et al, 18 April 2022, Artificial oxygen carriers in organ preservation: Dose dependency in a rat model of ex-vivo normothermic kidney perfusion, Artificial Organs. 2022;46:1783-1793. (https: / / onlinelibrary.wiley.com / doi / full / 10. Ill 1 / aor.14264 ) describes organ behaviour during ex vivo normothermic perfusion (EVNP) using artificial oxygen carriers based on albuminderived perfluorocarbon (A-AOCs), consisting of nanocapsules filled with perfluorodecalin derived from albumin, prior to transplantation, which would be a promising approach to avoid hypoxic tissue damage during organ storage. According to the Gibbs-Helmholtz equation, a major challenge in emulsion formation of a large phase interface. The large difference in surface tension that exists between the perfluorocarbon and albumin results from the lack of hydrogen bond acceptors, which increases the internal energy of the system. Emulsifiers can adhere to the interfacial surface and act as hydrogen bond acceptors to minimise the undesirable increase in internal energy during the manufacturing process. An important property of the albumin emulsifier used in this system is its anisotropy. It has a hydrophilic side and a hydrophobic side that try to escape from the aqueous phase. In this way, albumin acts as an excellent hydrogen bond acceptor on one side and covers the hydrophilic surface on the other side. In the method for producing the emulsion, the indicated document discloses emulsifying 4 ml of perfluorodecalin (Fluorochem Chemicals, Derbyshire, UK) in 20 ml of a 5% bovine serum albumin solution (BSA, AppliChem GmbH, Darmstadt, Germany) in a Ringer-saline solution, both cooled liquids are pre-emulsified for 20 s using Ultra- Turrax® (IKA®-Werke GmbH & Co. KG, Staufen, Germany). The emulsion is then further emulsified using microfluidization technology (Microfluidics®, Westwood, MA) at 20000 PSI in single pass mode. The emulsion is then diluted with a Ringer-Saline solution containing 5% BSA to 2%, 4%, and 8% A-AOCs, respectively.

[0028] Document Hosgood, Sarah A.; Nicholson, Michael L., 27 May 2010, The Role of Perfluorocarbon in Organ Preservation, Transplantation 89(10):p 1169-1175, 2010, DOI: 10.1097 / TP.0b013e3181 da6064(https: / / j ournals.lww.com / transplantj ournal / fulltext / 2010 / 0527 0 / the_role_of_perfluorocarbon_in_organ_p reservation.1.aspx) discloses perfluorocarbon (PFC) emulsions used in organ storage, dividing them into first generation (e.g. FX-80, FC-43, Fluosol-DA, Oxypherol, Perftoran) and second generation (e.g. Perftoran 1989 - 1996, Oxygent, Pher O2), with various components and emulsions such as Pluronic F-68, Proxanol-268, phospholipids, perfluorodecalin and perfluorotributylamine. The document discloses that the most successful application of PFCs is TLM (perfusion therapy) for pancreatic preservation, which has significant evidence of its benefits in clinical practice. However, the document indicates that questions remain regarding how effectively oxygen can diffuse through tissue under these conditions. Additionally, the usefulness of this technique for organs other than the pancreas has not been thoroughly investigated. Evidence suggests that perfusion or flushing techniques using PFC emulsions are beneficial for hypothermic storage and appear to be superior to static techniques. However, most of these mainly historical, isolated trials have not been introduced into clinical practice, probably due to the instability and adverse reactions associated with first-generation emulsions at the time. Recent advances and the trend toward machine perfusion and the development of improved second-generation PFCs suggest that PFCs may have a significant role in organ preservation techniques in the future.

[0029] The publication entitled “A Comparison of Lipoprotein Secretion, Bile Production and Hepatic Morphology in Isolated Rat Livers Perfused with a Perfluorocarbon Emulsion or Rat Erythrocytes”, discloses perfusion fluids, including a perfluorocarbon emulsion FC-43 (Oxypherol, Alpha Therapeutics Corp.) and a medium containing rat erythrocytes (RBC) in combination with Krebs-Henseleit buffer (25% v / v RBC and 75% v / v KH buffer + glucose 1.5 mg / mL (8.8 mmol / L)). The FC-43 (Oxypherol) emulsion consisted of the respiratory gas carrier FC-43 (perfluorotributylamine) at a concentration of 20% w / v (equivalent to 10% v / v), a nonionic surfactant Pluronic F-68 (polyoxyethylene-polyoxypropylene copolymer) at 2.56% w / v, an oncotic agent hydroxyethyl starch at 3.0% w / v, an energy substrate glucose at 1.8 mg / mL (10 mmol / L), and electrolytes: NaCl (103 mmol / L), KC1 (4.6 mmol / L), CaCL (2.5 mmol / L), MgCL (2.1 mmol / L), andNaHCOs (25 mmol / L). The emulsions were prepared from stock solutions supplied by the manufacturer (Alpha Therapeutics Corp.) and filtered through a 1.2 pm filter prior to use. The pH of the emulsion was adjusted to 7.4 using a gas mixture of 95% O2 / 5% CO2. The nanoemulsion was characterized by particle size ranging from 82.0 ± 34.2 nm to 123.4 ± 38.3 nm and a density of 1.8 g / mL. Before use, the emulsion was filtered through a 1.2 pm filte. The study presented a comparison of the effects of two different perfusion media on the function of isolated rat livers. During liver perfusion, the following parameters were evaluated: Oxygen consumption - both media provided adequate oxygenation; Bile secretion - livers perfused with FC-43 secreted significantly less bile (approximately 40% compared to RBC); Liver morphology - FC-43 caused structural damage such as sinusoidal ballooning, endothelial detachment, numerous vacuoles in Kupffer cells, endothelial cells, and hepatocytes, loss of microvilli; Lipoprotein structure and secretion - perfusates from FC-43 contained markedly fewer lipoproteins, whereas livers perfused with RBC secreted lipoproteins of normal composition and structure. In summary, it was indicated that the FC-43 emulsion is not a suitable medium for studies on hepatic lipoprotein metabolism, and that Pluronic F-68 may leak from the emulsion, accumulate in cells, negatively affect their structure and lipoprotein composition. Despite adequate oxygen consumption, FC-43 emulsion causes significant cellular damage, ultimately leading to impaired liver function. The document did not disclose the full characterization of the perfusion fluid, nor the specific oncotic or osmotic pressure of the said composition.

[0030] Document WO 2007 / 139827 A2, discloses a stable perfluorocarbon emulsion for medical applications, primarily as an oxygen carrier in situations requiring blood transfusion reduction (e.g., surgery, trauma, anemia). The dispersed phase consists of perfluorocarbons: perfluorooctyl bromide (PF OB) - 58% w / v and perfluorodecyl bromide (PFDB) - 2% w / v, while the continuous (aqueous) phase contains an emulsifier - egg yolk phospholipid (EYP) at 3.6% w / v, antioxidant - d,a-tocopherol at 1% w / v (optional), chelator: EDTA at 0.02% w / v (optional), buffers and electrolytes: NaCl - 0.36% w / v, NaEEPCL FEO - 0.069% w / v, NazHPO^ EEO - 0.474% w / v. Average particle size of the emulsion according to the invention is < 0.18 pm (average), osmolality 300 - 310 mOsm / kg, pH 7.0 - 7.2, viscosity ~4 ePoise (at 1 / s), stability > 24 months at 5°C, sterilization by steam autoclaving. According to the invention, advantageous features of the PFOB / PFDB formulation include: PFDB stabilizes the emulsion by limiting Ostwald ripening (particle growth); low organ retention of PFDB (~23 days) at low concentration (1 - 3% w / v); no PFDB crystallization at low concentration (high melting point: 55°C); palanced particle size - reduced risk of intercellular space penetration; possibility of repeated dosing - critical for multi-stage therapy. Pharmacokinetics were evaluated after nanoemulsion administration in rats and humans. Therapeutic applications indicated by the invention include surgery (orthopedic, cardiac, oncologic), treatment of anemia, brain injury therapy, stroke, carbon monoxide poisoning treatment, organ preservation, and supportive radiotherapy and chemotherapy. The document does not disclose specific oncotic or osmotic pressure, pH of the composition(s), nor the osmotic agent

[0031] Document WO 2015 / 138999 Al, discloses perfluorocarbon (PFC) emulsions for use as blood substitutes and media for tissue and organ conservation, particularly for the heart. The main innovation involves the use of purified phospholipids with low content of lysophosphatidyl compounds (LPC, LPE), which at higher concentrations exhibit cytotoxic effects and impair cardiac function after conservation. The dispersed phase of the nanoemulsion consists of PFCs, which may include perfluoroperhydrophenanthrene (providing the best results in heart perfusion), perfluorodecalin, perfluorotrialkylamines, perfluorooctyl bromide, perfluoromethyldecalin, perfluoromethyladamantane, at concentrations of 10 - 50% v / v (optimally 35 - 45% v / v). The continuous (aqueous) phase of the nanoemulsion contains an emulsifier - phospholipids (e.g., egg yolk) with <5 mol% lysophosphatidyl compounds (LPC, LPE), optimally 0.5 - 7% w / v (preferably 0.5 - 4% w / v), crystalloid solution: NaCl, KC1, CaCL, MgCb, KH2PO4, NaHCCh, metabolic substrates: glucose, pyruvate, mannitol, pharmacological agents: lidocaine, heparin, oncotic additives: albumin, hydroxy ethyl starch, optional additives: antioxidants (vitamin E, ascorbic acid), buffers: THAM. The nanoemulsion is characterized by an average particle size of 0.09 - 0.15 pm (max. 0.4 pm), viscosity 1.5 - 17 cP (optimally 1.7 -2.3 cP at 11°C), density 1.1 - 1.5 g / mL, pH 6.5 - 8.5. The emulsion can be stored at room temperature. It is obtained by microfluidization at pressures up to 14,500 psi and temperatures <35°C (preferably <20°C), involving 70 - 80 passes through the reactor. The patent indicates the possibility of conservation of a rat heart with an emulsion containing 10% PFC and 4.5 mol% LPC / LPE, which maintained 100% function after 12 hours of conservation, and a rabbit heart that retained full function after 24 hours. Clinical applications of the emulsion according to the invention include organ conservation for transplantation (heart, lungs, kidneys, liver, etc.), cardioplegia during cardiac surgery, angioplasty and atherectomy - oxygen delivery to ischemic tissues, reperfusion after injuries (e.g., spinal cord ischemia, infarction), and use as a blood substitute in transfusions. However, the document does not disclose specific oncotic or osmotic pressure and discloses a broad pH range of 6.5 - 8.5 for the composition.

[0032] Document WO2015138999A1, discloses a perfusate composition for lung tissue conservation, comprising an aqueous Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KC1, 1.2 mM KH2PO4, 1.2 mM MgSCL, and 4.2 mM NaHCCh), 0.2 - 0.5% w / w D-glucose, 1 - 10% w / w dextran, 1 -15% w / w human albumin, and 1 - 20% w / w hetastarch (hydroxyethyl starch). The patent mentions the possibility of enriching the composition (Table 2) with various concentrations of glutamine, antibiotics, and amino acids; an antioxidant to neutralize reactive oxygen species (ROS), such as glutathione or N-acetyl cysteine; a cAMP analog (e.g., dibutyryl cAMP); insulin; hydrocortisone (as a membrane stabilizer); growth factors such as VEGF and FGF; red blood cells; perfluorocarbons; and hemoglobin-based oxygen carriers as artificial oxygen carriers. The document broadly discloses physiological values occurring in the human body under normal conditions, such as intrapleural pressure ranging from - 5 to - 8 cm JLO, arterial pressure around 13 mmHg, venous pressure around 6 mmHg, and interstitial pressure around -5 mmHg, among others. However, the document does not disclose specific concentrations of the said additives, only indicating the possibility of composition enrichment, and provides no implementation examples. As oxygen carriers, three compound types known in the literature are disclosed, with perfluorocarbons listed as one option. The document does not disclose specifications for the said fluid (pH, oncotic pressure, osmolality, particle size distribution) nor its application for perfusion of organs other than lungs.

[0033] In the publication by Dirks B., Krieglstein J., Lind H.H., “Fluorocarbon Perfusion Medium Applied to the Isolated Rat Brain” , the use of a fluorocarbon-based perfusate for isolated rat brains is disclosed. The document reveals a perfusion medium based on fluorocarbons containing 20% w / w FC-43 (perfluorotributylamine), 4.8% w / w Pluronic F-68, and 0.14 or 0.28% w / w glucose in Krebs-Henseleit buffer, filtered through a 0.2 pm membrane. The publication reports levels of substrates and metabolites (e.g., ATP, adenylate, glucose-6-P, lactate) in isolated rat brains perfused with the said perfusion fluid. The study measured parameters of the fluid such as osmolality, viscosity, particle size distribution, and oxygen capacity. Brains perfused with the perfluorotributylamine-containing fluid exhibited electrical impulses for over 7 hours, whereas brains perfused with simplified blood lasted only about 3 hours. The document discloses differences between perfusion fluids without an oxygen carrier versus those containing an oxygen carrier but does not disclose either the pH or the oncotic pressure of the fluid.

[0034] In patent EPO231091A1, an emulsion is disclosed comprising at least one perfluorinated compound dispersed in an oil phase, a surface active agent, and water. The perfluorinated compounds, in an amount of approximately 75% by volume, include perfluorocarbons with 9 - 18 carbon atoms, such as perfluorodecalin, perfluorotrimethylbicyclo(3.3.1)nonane, perfluoro-2, 2, 4, 4-tetram ethylpentane, perfluorooctyl bromide, perfluoroindan, 9 - 12 carbon perfluoroamines (e.g., perfluorotripropylamine, perfluorotributylamine, perfluorodimethyladamantane), perfluoro- 1 -azatricyclic amines, bromine- or iodinesubstituted perfluorocarbons, and F-4-methyloctahydroquinolizine, as well as their mixtures. Among oils, the patent discloses the use of approximately 15 - 20% by weight, either individually or as mixtures, including liquid fatty oils, hydrocarbons, waxes such as monoesters of fatty acids and monohydric alcohols, long-chain ethers, diglycerides, silicone oils, and nitriles. Examples include palmitoyl oleate, octylonitrile, dodecylonitrile, soybean oil, safflower oil, hexadecane, diglycerides with C12 - 18 carbon chains and one unsaturated bond, and mineral oil. Typically, 10 - 30% by weight of the non-fluorocarbon fraction of the emulsion consists of oil or oil mixtures. The disclosed surface active agents, in amounts of approximately 0.5 - 7% by weight relative to the non-fluorocarbon fraction, may be used individually or in combination. Preferred surfactants include nonionic types such as egg yolk phospholipids, lecithin, phospholipids, polyoxy ethylene-polyoxypropylene copolymer (Pluronic F-68), and alkyl salts of oleic acid, such as sodium oleate. The emulsion according to the invention contains approximately 2.5% by weight (relative to the non-fluorocarbon fraction) of an isotonic agent, typically glycerol, Tyrode’s solution, or similar, to adjust osmotic pressure. The emulsion is prepared by mixing the four main components - perfluorinated compounds, oil, surfactant, and water — in any order using conventional mixers and emulsifiers. The emulsions exhibit long-term stability at room temperature and can be sterilized by thermal treatment at 115°C for 15 minutes or under higher temperature conditions. The document describes efficacy testing of the developed emulsion by intravenous injection in mice at doses of 20 mL / kg body weight (or 40 mL / kg), with no observable signs of toxicity or gas / vapor embolism. The average clearance rate (elimination from the organism) for various nanoemulsions was evaluated (up to 3 -4 days).

[0035] Document WO2007 / 139827A2 discloses, a stable perfluorocarbon emulsion, consisting of a continuous aqueous phase and a dispersed perfluorocarbon phase containing two perfluorocarbons: PFDB and PFOB. A reference emulsion containing 90 g of PFOB, 4 g of egg yolk phospholipid (EYP), and physiological concentrations of salts and buffers was prepared by high-pressure homogenization according to Long’s method, yielding a PFOB emulsion at 90% w / v. Subsequently, the secondary perfluorocarbon perfluorodecyl bromide (PFDB) was added to the 90% w / v PFOB emulsion in amounts of 1%, 2%, 5%, and 10% w / w, creating one PFOB emulsion and four PFOB / PFDB emulsions. The document discloses the most stable nanoemulsion composition: CsFnBr (PFOB) 58% w / v, CwF2iBr (PFDB) 2% w / v, egg yolk phospholipid 3.6% w / v, d,a-tocopherol 1.0018% w / v, NaCl 0.36% w / v, NaEEPCL FEO 0.069% w / v, Na2HPO4-7H2O 0.474% w / v, EDTA 0.02% w / v, with the following characteristics: osmolality 300 - 310 mOsm / kg, pH 7.0 - 7.2, and viscosity approximately 4 ePoise (at shear rate 1 / s). Stability tests confirmed 24-month stability. The efficacy and safety of the nanoemulsion were validated by pharmacokinetic and tissue distribution studies in animals and humans.

[0036] In the field of perfusion fluids used for rinsing, storage, and improving the condition of organs intended for transplantation, as well as in the area of blood substitute preparations based on perfluorocarbon emulsions used as oxygen carriers, various formulations and stabilization methods are known. Patent documents disclose emulsions containing perfluorocarbons combined with suitable surfactants for medical applications. However, these solutions exhibit significant limitations regarding fluid composition, physical stability, manufacturing processes, sterilization parameters, and other technological aspects. The current state of the art does not satisfactorily describe formulations enabling effective reconditioning and regeneration of organs during ex vivo perfusion. This is particularly relevant for organs retrieved from donors after irreversible circulatory arrest (DCD), which represent a real opportunity to increase the number of organs available for transplantation and reduce dialysis duration and mortality among patients awaiting transplants. The prior art also fails to disclose an effective blood substitute that could realistically support resuscitation after haemorrhagic shock or other situations where such a preparation would be useful. Therefore, there is a clear need for the development of new formulations meeting these requirements.

[0037] Technical problem

[0038] With the need to increase the pool of potential organs eligible for transplantation, the use of mechanical kidney perfusion methods under subnormothermic and normothermic conditions could realistically increase the number of transplants performed, reduce the duration of dialysis use and reduce the number of deaths of patients awaiting transplantation.

[0039] The solution to this problem is to carry out the perfusion process using an acellular perfusion fluid capable of transporting respiratory gases, in the form of an oil-in-water nanoemulsion in which the dispersed phase consists of perfluorinated compounds — the subject of the present invention. Unexpectedly, it turned out that careful development of the preparation method and optimisation of the composition lead to a perfluorocarbon emulsion giving the possibility of perfusing organs, i.e. kidney, liver, heart, or its use as a potential blood substitute. The fluid ensures adequate oxygenation and the necessary nutrients for the organ awaiting transplantation. Perfluorocarbon-based perfusion fluid according to the invention also has a significant advantage over products based on blood morphotic elements, as it avoids phenomena such as the release of leukotrienes from them, as well as other inflammatory and vasoconstrictive substances.

[0040] Object of the invention

[0041] The subject matter of the invention is a perfusion fluid in the form of an oil-in-water PFC nanoemulsion including:

[0042] the dispersed phase, which includes:

[0043] - an oxygen carrier, preferably in an amount of 100.00 - 800.00 g / L, more preferably in an amount of 100 - 400 g / L, which is one or more compounds selected from: perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane,

[0044] the water phase, which includes:

[0045] - a surfactant which is the main surfactant or at least one main surfactant and at least one cosurfactant, wherein:

[0046] a main surfactant, preferably in an amount of 23.80 - 40.08 g / L, more preferably in an amount of 23.80 - 34.00 g / L, which is Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F-127, or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof, diblock / triblock / multiblock Pluronics, such as, for example Pluronics (condensation polymers of polypropylene glycols with polyoxyethylene glycols) and derivatives thereof, perfluorinated copolymers of the hydrocarbon-fluorocarbon type, gelatine and derivatives thereof,

[0047] a co-surfactant, preferably in an amount of 0.35 - 0.60 g / L, more preferably in an amount of 0.35 - 0.50 g / L, which is: an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or 1,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)- l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, perfluorinated phospholipids (e.g. phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids;

[0048] - an oncotic agent, preferably in an amount of 20.00 - 40.00 g / L, more preferably in an amount of 40.00 g / L, which is one oncotic agent selected from the group comprising: of bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch or recombinant human serum albumin or derivatives thereof, Dextran 30 kDa, Dextran 50 kDa or derivatives thereof, gelatine derivatives, cyclodextrins, preferably bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch, - a main osmotic agent, preferably in an amount of 10.00 - 100.00 mM, most preferably in an amount of 21.40 - 50.00 mM, which is mannitol or raffinose or sucrose or glucose or citrate chelates or salts of lactobionic acid or gluconic acid, preferably mannitol, most preferably mannitol in an amount of 21.40 - 50.00 mM,

[0049] - the main components of the buffer are one or more compounds selected from the

[0050] - sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O preferably in an amount of 5.00 - 20.00 mM, most preferably in an amount of 10.00 mM; - sodium salt of DL-P-hydroxybutyric acid, preferably in an amount of 0.10 - 0.50 mM, most preferably in an amount of 0.20 mM;

[0051] - antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N-acetyl cysteine or catalase or peroxidase, preferably glutathione, preferably in an amount of 1.50 - 6.00 mM, most preferably in an amount of 3.00 mM, and

[0052] - pharmacologically active substances - allopurinol or adenine, adenosine or phosphates or hormones: insulin, steroids i.e. dexamethasone, preferably allopurinol, preferably in an amount of 0.50 - 3.00 mM, most preferably in an amount of 1.00 mM;

[0053] - buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more of NaCl, preferably in an amount of 20,00 - 100.00 mM, most preferably in an amount of 60.00 mM, KC1, preferably in an amount of 2.00 - 10.00 mM, most preferably in an amount of 4.60 mM, CaCL, preferably in an amount of 0.30 - 2.00 mM, most preferably in an amount of 0.60 mM, MgCh • 6H2O, preferably in an amount of 0.20 - 2.00 mM, most preferably in an amount of 0.40 mM; - buffering components maintaining the pH in the range 7.1 - 7.6, preferably one or more compounds selected from the group Na2HPO4 and NaH2PO4, buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine-HCl and L-histidine, preferably these are Na2HPO4 and / or preferably Na2HPO4, most preferably these are: Na2HPO4 in an amount of 6.00 - 15.00 mM, most preferably in an amount of 8.00 mM and NaH2PO4, preferably in an amount of 0.50 - 3.00 mM, most preferably in an amount of 1.5 mM;

[0054] wherein, preferably, the perfusion fluid exhibits osmotic pressure in the range of: 280 - 400 mOsm / kgJLO, pH in the range of 7.1 - 7.6, oncotic pressure in the range of 15 - 36 mmHg, concentration of the main ions: sodium in the range of 80 - 200 mM, potassium in the range of 1 - 15 mM, calcium in the range of 0.1 - 2.5 mM and chloride in the range of 50 - 170 mM; furthermore, the average diameter of the dispersed phase nanoparticles does not exceed 300 nm, preferably is below 220 nm, most preferably is below 130 nm.

[0055] Preferably, the aqueous phase further comprises amino acids, including one or more amino acids selected from the group: taurine, L-arginine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine.

[0056] Preferably, the aqueous phase further comprises amino acids including preferably one or more amino acids selected from the group of: taurine preferably in an amount of 5.00 - 15.00 mM, most preferably in an amount of 10.00 mM; L-arginine preferably in an amount of 0.05 - 0.20 mM, most preferably in an amount of 0.10 mM; L-tryptophan preferably in an amount of 1.00 -3.00 mM, most preferably 2.00 mM; L-glutamine preferably in an amount of 0.10 - 1.00 mM, most preferably in an amount of 0.50 mM; L-isoleucine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.52 - 4.19 mM; L-leucine preferably in an amount of 1.00 - 10.00 mM, most preferably in an amount of 3.05 - 6.86 mM; L-lysine preferably in an amount of 1.00 - 10.00 mM, most preferably in an amount of 2.05 - 4.79 mM; L-methionine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.34 -3.35 mM; L-threonine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.69 -3.78 mM; L-v aline preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 2.56 - 5.55; L-histidine preferably in an amount of 0.10 - 10.00 mM, most preferably in an amount of 0.64 - 2.26 mM; L-alanine preferably in an amount of 1.00 - 20.00 mM, most preferably in an amount of 5.61 - 12.35 mM; L-glycine preferably in an amount of 1.00 - 20.00 mM, most preferably in an amount of 6.66 - 17.32 mM; L-aspartic acid preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.88 -4.51 mM; L-proline preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 2.17 - 5.21 mM; L-serine preferably in an amount of 0.10 - 10.00 mM, most preferably in an amount of 0.95 - 2.38 mM; L-tyrosine preferably in an amount of 0.01 - 1.00 mM, most preferably in an amount of 0.06 -0.28 mM.

[0057] Preferably, the aqueous phase comprises taurine in an amount of 5.00 - 15.00 mM, most preferably 10.00 mM; L-arginine in an amount of 0.05 - 0.20 mM, most preferably 0.10 mM; L-tryptophan in an amount of 1.00 - 3.00 mM, most preferably 2.00 mM; and L-glutamine in an amount of 0.10 - 1.00 mM, most preferably 0.50 mM.

[0058] Preferably, the oxygen carrier is one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin.

[0059] Preferably, the main surfactant is Pluronic F-68 (Kolliphor P188), or Pluronic F-108, or Pluronic F-127, or bovine serum albumin (BSA), or human serum albumin (HSA), or recombinant human serum albumin, or derivatives thereof.

[0060] Preferably, the co-surfactant is an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or 1,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)- l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt.

[0061] Preferably, the oncotic agent is bovine serum albumin (BSA), or human serum albumin (HSA), or recombinant human serum albumin, or succinylated gelatine, and more preferably bovine serum albumin (BSA), human serum albumin (HSA), or recombinant human serum albumin, in an amount of: 20 - 40 g / L, most preferably 40 g / L.

[0062] Preferably, the main surfactant is Pluronic F-68 or homologs thereof or derivatives thereof, or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin and derivatives thereof, and the co-surfactant is (1H,1H,2H,2H-perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains.

[0063] Preferably, the main buffer component is one or more compounds selected from

[0064] - pharmacologically active substances, preferably allopurinol; and / or - antioxidants, preferably glutathione, N-acetylcysteine, vitamin C and vitamin E; whereas the buffer components constituting salts are salts selected from Na+, K+, Ca2+, Mg2+. Preferably, the buffer is mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine, and Na2HPO4, NaEbPC , NaCl, KC1, CaCh, MgCh • 6H2O and more preferably mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L- tryptophan, L-glutamine, and Na2HPO4, NaH2PO4, NaCl, KC1, CaCl2, MgCh • 6H2O.

[0065] Preferably, the perfusion fluid contains perfluorooctyl bromide (PFOB) in an amount of 187.21 g / L; perfluorodecyl bromide (PFDB) in an amount of 5.79 g / L; Pluronic F-68 (Kolliphor P188) in an amount of 27.20 g / L; 1H, 2H, 2H-Perfluorooctyl)phosphocholine in an amount of 4.00 g / L; bovine albumin in an amount of 40.00 g / L; L-arginine in an amount of 0.10 mM; allopurinol in an amount of 1.00 mM; mannitol in an amount of 50.00 mM; taurine in an amount of 10.00 mM; L-tryptophan in an amount of 2.00 mM; D-glucose • H2O in an amount of 10.00 mM; NaCl in an amount of 60.00 mM; KC1 in an amount of 4.60 mM; Na2HPO4in an amount of 8.00 mM; NaFLPCU in an amount of 1.50 mM; CaCh in an amount of 0.60 mM; MgCh • 6H2O in an amount of 0.40 mM; L-glutamine in an amount of 0.50 mM; glutathione in an amount of 3.00 mM; DL-P -hydroxybutyric acid sodium salt in an amount of 0.20 mM; NaOH to adjust a pH in the range of 6.8 - 7.4.

[0066] Preferably, the perfusion fluid contains perfluorooctyl bromide (PFOB) in an amount of 374.42 g / L; perfluorodecyl bromide (PFDB) in an amount of 11.58 g / L; Pluronic F-68 (Kolliphor P188) in an amount of 34.00 g / L; 1H, 2H, 2H-Perfluorooctyl)phosphocholine in an amount of 5.00 g / L; bovine albumin in an amount of 40.00 g / L; L-arginine in an amount of 0.10 mM; allopurinol in an amount of 1.00 mM; mannitol in an amount of 21.40 mM; taurine in an amount of 10.00 mM; L-tryptophan in an amount of 2.00 mM; D-glucose • H2O in an amount of 10.00 mM; NaCl in an amount of 60.00 mM; KC1 in an amount of 4.60 mM; Na2HPO4in an amount of 8.00 mM; NaH2PO4in an amount of 1.50 mM; CaCh in an amount of 0.60 mM; MgCh • 6H2O in an amount of 0.40 mM; L-glutamine in an amount of 0.50 mM; glutathione in an amount of 3.00 mM; DL-P -hydroxybutyric acid sodium salt in an amount of 0.20 mM; NaOH to adjust a pH in the range of 6.8 - 7.4.

[0067] The subject matter of the invention is a method for producing a perfusion fluid in the form of a PFC oil-in-water nanoemulsion, comprising the steps of

[0068] 1. preparation of the components of the primary perfusion fluid including

[0069] la. preparation of the solution of surfactant or surfactants,

[0070] lb. preparation of perfluorinated phase,

[0071] lc. preparation of the buffer

[0072] 2. mechanical homogenization of the components prepared in step 1. to obtain a primary perfusion fluid;

[0073] 3. microfluidization of the primary perfusion fluid obtained in step 2. to obtain the perfusion fluid,

[0074] wherein:

[0075] in step la. the solution of surfactant or surfactants is: ultrapure water and

[0076] the main surfactant or at least one main surfactant and at least one co-surfactant wherein: the main surfactant is Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F-127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof, diblock / triblock / multiblock pluronics, such as e.g. pluronics (condensation polymers of polypropylene glycols with polyoxyethylene glycols) and derivatives thereof, perfluorinated copolymers of the hydrocarb on-fluorocarb on type, gelatine and derivatives thereof, preferably Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F-127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof;

[0077] co-surfactant is: an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or 1,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)- l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, perfluorinated phospholipids (e.g. phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids, preferably (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt or 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt

[0078] in step lb. the perfluorinated phase is one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane, preferably perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin

[0079] in step 1c. the solid component of the buffer is:

[0080] a. an oncotic agent which is one oncotic agent selected from the group comprising of bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch or recombinant human serum albumin or derivatives thereof, Dextran 30 kDa, Dextran 50 kDa, or derivatives thereof, gelatine derivatives, cyclodextrins, preferably bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch;

[0081] b. the main osmotic agent, which is mannitol or raffinose or sucrose or glucose or citrate chelates or salts of lactobionic acid or gluconic acid, preferably mannitol;

[0082] c. the main component of a buffer which is one or more compounds such as

[0083] - sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O, - DL-P -hydroxybutyric acid sodium salt, - antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N-acetyl cysteine or catalase or peroxidase, preferably glutathione, and

[0084] - pharmacologically active substances - allopurinol or adenine or adenosine or phosphates or hormones, preferably insulin or steroids preferably dexamethasone, most preferably allopurinol;

[0085] d. buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more, most preferably all, of: NaCl, KC1, CaCh, MgCh • 6H2O;

[0086] e. buffering components maintaining the pH in the range 7.1 - 7.6, preferably one or more compounds selected from the group: Na2HPO4 and NaH2PC>4, buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine HCl and L-histidine, preferably Na2HPO4 and / or Na2HPO4; f. possibly other components, that are amino acids including preferably one or more amino acids selected from the group: taurine, L-arginine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine.

[0087] Preferably, in step 3. the primary perfusion fluid obtained in step 2. is pumped at a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled using a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C;

[0088] and / or

[0089] in step 3. the preliminary perfusion fluid obtained in step 2. is passed 1 - 15 times through the microfluidizer chamber, most preferably 3 - 9 passes through the microfluidizer chamber. The subject matter of the invention is a method for producing a perfusion fluid in the form of a PFC oil-in-water nanoemulsion, comprising the steps of

[0090] 1. preparation of the primary perfusion fluid components comprising

[0091] I. preparation of the pre-emulsion components comprising

[0092] la. preparation of the solution of surfactant or surfactants

[0093] lb. preparation of the perfluorinated phase

[0094] II. mechanical homogenization of the components prepared in step I to obtain a primary preemulsion,

[0095] III. microfluidization of the primary pre-emulsion obtained in step II to obtain the preemulsion,

[0096] IV. the preparation of the buffer comprising

[0097] IVa. preparation of buffer components

[0098] IVR. pH adjustment

[0099] IVQ. quality control of the buffer obtained 2. mixing the pre-emulsion obtained in step III with the buffer obtained in step IV to obtain the primary perfusion fluid, wherein the components being preferably mixed in a ratio of preemulsion to buffer from 1:1 to 1:2;

[0100] 3. microfluidization of the primary perfusion fluid obtained in step 2. to obtain the perfusion fluid;

[0101] wherein:

[0102] in step la. the solution of surfactant or surfactants is:

[0103] ultrapure water and

[0104] the main surfactant or at least one main surfactant and at least one co-surfactant wherein: the main surfactant is Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F- 127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof, diblock / triblock / multiblock Pluronics, such as e.g. Pluronics (condensation polymers of polypropylene glycols with polyoxyethylene glycols) and derivatives thereof, perfluorinated copolymers of the hydrocarb on-fluorocarb on type, gelatine and derivatives thereof, preferably Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F- 127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof

[0105] co-surfactant is: an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or 1,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)- l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, perfluorinated phospholipids (e.g. phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids, preferably (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt or 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt

[0106] in step lb. the perfluorinated phase is of one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane, preferably perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin

[0107] in step 1c. the solid component of the buffer is:

[0108] a. an oncotic agent, which is one oncotic agent selected from the group comprising of bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch or recombinant human serum albumin or derivatives thereof, Dextran 30 kDa, Dextran 50 kDa or derivatives thereof, gelatine derivatives, cyclodextrins, preferably bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch;

[0109] b. the main osmotic agent, which is mannitol or raffinose or sucrose or glucose or citrate chelates or salts of lactobionic acid or gluconic acid, preferably mannitol;

[0110] c. the main component of the buffer, which is one or more compounds selected from:

[0111] - sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O, - DL-P -hydroxybutyric acid sodium salt,

[0112] - antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N-acetyl cysteine or catalase or peroxidase, preferably glutathione, and

[0113] - pharmacologically active substances - allopurinol or adenine or adenosine or phosphates or hormones, preferably insulin or steroids preferably dexamethasone, most preferably allopurinol;

[0114] d. buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more, most preferably all, of: NaCl, KC1, CaCl2, MgCh • 6H2O;

[0115] e. buffering components maintaining the pH in the range 7.1 - 7.6, preferably one or more compounds selected from the group: ISfeHPCL and NaH2PO4, buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine HCl and L-histidine, preferably ISfeHPCU and / or ISfeHPCU; f. possibly other components, that are amino acids including preferably one or more amino acids selected from the group: taurine, L-arginine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine.

[0116] Preferably, in step III. preferably the primary pre-emulsion obtained in step (II) is pumped at a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled by means of a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C;

[0117] and in step 3. preferably the primary perfusion fluid obtained in step 2. is pumped at a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled by means of a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C;

[0118] and / or

[0119] in step III. microfluidization of the primary pre-emulsion obtained in step II to obtain the preemulsion is carried out in 1 - 10 passes of the pre-emulsion PFC through the microfluidizer chamber, most preferably 8 passes

[0120] whereby

[0121] in step 3. microfluidization of the primary perfusion fluid obtained in step 2. is carried out in 1-10 passes of the pre-emulsion obtained in step 2. through the microfluidizer chamber, most preferably 1-5 passes.

[0122] Preferably, the oncotic agent is bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin, or succinylated gelatine, and more preferably bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin, in an amount of 20 - 40 g / L, most preferably 40 g / L.

[0123] Preferably, other buffer components which are amino acids include taurine, L-arginine, L-tryptophan, and L-glutamine.

[0124] Preferably, the perfluorinated phase is one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin.

[0125] Preferably, the main surfactant is Pluronic F-68 (Kolliphor P188), or Pluronic F-108, or Pluronic F-127, or bovine serum albumin (BSA), or human serum albumin (HSA), or recombinant human serum albumin, or derivatives thereof.

[0126] Preferably, the co-surfactant is an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol-2000)-l,2-distearoyl-sn-glycero-3 -phosphoethanolamine sodium salt.

[0127] Preferably, the main surfactant is Pluronic F-68 or homologs thereof or derivatives thereof, or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin and derivatives thereof, and the co-surfactant is (1H,1H,2H,2H-perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains.

[0128] Preferably, the main buffer component is one or more compounds selected from

[0129] - pharmacologically active substances, preferably allopurinol; and / or

[0130] - antioxidants, preferably glutathione, N-acetylcysteine, vitamin C and vitamin E; whereas the buffer components constituting salts are salts selected from Na+, K+, Ca2+, Mg2+. Preferably, the buffer is mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine, and Na2HPO4, NaftPCU, NaCl, KC1, CaCh, MgCL • 6H2O, more preferably mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L-tryptophan, L-glutamine, and Na2HPO4, NaH2PO4, NaCl, KC1, CaCl2, MgCL • 6H2O.

[0131] Preferably, the primary surfactant is: Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic Fl 27 or bovine serum albumin or human serum albumin or recombinant human serum albumin, in an amount of: 23.80 - 40.08 g / L, most preferably 23.80 - 34.00 g / L, and the cosurfactant is: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-Maltopyranoside or Sphingomyelin or modified lipid: 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt orN-(carbonyl-methoxy polyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, in amounts: 0.35 - 0.60 g / L, preferably 0.35 - 0.50 g / L.

[0132] Preferably, steps 1 to 3 of the method are carried out under sterile / sterile conditions and / or step 3 shall be followed by step 4 involving sterilization of the perfusion fluid obtained in step 3

[0133] to obtain sterile perfusion fluid,

[0134] wherein sterilization of the perfusion fluid obtained in step 3 is carried out by sterilization filtration, gamma radiation, sterilization by ethylene oxide, most preferably by sterilizing filtration,

[0135] alternatively, after steps 1 - 3 or after step 4, packaging of the resulting sterile perfusion fluid is carried out.

[0136] The subject matter of the invention is a perfusion fluid obtained by the method as disclosed above, preferably having a qualitative and quantitative composition and physicochemical properties as disclosed in the present application.

[0137] The subject matter of the invention is the perfusion fluid for use as a medicament.

[0138] The subject matter of the invention is also the perfusion fluid for use a blood substitute preparation.

[0139] The subject matter of the invention is as well the perfusion fluid for use in the storage of organs intended for transplantation.

[0140] The subject matter of the invention is a perfusion fluid for use in the treatment of acute renal failure classified under ICD-10 as N17, and chronic renal failure classified under ICD-10 as N18, in particular end-stage renal disease classified under ICD-10 as N18.0.

[0141] The subject matter of the invention is a perfusion fluid for use in the treatment of acute and subacute liver failure classified under ICD-10 as K72.0, liver fibrosis and cirrhosis classified under ICD-10 as K74, alcoholic liver disease classified under ICD-10 as K70, toxic liver injury classified under ICD-10 as K71, and liver failure associated with liver cell cancer (hepatocellular carcinoma) classified under ICD-10 as C22.0.

[0142] The subject matter of the invention is a perfusion fluid for use in the treatment of heart failure classified under ICD-10 as 150, including acute systolic (congestive) heart failure classified under ICD-10 as 150.21 and unspecified heart failure classified under ICD-10 as 150.9.

[0143] The subject matter of the invention is a perfusion fluid for use in the treatment or prevention of a pathological condition that can be prevented, alleviated, or eliminated by administration of blood substitute preparations.

[0144] Preferably, the pathological condition includes shock classified under ICD-10 as R57, including hypovolemic shock classified under ICD-10 as R57.1, traumatic shock classified under ICD-10 as T79.4, other shock classified under ICD-10 as R57.8, including haemorrhagic shock, and unspecified shock classified under ICD-10 as R57.9.

[0145] The subject matter of the invention is a perfusion fluid for use in ex vivo machine perfusion of organs intended for transplantation.

[0146] Preferably, the organs intended for transplantation include organs retrieved after brain death -DBD, organs retrieved from donors after irreversible circulatory arrest - DCD, organs retrieved from donors aged 60 years or older, and donors in the age range 50 - 59 years with at least two of the following risk factors: arterial hypertension, death caused by stroke, blood creatinine level at time of death above 1.5 mg / dL -ECD.

[0147] The subject matter of the invention is a perfusion fluid for use in a method of treatment by transplantation. The subject matter of the invention is a perfusion fluid for use in organ storage before and / or during transplantation.

[0148] The subject matter of the invention is a perfusion fluid for use in organ reconditioning before and / or during transplantation.

[0149] The subject matter of the invention is a perfusion fluid for use in ex vivo machine perfusion of organs, preferably kidney, liver, and heart.

[0150] The subject matter of the invention is a perfusion fluid for use in ex vivo machine perfusion of organs under subnormothermic and normothermic conditions, preferably under hybrid conditions comprising periods of hypothermic perfusion, periods of controlled linear temperature increase or decrease, and periods of subnormothermic or normothermic perfusion, within the entire application range of 4 - 37°C and mean arterial pressure - MAP of 30 - 120 mmHg.

[0151] The subject matter of the invention is a method of treatment of acute renal failure and chronic renal failure, in particular end-stage renal disease, using the perfusion fluid according to the invention.

[0152] The subject matter of the invention is also a method of treatment of acute and subacute liver failure, liver fibrosis and cirrhosis, alcoholic liver disease, toxic liver injury, and liver failure associated with liver cell cancer (hepatocellular carcinoma) using the perfusion fluid according to the invention.

[0153] The subject matter of the invention further relates to a method of treatment of heart failure, including acute systolic (congestive) heart failure and unspecified heart failure, using the perfusion fluid according to the invention.

[0154] The subject matter of the invention is a method of treatment, or prevention of a pathological condition that can be prevented, alleviated, or eliminated by administration of blood substitute preparations, using the perfusion fluid according to the invention.

[0155] Preferably, the pathological condition includes shock, including hypovolemic shock, traumatic shock, other shock including haemorrhagic shock, and unspecified shock.

[0156] Therefore, in response to the growing demand for transplantable organs and the limited pool of organs suitable for transplantation, a perfusion fluid has been developed in the form of a perfluorocarbon nanoemulsion intended for ex vivo perfusion under hypothermic, subnormothermic, normothermic, and mixed conditions, as well as for use as a blood substitute preparation.

[0157] The ex vivo perfusion process, enabling effective reconditioning and regeneration of organs, particularly those obtained from DCD donors (after circulatory arrest) and ECD donors (extended criteria donors), can significantly increase the number of organs eligible for transplantation. The realization of these assumptions is possible due to performing ex vivo perfusion under subnormothermic or normothermic conditions using an acellular perfusion fluid in the form of an oil-in-water nanoemulsion, in which the dispersed phase consists of perfluorinated compounds, which is the subject of the present invention.

[0158] Unexpectedly, it was found that careful development of the preparation method and optimization of the composition results in a perfluorocarbon emulsion that enables perfusion of organs such as kidney, liver, and heart, or its use as a potential blood substitute.

[0159] The fluid ensures adequate oxygenation and essential nutrients for the organ awaiting transplantation. The perfluorocarbon-based perfusion fluid according to the invention also offers a significant advantage over methods based on blood morphotic elements, as it avoids phenomena such as the release of leukotrienes and other inflammatory and vasoconstrictive substances. Due to small particle, the oxygen carrier freely penetrates damaged microcirculation, often compromised by ischemia, where erythrocytes may have difficulty passing.

[0160] According to the invention, perfusion fluids in the form of perfluorocarbon nanoemulsions (hereinafter also referred to as PFC nanoemulsions) of the oil-in-water type contain a dispersed phase in an amount of 100.00 - 800.00 g / L, preferably 100 - 400 g / L, comprising single compounds or mixtures of perfluorinated compounds such as perfluorooctyl bromide, perfluorodecyl bromide, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), and perfluorocyclohexane, exhibiting diverse physicochemical properties. The use of a mixture of perfluorinated compounds with varying chain lengths and branching enables both rapid oxygen saturation of the fluid by short-chain compounds (e.g., PFOB, PFD) and maintenance of a long-term oxygen partial pressure gradient through the use of longer-chain compounds (e.g., PFDB) or compounds with higher molecular weight and more complex structure (e.g., PFTBA). Such a combination ensures optimal oxygen and carbon dioxide transport, appropriate viscosity, stability, and uniform oxygenation of the entire organ, including areas with restricted flow. The most preferable combinations include perfluorooctyl bromide, perfluorodecyl bromide, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, and perfluorodecalin.

[0161] According to the invention, perfusion fluids in the form of perfluorocarbon nanoemulsions of the oil-in-water type contain an aqueous phase comprising surface active agents, an oncotic agent, an osmotic agent, and other auxiliary substances forming a buffer with pharmacological activity or bioactive components supporting organ reconditioning during perfusion.

[0162] According to the invention, surfactants are used in an amount of 23.80 - 40.08 g / L, including surface-active compounds such as Pluronic F-68 (also known as Kolliphor Pl 88, INCI name: Poloxamer 188; CAS 9003-11-6; structure: PEO75 - PPO30 - PEO75), Pluronic F-108 (Poloxamer 338; CAS 9003-11-6; structure: PEOui - PPO44 - PEO141), or Pluronic F-127 (Poloxamer 407; CAS 9003-11-6; structure: PEO100 - PPOes - PEO100), their derivatives, diblock / triblock / multiblockPluronics, such as condensation polymers of polypropylene glycols with polyethylene glycols. In some cases, bovine serum albumin (BSA), human serum albumin (HSA), recombinant human serum albumin, their derivatives, perfluorinated hydrocarbonfluorocarbon copolymers, or gelatine and derivatives thereof were used as surfactants at appropriate concentrations. The most preferable effects were observed when using 23.80 -34.00 g / L of surfactants such as Pluronic F-68 (Kolliphor P188), Pluronic F-108, Pluronic F-127, bovine serum albumin (BSA), human serum albumin (HSA), or recombinant human serum albumin and their derivatives.

[0163] According to the invention, as the co-surfactant in the nanoemulsion were used in an amount of 0.35 - 0.60 g / L perfluorinated compounds (i.e., organic compounds containing perfluorinated chains): (lH,lH,2H,2H-perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or perfluorinated phospholipids (e.g., phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine); perfluorinated glycolipids; or modified lipids such l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt, 1,2-dipalmitoyl- sn-glycero-3 -phosphate sodium salt, l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt, or N-(carbonyl-methoxypolyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt. The most preferable effects were observed when using 0.35 - 0.50 g / L.

[0164] The surfactant system employed in the invention ensures high emulsion stability, resulting from the synergy of hydrophilic-fluorophilic interact! ons / strong affinity to the perfluorocarbon phase (perfluorinated co-surfactants), steric stabilization (Pluronics, PEGylated lipids), and electrostatic stabilization - surface charge imparted by modified lipids and albumin. This system also enables reduction of the average perfluorocarbon nanoparticle size to below 200 nm, which promotes improved penetration of the microcirculation during organ perfusion. The nanoemulsion further exhibits enhanced resistance to coalescence and long-term maintenance of a homogeneous structure. Properly selected surfactants and co-surfactants improve biocompatibility and reduce cytotoxicity.

[0165] The most preferable effects were achieved using (1H,1H,2H,2H-perfluorooctyl)phosphocholine and homologs thereof and derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, as co-surfactants, and Pluronic F-68 and derivatives thereof as the primary surfactant, or alternatively bovine serum albumin (BSA), human serum albumin (HSA), or recombinant human serum albumin, as well as derivatives thereof.

[0166] According to the invention, the oncotic agent in the nanoemulsion is used in an amount of 20.00 - 40.00 g / L, selected from the group comprising bovine serum albumin (BSA), human serum albumin (HSA), PEG 35 kDa, PEG 20 kDa, Dextran 40 kDa, succinylated gelatine, hydroxy ethyl starch, recombinant human serum albumin and derivatives thereof, Dextran 30 kDa, Dextran 50 kDa, gelatine derivatives, or cyclodextrins. The use of oncotic substances maintains proper colloid-osmotic pressure, preventing tissue edema and supporting microcirculatory stability during perfusion. The most preferable effect was observed with 40.00 g / L of bovine serum albumin (BSA), human serum albumin (HSA) or succinylated gelatine. According to the invention, the osmotic agent in the nanoemulsion is used in an amount of 10.00 - 100.00 mM, selected from mannitol, raffinose, sucrose, glucose, citrate chelates, salts of lactobionic acid or gluconic acid. The most preferable effect was observed with 21.40 - 50.00 mM mannitol.

[0167] According to the invention, as the main component of the buffer a single substance or a mixture was used:

[0168] - sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O preferably in an amount of 5.00 - 20.00 mM, most preferably in an amount of 10.00 mM; - sodium salt of DL-P-hydroxybutyric acid, preferably in an amount of 0.10 - 0.50 mM, most preferably in an amount of 0.20 mM;

[0169] - antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N-acetyl cysteine or catalase or peroxidase, preferably glutathione, preferably in an amount of 1.50 - 6.00 mM, most preferably in an amount of 3.00 mM,

[0170] - pharmacologically active substances - allopurinol or adenine, adenosine or phosphates or hormones: insulin, steroids such as dexamethasone, preferably allopurinol, preferably in an amount of 0.50 - 3.00 mM, most preferably in an amount of 1.00 mM;

[0171] - buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more of NaCl, preferably in an amount of 20,00 - 100.00 mM, most preferably in an amount of 60.00 mM, KC1, preferably in an amount of 2.00 - 10.00 mM, most preferably in an amount of 4.60 mM, CaCL, preferably in an amount of 0.30 - 2.00 mM, most preferably in an amount of 0.60 mM, MgCh • 6H2O, preferably in an amount of 0.20 - 2.00 mM, most preferably in an amount of 0.40 mM; - buffering components maintaining the proper pH range, preferably one or more compounds selected from the group ISfeHPC and Na^PC , buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine HC1 and L-histidine, preferably these are ISfeHPC and / or preferably Na2HPO4, most preferably these are: ISfeHPC in an amount of 6.00 - 15.00 mM, most preferably in an amount of 8.00 mM and Na^PC , preferably in an amount of 0.50 - 3.00 mM, most preferably in an amount of 1.5 mM;

[0172] The main buffer components used perform essential functions in organ reconditioning processes: pharmacologically active substances (e.g., allopurinol) act as xanthine oxidase inhibitors, reducing uric acid concentration in plasma and urine and inhibiting the conversion of hypoxanthine, which generates free radicals;

[0173] antioxidants (e.g., glutathione, N-acetylcysteine, vitamins C and E) reduce oxidative stress; buffer components constituting salts (Na+, K+, Ca2+, Mg2+) - are indispensable for mitochondrial enzyme activity, thereby supporting metabolism and cellular protection during perfusion. Additionally, a beneficial effect was obtained when the perfusion fluid exhibits osmotic pressure in the range of: 280 - 400 mOsm / kgH2O; pH in the range of 7.1 - 7.6; oncotic pressure in the range of 15 - 36 mmHg; concentrations of the main ions: sodium in the range of 80 -200 mM, potassium in the range of 1 - 15 mM, calcium in the range of 0.1 - 2.5 mM, and chloride in the range of 50 - 170 mM; and the average diameter of the dispersed phase nanoparticles does not exceed 300 nm, preferably is below 220 nm, most preferably below 130 nm.

[0174] According to the invention, auxiliary substances forming part of the buffer and acting as bioactive components supporting organ reconditioning during perfusion comprise amino acids, individually or as a mixture, selected from the group of taurine preferably in an amount of 5.00 - 15.00 mM, most preferably in an amount of 10.00 mM, L-arginine preferably in an amount of 0.05 - 0.20 mM, most preferably in an amount of 0.10 mM, L-tryptophan preferably in an amount of 1.00 - 3.00 mM, most preferably in an amount of 2.00 mM, L-glutamine preferably in an amount of 0.10 - 1.00 mM, most preferably in an amount of 0.50 mM, L-isoleucine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.52 - 4.19 mM, L-leucine preferably in an amount of 1.00 - 10.00 mM, most preferably in an amount of 3.05 - 6.86 mM, L-lysine preferably in an amount of 1.00 - 10.00 mM, most preferably in an amount of 2.05 - 4.79 mM, L-methionine preferably in an amount of 0.50 -10.00 mM, most preferably in an amount of 1.34 - 3.35 mM, L-threonine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.69 - 3.78 mM, L-valine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 2.56 - 5.55 mM, L-histidine preferably in an amount of 0.10 - 10.00 mM, most preferably in an amount of 0.64 - 2.26 mM, L-alanine preferably in an amount of 1.00 - 20.00 mM, most preferably in an amount of 5.61 - 12.35 mM, L-glycine preferably in an amount of 1.00 - 20.00 mM, most preferably in an amount of 6.66 - 17.32 mM, L-aspartic acid preferably in an amount of 0.50 -10.00 mM, most preferably in an amount of 1.88 -4.51 mM, L-proline preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 2.17 - 5.21 mM, L-serine preferably in an amount of 0.10 - 10.00 mM, most preferably in an amount of 0.95 - 2.38 mM, L-tyrosine preferably in an amount of 0.01 - 1.00 mM, most preferably in an amount of 0.06 - 0.28 mM. The comprehensive content of bioactive components (amino acids) in the perfusion fluid supports organ reconditioning during perfusion by enhancing its ability to perform key metabolic processes such as cellular respiration, oxidative phosphorylation, and energy substrate management. These components also assist detoxification systems responsible for neutralizing toxic metabolites generated during ischemia or cellular stress. Furthermore, they help counteract damage caused by reactive oxygen species by activating antioxidant enzymes, redox systems, and cellular repair mechanisms, as confirmed by proteomic and metabolomic studies described in the examples.

[0175] According to the invention, the perfusion fluid in the form of a perfluorinated nanoemulsion is preferably produced by a method comprising: preparation of a solution of the surfactant or surfactants, preparation of the perfluorinated phase and preparation of the buffer, mechanical homogenization of the components to obtain a preliminary perfusion fluid; microfluidization of the preliminary perfusion fluid and sterilizing filtration.

[0176] According to the invention, the perfusion fluid in the form of a perfluorinated nanoemulsion is also preferably produced by a method comprising: preparation of a pre-emulsion and its preliminary microfluidization and preparation of the buffer, mechanical homogenization of the components to obtain a preliminary perfusion fluid; microfluidization of the preliminary perfusion fluid and sterilizing filtration.

[0177] The most preferable effect of the perfusion fluid was obtained when microfluidization of the preliminary perfusion fluid was carried out by pumping (preferably the number of pumping cycles is 1-15, most preferably 3-9) under a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an internal microchannel diameter of 75 pm and cooled by a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30°C.

[0178] Preferably, sterilization of the perfusion fluid obtained in the microfluidization step is carried out by sterilizing filtration, gamma irradiation, ethylene oxide sterilization, most preferably by sterilizing filtration.

[0179] Conducting the process of producing the perfusion fluid according to the invention enables obtaining a stable perfluorinated nanoemulsion with an average diameter of dispersed phase nanoparticles not exceeding 300 nm, preferably below 220 nm, most preferably below 130 nm. The carefully developed composition of the perfusion fluid and the controlled manufacturing process allow reduction of PFC particle size to below 200 nm, which improves penetration of the microcirculation during organ perfusion, as well as increases the resistance of the nanoemulsion to aging processes and ensures long-term maintenance of its homogeneity. According to the invention, the perfusion fluid in the form of a perfluorinated nanoemulsion is characterized by preferable long-term stability, confirmed by storage results for 6 months and extended periods up to 12 months, during which the average diameter of nanoparticles remains below 350-400 nm.

[0180] According to the invention, the perfusion fluid has been used for ex vivo machine perfusion of organs intended for transplantation, preferably organs donated after brain death - DBD and organs retrieved from donors after irreversible circulatory arrest -DCD, or donors aged 60 years and older, as well as donors aged 50-59 years with at least two of the following risk factors: arterial hypertension, death caused by stroke, blood creatinine level at the time of death above 1.5 mg / dL - ECD, with the final effect of the improved preservation and reconditioning of the organ prior to transplantation.

[0181] The perfusion fluid has been used for ex vivo machine perfusion of organs, most preferably under subnormothermic and normothermic conditions, and in particular under hybrid conditions comprising periods of hypothermic perfusion, periods of controlled linear temperature increase or decrease, and periods of subnormothermic or normothermic perfusion, within the entire application range of 4-37°C and mean arterial pressure - MAP of 30-120 mmHg. As a result, during perfusion, the components of the fluid and its physicochemical parameters exert a preferable effect on key metabolic parameters of the organ. Studies conducted on a porcine kidney model demonstrated that the fluid according to the invention improves biochemical indicators (ALT, AST, LDH, perfusion flow) and enables successful autotransplantation while maintaining organ function. The perfusion fluid has been used for ex vivo machine perfusion of organs, most preferably kidneys, liver, and heart.

[0182] The perfusion fluid may be used in the treatment of, among others, acute and subacute liver failure classified in ICD-10 as K72.0, liver fibrosis and cirrhosis classified in ICD-10 as K74, alcoholic liver disease classified in ICD-10 as K70, toxic liver injury classified in ICD-10 as K71, and liver failure associated with liver cell cancer (hepatocellular carcinoma) classified in ICD-10 as C22.0; as well as heart failure classified in ICD-10 as 150, including acute systolic (congestive) heart failure classified in ICD-10 as 150.21 and unspecified heart failure classified in ICD-10 as 150.9.

[0183] The perfusion fluid may be used in the treatment or prevention of a pathological condition that is prevented, alleviated, or eliminated by administration of blood substitute preparations, the pathological condition including, among others, shock classified in ICD-10 as R57, including hypovolemic shock classified in ICD-10 as R57.1, traumatic shock classified in ICD-10 as T79.4, and other shock classified in ICD-10 as R57.8, including haemorrhagic shock, as well as unspecified shock classified in ICD-10 as R57.9.

[0184] According to the invention, the perfusion fluid in the form of a perfluorinated nanoemulsion exhibits the ability to bind and transport oxygen, maintains oncotic stability, and effectively supports microcirculation. Due to these properties, it can perform functions similar to physiological oxygen transport by blood, enabling its use in situations requiring supplementation or replacement of natural blood functions. The fluid may preferably be used in resuscitation following haemorrhagic shock as a blood substitute and as a supplement to blood bank resources, in military, hospital, and other emergency applications.

[0185] Unless otherwise indicated, all disclosed features, variants, parameters, components, as well as preferable elements and solutions presented in this description may be used both in combination and individually. The disclosure also encompasses all combinations of these features, provided they are consistent with the essence of the invention.

[0186] Detailed description of the solution

[0187] Method

[0188] Perfusion fluid is prepared according to the methods shown in Fig. 1 and Fig. 2.

[0189] Method I for producing of a perfusion fluid in the form of an oil-in-water PFC nanoemulsion as is shown in Fig. 1 includes the steps of

[0190] 1. preparation of the primary perfusion fluid components including: la. preparation of the solution of surfactant or surfactants.

[0191] Suitable surface active agents are weighed on an analytical balance, and are dissolved in ultrapure water.

[0192] The appropriate compounds and their quantities are indicated in Table 1, with the compounds used being the main surfactant or at least one main surfactant and at least one co-surfactant. The solution is placed on a magnetic stirrer at room temperature for about 24 h. The clear solution of the surfactant mixture is then transferred to a volumetric flask and made up to volume with ultrapure water.

[0193] lb. preparation of perfluorinated phase,

[0194] The perfluorinated phase is a mixture or a single compound. The mixture is prepared by dissolving the appropriate compound(s) and stirring mechanically, at room temperature for approximately 0.25 h.

[0195] The appropriate compounds and their quantities are indicated in Table 1.

[0196] lc. preparation of the buffer.

[0197] The buffer components are weighed on an analytical balance and dissolved in ultrapure water. The appropriate compounds and their amounts are indicated in Table 1.

[0198] The buffer is mixed on a magnetic stirrer using 350 rpm at 37 °C for about 2 h. Once all components are dissolved, pH adjustment is performed (correction / elevation with 5 M NaOH to a value of 7.4). The solution is then transferred to a 2 L volumetric flask and topped up with an appropriate amount of ultrapure water. A preliminary quality check of the resulting buffer is also performed, including: measurement of pH, osmotic pressure and ion concentration -sodium, potassium, chloride and calcium.

[0199] 2. mechanical homogenization of the components prepared in step 1. to obtain the primary perfusion fluid;

[0200] The primary perfusion fluid is obtained by measuring into a 250 mL beaker using graduated cylinders and automatic pipettes the respective volumes of the surfactant solution (la), perfluorinated phase (lb) and buffer (1c) prepared in step 1.

[0201] The mixture is emulsified by mechanical homogenization to obtain the primary perfusion fluid (2).

[0202] In the examples below, this step uses:

[0203] - PRO25D mechanical homogenizer with 20 x 115 mm homogenizing tip,

[0204] - Process parameters: the speed of the homogenizer motor was approximately 12000 rpm, the duration of the process - 1 min homogenization process: 1 minute interval / 100 mL preemulsion.

[0205] - The homogenization process was carried out for about 4 minutes.

[0206] However, this step is not limited to this device and the above parameters.

[0207] 3. microfluidization of the primary perfusion fluid obtained in step 2. to obtain the perfusion fluid;

[0208] Essentially, the microfluidic technique used in the present invention makes it possible to obtain more stable nanoparticle systems. The technique involves forcing particles of a given mixture / solution to collide under high pressure of about 2000 bar in a diamond interaction chamber of specific design, resulting in an effective reduction in the size of the nanoparticles obtained and a reduction in their polydispersity index. The technique is typical for a variety of nanoparticle solutions, including PFCs, but is not typical for other perfusion fluids.

[0209] In this step, the primary perfusion fluid obtained in step (2) is microfluidized (3) using a high-pressure homogenizer to obtain the perfusion fluid:

[0210] In the examples below, this step uses

[0211] - a high-pressure homogenizer (microfluidizer),

[0212] - the primary perfusion fluid obtained in step (2) is pumped, preferably at a pressure of 2065 bar, through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and it is cooled by means of a water bath (so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C).

[0213] - the number of repetitions of pumping the total volume of the primary perfusion fluid through the microfluidizer chamber is 1-15 passes / cycles, preferably 9 passes / cycles,

[0214] However, this step is not limited to this device and the above parameters.

[0215] In this step, samples are taken to test the pH and osmolality and the ion concentration; if the measurements indicated an inadequate pH, it is adjusted by the addition of a solution of alkali (5M NaOH) or acid (IM HC1), to a value of 7.4 at T = 21 - 23° C. A test of the osmolality of the preparation is also performed (it must not exceed 400 mOsmol / kgH2O), and the ion concentration is tested (concentration in the range: 80 - 200 mmol / L for Na+, 1 - 15 mmol / L for K+, 0.1 - 2.5 mmol / L for Ca2+, 50 - 170 mmol / L for Cl" ).

[0216] 4. sterilization of the perfusion fluid obtained in step 3.;

[0217] The perfusion fluid is subjected to sterilization, by means known to the expert in the field, for example by sterilizing filtration, gamma radiation or ethylene oxide sterilization. Preferably, as illustrated in the following examples, the fluid is subjected to sterilization filtration, carried out under sterile conditions (under a chamber with laminar air flow, provided with HEPA filters). The filtration process aims to remove possible contaminants from mixing and microfluidization (metal filings, fragments of equipment seals) and microbial infections. In the examples shown below, ready-to-use filter sets are used for sterilization by this method, consisting of a funnel with a 0.22 pm pore diameter membrane and a 250 mL receiver, MerckMillipore, USA. Perfusion fluid is placed on the filter and filtered using a vacuum pump into a sterile container. Alternatively, as also mentioned above, the method can be carried out under sterile / sterile conditions as a possible alternative to the sterilization step.

[0218] 5. Confection of the sterile perfusion fluid obtained in step 4.;

[0219] The ways of packaging are known to the expert in the field and the way should not be limited to them.

[0220] In the examples shown below, the resulting perfusion fluid is transferred from an intermediate pack, to a sterile target pack under a laminar chamber. The fluid is bottled into sterile 250 mL or 100 mL plastic bottles fitted with septa stoppers, Nalgene, Thermo Fischer. Each bottle is labelled with batch number, date of production and storage conditions. In addition, samples are also bottled for perfusion fluid quality control (5Q) tests: physicochemical tests (measurement of particle size distribution, zeta potential, osmolality, pH, concentration of selected ions, viscosity, oncotic pressure, biological tests (cytotoxicity). Method II for producing of the perfusion fluid in the form of an oil-in-water PFC nanoemulsion as shown in Fig. 2 includes the steps:

[0221] 1. preparation of the primary perfusion fluid components

[0222] I. Preparation of pre-emulsion components

[0223] la. Preparation of the solution of surfactant or surfactants

[0224] Appropriate surface active agents are weighed on an analytical balance.

[0225] The appropriate compounds and their quantities are indicated in Table 1, with the compounds used being the main surfactant or at least one main surfactant and at least one co-surfactant. They are then placed in suitable beakers and dissolved in ultrapure water. The solutions are stirred on a magnetic stirrer at room temperature, approximately 24 hours. The clear solution is then transferred to suitable volumetric flasks and topped up with ultrapure water to the appropriate volume, and when combined, a surfactant or surfactants solution is obtained. lb. Preparation of the perfluorinated phase

[0226] The perfluorinated phase is a mixture or a single compound. The mixture is prepared by dissolving the appropriate compound(s) and stirring mechanically, at room temperature, for about 15 min.

[0227] The appropriate compounds and their quantities are indicated in Table 1.

[0228] II. Mechanical homogenization of the ingredients prepared in step I to obtain a primary preemulsion

[0229] In this step, the resulting perfluorinated phase Ib is combined with the surfactant solution la by mechanical homogenization to obtain a primary pre-emulsion.

[0230] In the examples below, this step uses:

[0231] - PRO25D mechanical homogenizer with 20 x 115 mm homogenizing tip,

[0232] - and the process parameters: the homogenizer motor speed is approximately 12,000 rpm, the process duration is 1 min homogenization process: 1 minute interval / 100 mL pre-emulsion,

[0233] However, this step is not limited to this device and the above parameters.

[0234] III. Microfluidization of the primary pre-emulsion obtained in step II to obtain the pre-emulsion In this step, microfluidization of the primary pre-emulsion obtained in step II is carried out using a high-pressure homogenizer:

[0235] In the examples below, this step uses

[0236] - a high-pressure homogenizer (microfluidizer),

[0237] - the primary pre-emulsion obtained in step II is pumped, preferably at a pressure of 2065 bar, through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled using a water bath (so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C). - the number of times the total volume of emulsion is forced through the microfluidizer chamber was 1 - 10 passes / cycles, preferably 8 passes / cycles.

[0238] However, this step is not limited to this device and the above parameters. Basically, with a certain number of permeations / microfluidization cycles, a certain size of nanoparticles is obtained. The higher the number of repetitions, the better the particle size distribution of the pre-emulsion.

[0239] In this step, a pre-emulsion is obtained.

[0240] IV. Preparation of the buffer

[0241] IVa. preparation of buffer components

[0242] In this step, the buffer ingredients will be prepared. In the non-restrictive examples shown below, the buffer components are weighed on an analytical balance and placed in a beaker. The appropriate compounds and their amounts are indicated in Table 1. The buffer is mixed on a magnetic stirrer using 350 rpm at 37 °C for approximately 2 hours.

[0243] IVR. pH adjustment

[0244] Once all components have dissolved, pH adjustment is performed (correction / elevation with 5 M NaOH to a value of 7.4).

[0245] The pH adjustment at this step affects the stability of the final obtained emulsion, as a significant change in pH may cause or accelerate ageing of the nanoemulsion and separation of two nanoemulsion phases. The final aim is a pH in the range of 7.1 - 7.6. Adjustment of the pH is necessary to obtain a fluid with the correct pH. At this step, it is also possible to dissolve the buffer components more quickly.

[0246] IVO. Quality control of the buffer obtained.

[0247] A preliminary quality control of the resulting buffer (4Q) is also performed at this step, including: measurement of pH, osmotic pressure and ion concentration - sodium, potassium, chloride and calcium.

[0248] This is a typical preparation for the final microfluidization and is an activity to check that the buffer obtained has the right parameters and can be used for the further process according to standard quality control at production. This activity is intended to exclude possible coarse weighing errors.

[0249] 2. Mixing of the pre-emulsion with the buffer

[0250] The primary perfusion fluid is obtained by mixing the pre-emulsion and the buffer in appropriate proportions. In the examples shown below, the volume of the pre-emulsion and the buffer was measured using a measuring cylinder in the ratio: 1:2 (for 1 L of the pre-emulsion, 2 L of the buffer was added) or the pre-emulsion was mixed with buffer in the ratio: 1 : 1 (for 1.5 L of the pre-emulsion, 1.5 L of the buffer was added).

[0251] The primary perfusion fluid is mixed on a magnetic stirrer at 250 rpm, at room temperature, approximately Ih.

[0252] 2R. Adjustment of pH and osmolality and ion concentration

[0253] Samples are then taken to test pH and osmolality and ion concentration (2Q), if measurements indicated an inadequate pH, in the range 7.1 - 7.6 it is adjusted by the addition of an alkali solution (5M NaOH) or acid (IM HC1), to a value of 7.4 at T = 21 - 23 °C (2R).

[0254] However, care is taken to ensure that the osmolality of the primary perfusion fluid does not exceed 400 mOsmol / kgH2O and that ion concentrations are in the range: 80 - 100 mmol / L for Na+, 1 - 15 mmol / L for K+, 0.1 - 2.5 mmol / L for Ca2+, 50 - 170 mmol / L for Cl."

[0255] 3. Microfluidization of the primary perfusion fluid to obtain the perfusion fluid The primary perfusion fluid obtained in step (2) is microfluidized (3) using a high-pressure homogenizer to obtain the perfusion fluid:

[0256] In the examples below, this step uses:

[0257] - a high-pressure homogenizer (microfluidizer),

[0258] - the primary perfusion fluid obtained in step (2) is pumped, preferably at a pressure of 2065 bar, through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled by means of a water bath (so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C), - the number of times the total volume of emulsion is forced through the microfluidizer chamber was 1-5 passes / cycles.

[0259] However, this step is not limited to this device and the above parameters.

[0260] At the higher PFC concentration characteristic of the perfluorinated phase, a so-called thick emulsion is formed, in which the PFC nanoparticles are larger due to its concentration. At this step, at a given target concentration, the microfluidization step allows the smallest possible nanoparticles to be obtained at a given PFC and surfactant concentration. This step may be called final microfluidization.

[0261] The parameters for both microfluidizations (in step (3) and in step III.) are the same, they differ in the composition of the mixture that is microfluidized.

[0262] 4. Sterilizing filtration

[0263] The perfusion fluid is subjected to sterilization, by methods known to the expert in the field, as already indicated above, for example by sterilizing filtration, gamma radiation or sterilization with ethylene oxide. Preferably, as shown in the following examples, the perfusion fluid obtained in step (3) is subjected to a sterilization filtration step (4). The sterilization filtration step (4) is carried out under sterile conditions (under a chamber with laminar air flow, provided with HEPA filters). The sterilization filtration process (4) is carried out in order to get rid of possible contaminants generated after mixing and microfluidization (metal filings, fragments of equipment seals) and microbial infections.

[0264] In the examples shown below, ready-made filter sets were used for sterilization by this method, consisting of a funnel with a 0.22 pm pore diameter membrane and a 250-500 mL receiver. The perfusion fluid obtained in step (3) is placed on filters and filtered using a vacuum pump into sterile containers.

[0265] Alternatively, as also mentioned above, the method can be carried out under sterile / sterile conditions as a possible alternative to the sterilization step.

[0266] 5. Confection

[0267] The ways of packaging are known to the expert in the field and the way should not be limited to them.

[0268] In the examples shown below, after the perfusion fluid has been cleared of particulates and microorganisms using the sterile filtration process (4), the next step is to transfer the perfusion fluid from the intermediate package, to the sterile target package under the laminar chamber. The perfusion fluid is bottled into sterile 1000 mL, 500 mL, 250 mL or 100 mL plastic bottles fitted with septa stoppers, Nalgene, Thermo Fischer or into 10 mL, 20 mL, 50 mL or 100 mL glass vials capped with aluminium septa caps. Appropriate volumes of perfusion fluid are measured using graduated cylinders or an automatic pipettor. Each bottle is labelled with the batch number, date of manufacture and storage conditions.

[0269] 5Q. Quality control

[0270] In addition, samples are also bottled for perfusion fluid quality control (5Q) tests: physicochemical tests (measurement of: particle size distribution, zeta potential, osmolality, pH, concentration of selected ions, oncotic pressure, viscosity and cytotoxicity tests).

[0271] Perfusion fluid

[0272] The perfusion fluid which is the subject of this invention is in the form of an emulsion (o / w type) in which the dispersed phase is perfluorinated compounds having the ability to dissolve and transport the respiratory gases oxygen and carbon dioxide. The perfluorinated phase is composed of mixtures or single PFCs from among perfluorooctyl bromide, perfluorodecyl bromide, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin (based on examples of the implementation of the invention) and based on literature data (DOI: 10,2217 / nnm-2019-0260) perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane.

[0273] The PFC nanoparticles are stabilised by the addition of biocompatible surface active agents. As the main surfactant are used: Pluronic F-68 (Kolliphor P188, numer wg INCI: Poloxamer 188; CAS 9003-11-6, Index F-68, PEO75-PPO30-PEO75) or Pluronic F-108 numer wg INCI: Poloxamer 338; CAS 9003-11-6, Index F-108; PEO141-PPO44-PEO141) or Pluronic Fl 27 (numer wg INCI: Poloxomer-407: CAS 9003-11-6, Index F-127; PEOioo-PPOes-PEOioo or their diblock / triblock / multiblock copolymers or condensation polymers of polypropylene glycols with polyoxyethylene glycols (https: / / doi.org / 10.1166 / jnn.2006.449) or perfluorinated copolymers of the hydrocarbon-fluorocarbon type or perfluorinated copolymers of the hydrocarbon-fluorocarbon type (https: / / doi.org / 10.1002 / macp.201700558) or mammalian albumin: bovine serum albumin (BSA), human serum albumin (HSA) and recombinant human serum albumin or their derivatives, gelatine and its derivatives. Perfluorinated surfactants were used as co-surfactant: (1H, 1H, 2H, 2H-perfluorooctyl)phosphocholine, or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, (1H, 1H, 2H, 2H-perfluorooctyl)-P-D-maltopyranoside, Sphingomyelin, modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt, l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt, N-(carbonyl-methoxypolyethyleneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt or perfluorinated phospholipids (e.g. Phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids.

[0274] The continuous phase of the emulsion is a buffer solution, ensuring physiological (for human blood) pH, osmotic pressure and oncotic pressure of the perfusion fluid. To ensure adequate perfusion fluid parameters, mammalian albumin is used as the oncotic agent: bovine serum albumin (BSA), human serum albumin (HSA), recombinant human serum albumin or their derivatives, gelatine and its derivatives or PEG 35 kDa or PEG 20 kDa or Dextran 70 kDa, Dextran 40 kDa, Dextran 30 kDa, Dextran 50 kDa or succinylated gelatine or hydroxy ethylated starch or cyclodextrin derivatives. Preferably, mannitol is used as the osmotic agent, but other osmotic agents described in the literature are possible, i.e. raffinose, sucrose, glucose, citrate chelates, lactobionic acid salts and gluconic acid.

[0275] In addition to oxygen-carrying substances, preferably the perfusion fluid contains a source of energy for the cells (i.e. D-glucose • H2O, fructose, sucrose, dextran), compounds that enable the regeneration of the high-energy phosphate compound - ATP (i.e. sodium salt of D-P-hydroxybutyric acid), substances that reduce / neutralise the formation of free radicals and peroxides and prevent intracellular acidosis (i.e. glutathione, vitamin C, vitamin E, deferoxamine, N-acetyl cysteine, catalase, peroxidase or xanthine oxidase inhibitors, i.e. allopurinol, adenine, adenosine, phosphates; hormones: insulin, steroids - dexamethasone). Preferably the perfusion fluid also contains amino acids with various functions. These include: stimulation of nitric oxide synthesis, prevention of epithelial cell damage, antioxidant, osmotic pressure regulation, anti-inflammatory and anti-apoptotic effects, protection of cells against lipid peroxidation and harmful changes to membrane structure, substrates for protein and coenzyme synthesis, participation in protein synthesis, role in nitrogen metabolism and in the synthesis of purines, pyrimidines and other amino acids. In the perfusion fluid are used: L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine. Buffering ingredients and electrolytes that affect the osmotic pressure of the perfusion fluid are also used, i.e.: ISfeHPCU, NaEbPC , NaCl, KC1, CaCh, MgCh • 6H2O, NaOH. It is possible to use other buffer systems and salts described in the literature, i.e. buffer systems: bicarbonate (mixture of NaHCOs and H2CO3), phosphate (containing KH2PO4 and K2HPO4), citrate (containing NasCeEEOz ^EbO and C6H8O7-H2O), Histidine (containing histidine-HCl and L-histidine), or sodium, potassium, calcium, magnesium and chloride salts: sulphates(VI), phosphate, lactobionate, citrate, gluconate.

[0276] Table 1 shows the overall composition of the fluid according to the invention, including the role of the individual components and the range of concentrations.

[0277] Table 1. General composition of the perfusion fluid.

[0278] Individual Examples of compounds used in the

[0279] component Concentration Benefits of the present invention Units perfusion fluid groups

[0280] A mixture of perfluorooctyl bromide

[0281] and perfluorodecyl bromide a mixture

[0282] of perfluorotributylamine and

[0283] perfluorodecalin;

[0284] PFC - oxygen a mixture of perfluorooctyl bromide, 96.51 - 772.00 The main component carrier, perfluorodecyl bromide, and preferably 96.51 - of the nanoemulsion mixture or perfluorotributylamine; 386.00 g / L responsible for the single or a mixture of perfluorooctyl bromide most preferably transport of respiratory compound and perfluorotributylamine or 193.00 - 386.00 gases perfluoropentane or perfluorohexane or

[0285] perfluoroheptane or

[0286] perfluorotributylamine or

[0287] perfluorodecalin

[0288] Main surfactant: Pluronic F-68 The surfactant is Surface active 0.0 -47.6

[0289] (Kolliphor P188) or Pluronic F-108 or responsible for agent Pluronic F-127 orbovine / human serum preferably 23.8 - 34.0

[0290] lowering the surface (Surfactant), most preferably g / L

[0291] albumin or recombinant human serum tension of the liquid, mixture: 27.2 - 34.0

[0292] albumin. enables the formation selected main

[0293] of a stable surfactant and Co-surfactant: An organic compound 3.5 -7.0 nanoemulsion, slows co-surfactant containing perfluorinated chains: (1H,

[0294] preferably 3.5 - 5.0 g / L

[0295]

[0296] 1H, 2H, 2H- down the ageing Individual

[0297] component Examples of compounds used in the Concentnition Units Benefits of the present invention perfusion fluid groups

[0298] Perfluorooctyl)phosphocholine or most preferably 4.0 - processes e.i. Ostwald 5.0 ripening homologs thereof or derivatives

[0299] thereof, including phosphocholine

[0300] derivatives containing, in various

[0301] structural configurations,

[0302] perfluorocarbon chains, or (1H, 1H,

[0303] 2H, 2H-Perfluorooctyl)-P-D- maltopyranoside or Sphingomyelin or

[0304] modified lipids: 1,2-Dioleoyl-sn- glycero-3-phospho-L-serine sodium

[0305] salt or l,2-dipalmitoyl-sn-glycero-3- phosphate sodium salt or 1,2- dimyristoyl-sn-glycero-3-phospho-rac- glycerol sodium salt or N-(carbonyl- methoxy polyethylene glycol-2000)- 1 ,2-distearoyl-sn-glycero-3 - phosphoethanolamine sodium salt

[0306] Bovine / human serum albumin or

[0307] Substances that recombinant human serum albumin or

[0308] Oncotic regulate oncotic PEG 35 kDa or PEG 20 kDa or Dextran 20.0 - 70.0

[0309] factor, single pressure, preventing g / L Dextran 70 kDa or Dextran Dextran 40 preferably 40.0 organ swelling during compound

[0310] kDa or succinylated gelatine or perfusion hydroxy ethylated starch

[0311] The main osmotic agent, in addition to preventing cell 10.0 - 100.0 Main osmotic mM swelling, protects mannitol

[0312] agent preferably 21.4 - 50.0

[0313] against the formation of reactive oxygen species (antioxidant) 5.00 - 20.00 Main source of energy mM D-glucose ■ H2O

[0314] preferably 10.00 for cells

[0315] 0.10 - 0.50 sodium salt of DL-p-hydroxybutyric mM Energy source for cells acid preferably 0.20

[0316] Main antioxidant - antioxidant (prevents Basic buffer the formation of free components 1.50 - 6.00 radicals), in addition to mM glutathione

[0317] preferably 3.00 being an important substrate and cofactor in many metabolic reactions

[0318] 0.50 - 3.00 Xanthine oxidase mM allopurinol

[0319] preferably 1.00 inhibitor, antioxidant 5.00 - 15.00 Amino acids have mM taurine

[0320] preferably 10.00 various functions 0.05 - 0.20 including.: stimulation mM L-arginine

[0321] preferably 0.10 of nitric oxide 1.00 - 3.00 synthesis, prevention L-tryptophan mM

[0322] preferably 2.00 Buffer of epithelial cell 0.10 - 1.00 components - damage, antioxidants, mM L-glutamine regulation of osmotic Amino acids preferably 0.50

[0323] pressure, anti0.50 - 10.00

[0324] mM L-isoleucine inflammatory and anti- preferably 1.52 - 4.19

[0325] apoptotic effects, 1.00 - 10.00

[0326] mM L-leucine protection of cells preferably 3.05 - 6.86

[0327] against lipid 1.00 - 10.00 mM L-lysine

[0328]

[0329] Individual

[0330] component Examples of compounds used in the Concentration Benefits of the present invention Units perfusion fluid groups

[0331] preferably 2.05 - 4.79 peroxidation and 0.50 - 10.00 harmful changes in L-methionine mM

[0332] preferably 1.34 - 3.35 membrane structure, 0.50 - 10.00 substrates for protein L-threonine mM

[0333] preferably 1.69 - 3.78 synthesis and 0.50 - 10.00 coenzymes,

[0334] L-valine mM

[0335] preferably 2.56 - 5.55 participation in protein 0.10 - 10.00 synthesis, role in L-histidine mM

[0336] preferably 0.64 - 2.26 nitrogen metabolism 1.00 - 20.00 and in the synthesis of L-alanine preferably 5.61 mM purines, pyrimidines 12.35 and other amino acids, 1.00 - 20.00 a mixture of L-glycine preferably 6.66 mM compounds 17.32

[0337] 0.50 - 10.00

[0338] L-aspartic acid mM

[0339] preferably 1.88 - 4.51

[0340] 0.50 - 10.00

[0341] L-proline mM

[0342] preferably 2.17 - 5.21

[0343] 0.10 - 10.00

[0344] L-serine mM

[0345] preferably 0.95 - 2.38

[0346] 0.01 - 1.00

[0347] L-tyrosine mM

[0348] preferably 0.06 - 0.28

[0349] 6.00 - 15.00 Buffering components Na2HPO4mM

[0350] preferably 8.00 and electrolytes, affect the osmotic pressure of 0.50 - 3.00

[0351] NaH2PO4mM the perfusion fluid, a preferably 1.50 mixture of compounds 20.00 - 100.00 Inorganic salts, Buffer NaCl mM

[0352] preferably 60.00 containing the most components 2.00 - 10.00 important ions needed and KC1 mM

[0353] preferably 4.60 by the organism electrolytes (sodium, potassium,

[0354] 20.30 - 2.00

[0355] CaCl mM

[0356] preferably 0.60 calcium, magnesium 0.20 - 2.00 and chloride), also MgCl2■ 6H2O mM

[0357] preferably 0.40 affect the osmotic pressure of the 0.50 - 20.00

[0358] NaOH mM perfusion fluid, a preferably 1.4 - 14.00

[0359]

[0360] mixture of compounds

[0361] Table 2 shows the parameters characterising the perfusion fluid according to the invention.

[0362] Table 2. Parameters characterising perfusion fluid

[0363] Test parameter Acceptance criteria

[0364] Average diameter <200 - 300 nm

[0365] Size distribution

[0366] Pdl <0.2

[0367] Zeta potential #) mV

[0368] Osmolality 280 - 400 mOsm / kgH2O

[0369] pH 7.1 -7.6

[0370] Na+[mM]: 80 - 200

[0371] K+[mM]: 1 - 15

[0372] Concentration of selected ions

[0373] Ca2+[mM]: 0.1 - 2.5

[0374] Cl’ [mM]: 50 - 170

[0375]

[0376] Oncotic pressure For blood: 15 - 36 mmHg Test parameter Acceptance criteria

[0377] Cell viability f%] Liquid concentration in culture medium f%] : 50 Cytotoxicity

[0378]

[0379] > 70 % L929 f%]

[0380] The perfusion fluid produced according to the manufacturing methods in question enables the formation of stable nanoparticles with the parameters shown in Table 2.

[0381] The method of producing the perfusion fluid according to present invention, consisting of a combination of mechanical homogenization techniques and microfluidization of a PFC mixture having an appropriate composition, makes it possible to obtain stable nanoparticles with a size below 300 nm, preferably below 220 nm, most preferably below 130 nm.

[0382] The key steps in the production of perfusion fluid are, according to mode I of production: mechanical homogenization and microfluidization, and according to mode II of production: mechanical homogenization and microfluidization of the pre-emulsion and microfluidization of the primary perfusion fluid.

[0383] The size of the PFC nanoparticles obtained in the perfusion fluid, as well the stability of the perfusion fluid, are determine by the microfluidization step, which uses a pump to generate high pressure to break up the PFC mixture by shear forces. The PFC suspension is fed into the inlet and then pumped at high pressure through the microchannels of a Y-type diamond chamber, resulting in a high shear rate and ultimately resulting in the formation of nanoparticles. One of the main advantages of microfluidization is the high reproducibility of the process due to the fixed geometry and easy scalability.

[0384] There is a sterilizing filtration step in the perfusion fluid manufacturing process (Methods I and II), which can be replaced by carrying the process under sterile conditions or realising the sterilization of the perfusion fluid by other commercial sterilization methods.

[0385] In the examples of the implementation of the invention, it has been confirmed that the perfusion fluid_02 and the perfusion fluid_04 obtained according to production methods I and II have the same parameters. Therefore, both methods are equally effective and lead to an equivalent perfusion fluid.

[0386] All perfusion fluid formulations, prepared according to the invention, are characterised by physicochemical properties similar to human blood, in terms of: osmotic pressure (in the range: 280 - 400 mOsm / kgJUO), pH (7.1 - 7.6), oncotic pressure (15 - 36 mmHg), concentration of the main ions: sodium (80-200 mM), potassium (1 - 15 mM), calcium (0.1-2.5 mM) and chloride (50 - 170 mM). Furthermore, the average diameter of the nanoparticles of the dispersed (perfluorinated) phase of the present invention does not exceed 300 nm, and the homogeneity of the described emulsions, expressed by the polydispersity index (Pdl), does not exceed 0.1 (over a period of 3 months after manufacture). In addition, the zeta potential value of the nanoparticles is different from zero, and is at least: -5 mV or +5mV, which prevents coalescence and aggregation of the particles of the dispersed phase of the emulsion. The perfusion fluid formulations presented in the present invention, are nontoxic towards L929 mouse fibroblast cells, as indicated by a cell viability above 70%, for a fluid concentration in the culture medium equal to 50%.

[0387] The above-mentioned composition of the perfusion fluid according to the invention, enables the use of warm machine perfusion technology to improve the storage conditions of organs for transplantation. Existing procedures for storing an organ in a static cold room on ice after rinsing with crystalline solutions, such as the University of Wisconsin solution or Custodiol, do not completely stop cellular metabolism in organs; they only lead to a slowing down of enzymatic reactions and a delay in cell death. In contrast, the use of perfusion fluid in warm ex vivo machine perfusion of an organ allows the organ to be maintained under full metabolic conditions, with the direct result that the organ is in better condition and the organ can be stored for a longer period of time (up to about 30 h). In addition, the perfusion fluid according to the invention used for ex vivo machine perfusion of organs intended for transplantation, i.e. donations of organs after brain death - DBD and organs taken from donors after irreversible cardiac arrest - DCD or donors aged 60 and over and donors aged 50-59 with at least two of the following risk factors: arterial hypertension, death caused by stroke, blood creatinine level at the time of death above 1.5 mg / dL - ECD, enables better storage and reconditioning of the organ before transplantation.

[0388] This is further is confirmed by the perfusion results of isolated organs damaged during controlled DCD, autotransplantation results and proteomic-metabolomic results.

[0389] The above-mentioned composition of the perfusion fluid according to the invention enables warm ex vivo machine perfusion of organs, i.e. kidney, liver, heart, as confirmed in the examples.

[0390] The above composition of the perfusion fluid allows it to be used in the resuscitation process after haemorrhagic shock as a blood substitute, as confirmed in the examples.

[0391] Examples of the implementation of the invention

[0392] The following examples are included only to illustrate the invention and to clarify particular aspects of the invention, not to limit the invention, and should not be equated with the entire scope of the invention as defined in the appended claims. In the following examples, unless otherwise indicated, standard materials and methods used in the field have been used or manufacturers' recommendations for specific reagents and methods have been followed.

[0393] Example I: Preparation of PEC nanoemulsions (organ perfusion fluids):

[0394] Example LI. Obtaining perfusion fluid_02_II production method

[0395] Table 3. Quantitative composition of the perfusion fluid_02_II production method.

[0396] Component Concentration Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0397] 9048-46-8

[0398] Bovine serum albumin 40.00

[0399] 90604-29-8 g / L

[0400] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0401]

[0402] L-tryptophan 73-22-3 2.00 mM Component Concentration Units D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM

[0403] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0404] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0405]

[0406] NaOH 1310-73-2 to determine the pH

[0407] The method for obtaining the perfusion fluid involves the formation of a pre-emulsion from a surfactant solution and a perfluorinated phase.

[0408] For this purpose, the components (surfactant solution and perfluorinated phase, in the block diagram for obtaining perfusion fluid - Fig. 2., marked with No. (I)) were weighed using an analytical balance.

[0409] The surfactant solution was obtained by dissolving the surface active agents in ultrapure water in two beakers: a volume of 500 mL (amounts: 58.2857 g Pluronic F-68 and 8.5714 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine and a volume of 200 mL (amounts: 23.3143 g Pluronic F-68 and 3.4286 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine. The solutions were stirred on a magnetic stirrer at room temperature for about 24 hours. Then, the clear solutions were transferred to two volumetric flasks (500 mL and 200 mL) and made up to volume with ultrapure water. The two solutions were combined to give a 700 mL solution of the surfactants mixture.

[0410] The perfluorinated phase, i.e. a mixture of perfluorocarbons: perfluorooctyl bromide with perfluorodecyl bromide, was prepared by dissolving 17.37 g of perfluorodecyl bromide in 561.63 g of perfluorooctyl bromide, (mechanical stirring, at room temperature, approximately 0.25 hours).

[0411] This yielded approximately 300 mL of perfluorinated phase, which was combined with 700 mL of aqueous phase (surfactanst solution) by mechanical homogenization (primary preemulsification) (II). APRO25D mechanical homogenizer with a 20 x 115 mm homogenizing tip, PRO Scientific Inc., USA, was used. Process parameters: the speed of the homogenizer motor was approximately 12 000 rpm, process duration - 1 min homogenization process: 1 minute interval / 100 mL primary pre-emulsion. After approximately 20 minutes, the primary pre-emulsification process was completed.

[0412] The next step was a primary pre-emulsion (III) microfluidization process, carried out using a high-pressure homogenizer: Microfluidizer® LM20 processor, Microfluidics (IDEX Health & Science, LLC), Canada. The primary pre-emulsion was pressed at 2065 bar through an F12Y type diamond chamber cooled by a water bath. The number of times the total volume of emulsion was forced through the microfluidizer chamber was 8.

[0413] The final sub-step was the preparation of a 2 L buffer by weighing and dissolving the ingredients in ultrapure water (IVa), according to the following order (the order of addition of the ingredients being irrelevant): 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 gNaCl, 1.0288 gKCl, 3.4070 gNa2HPO4, 0.5399 gNaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium. The buffer was stirred on a magnetic stirrer using 350 rpm at 37° C for approximately 2 hours. Once all components were dissolved, pH adjustment (correction / elevation with 5 M NaOH to 7.4) was performed (IVR). Then, the solution was placed in a 2 L volumetric flask and topped up with an appropriate amount of ultrapure water. At this step, a preliminary quality control of the resulting buffer (IVQ) was also performed, including: measurement of pH, osmotic pressure and ion concentration - sodium, potassium, chloride and calcium.

[0414] The primary perfusion fluid is formed when the pre-emulsion and the buffer are mixed in the correct proportions (2). A measuring cylinder was used to measure the volume of the preemulsion and the buffer in the ratio: 1:2 (for 1 L of the pre-emulsion, 2 L of the buffer was added). The preparation was stirred at 250 rpm, at room temperature, for about one hour, on a magnetic stirrer. Samples were then taken for pH and osmolality and ion concentration testing (2R), if measurements indicated inadequate pH, it was adjusted by the addition of an alkali solution (5M NaOH) or acid (IM HC1), to a value of 7.4 at T = 21 - 23° C (2R) Special attention was paid to ensure that the osmolality of the preparation did not exceed 400 mOsmol / kgH2O, and that the ion concentration was in the range: 80 - 200 mmol / L for Na+, 1 - 15 mmol / L for K+, 0.1 - 2.5 mmol / L for Ca2+, 50 - 170 mmol / L for Cl."

[0415] The obtained primary perfusion fluid was microfluidized (final microfluidization) (3) using a high-pressure homogenizer: Microfluidizer® LM20 processor, Microfluidics (IDEX Health & Science, LLC), Canada. The primary perfusion fluid was pressed once at a pressure of 2065 bar through a type F12Y diamond chamber cooled by a water bath.

[0416] The sterilizing filtration step (4) took place under sterile conditions (under a chamber with laminar air flow, equipped with HEPA filters). The filtration process was carried out to get rid of possible contaminants from mixing and microfluidization (metal filings, fragments of equipment seals) and microbial infections. For sterilization by this method, off-the-shelf filter sets were used, consisting of a funnel with a 0.22 pm pore diameter membrane and a 250 - 500 mL receiver, MerckMillipore, USA. Perfusion fluid was placed on the filters (6 sets of 500 mL each were used) and passed through the membrane pore using a vacuum pump into sterile containers.

[0417] After the perfusion fluid was cleared of particulates and microorganisms by means of a filtration process, the next step was to transfer the obtained perfusion fluid from the intermediate package, into a sterile target package under a laminar chamber (5). The resulting perfusion fluid_02_II production method was bottled into sterile 1000 mL, 500 mL, 250 mL or 100 mL plastic bottles fitted with septa stoppers, Nalgene, Thermo Fischer, or into 10 mL, 20 mL, 50 mL or 100 mL glass vials capped with aluminium septa caps. Corresponding volumes of perfusion fluids were measured using graduated cylinders or an automatic pipettor. Each bottle was labelled with the batch number, date of manufacture and storage conditions. In addition, samples were also spilled for perfusion fluid quality control tests (5Q): physicochemical tests (measurement of particle size distribution, zeta potential, osmolality, pH, concentration of selected ions, viscosity, oncotic pressure, biological tests (cytotoxicity). Example 1.2. Obtaining perfusion fluid_04_II production method

[0418] Table 4. Quantitative composition of perfusion fluid_04_II production method.

[0419] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 374.42 g / L Perfluorodecyl bromide (PFDB) 307-43-7 11.58 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 34.00 g / L

[0420] (1H, 1H, 2H, 2H- 313997-22-7 5.00 g / L Perfluorooctyl)phosphocholine

[0421] 9048-46-8

[0422] Bovine albumin 40.00 g / L 90604-29-8

[0423] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM Taurine 107-35-7 10.00 mM

[0424] L-tryptophan 73-22-3 2.00 mM

[0425] D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM

[0426] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0427] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0428]

[0429] NaOH 1310-73-2 to determine the pH

[0430] The perfusion fluid_04_II production method was obtained according to the method of obtaining the perfusion fluid_02_ II production method described in example 1.1.

[0431] The surfactants solution was obtained by dissolving the surface active agents in ultrapure water in three beakers: one with a volume of 500 mL (amounts: 56.6667 g Pluronik F-68 and 8.3333 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine and two with a volume of 200 mL each (amounts: 22.6667 g Pluronik F-68 and 3.3333 g (1H, 1H, 2H, 2H- Perfluorooctyl)phosphocholine.

[0432] A mixture of perfluorocarbons: perfluorooctyl bromide with perfluorodecyl bromide, was prepared by dissolving 34.74 g of perfluorodecyl bromide in 11,232.60 g of perfluorooctyl bromide, (mechanical stirring, at room temperature, approximately 0.25 hours).

[0433] This yielded approximately 600 mL of perfluorinated phase, which was combined with 900 mL of aqueous phase (surfactants solution) by mechanical homogenization (primary pre-emulsification) (II). APRO25D mechanical homogenizer with a 20 x 115 mm homogenizing tip, PRO Scientific Inc., USA, was used. Process parameters: the speed of the homogenizer motor was approximately 12 000 rpm, process duration - 1 min homogenization process: 1 minute interval / 100 mL primary pre-emulsion. After approximately 30 minutes, the primary pre-emulsification process was completed. The primary pre-emulsion microfluidization process (III) was carried out according to the perfusion fluid_02_II production method, as described in example 1.1

[0434] The buffer was obtained by weighing and dissolving the ingredients in ultrapure water, using mechanical stirring (IV), according to the following order (whereby the order of addition of the ingredients does not matter): 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 gNaCl, 1.0288 gKCl, 3.4070 gNa2HPO4, 0.5399 gNaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium.

[0435] According to the perfusion fluid_02_II production method, a primary perfusion fluid was obtained, except that the ratio of the pre-emulsion to the buffer was 1:1 (for 1.5 L of the preemulsion, 1.5 L of the buffer was added).

[0436] The obtained primary perfusion fluid was microfluidized (final microfluidization) (3) using a high-pressure homogenizer: Microfluidizer® LM20 processor, Microfluidics (IDEX Health & Science, LLC), Canada. The primary perfusion fluid was pressed five times at a pressure of 2065 bar through an F12Y type diamond chamber, cooled by a water bath.

[0437] The steps of sterilizing filtration (4) and confectioning (5) were carried out according to the method for obtaining perfusion fluid_02_ II production method, described in example 1.1. Perfusion fluid_04_II production method was obtained.

[0438] Example 1.3 Obtaining perfusion fluid Ol I method of production (hereafter referred to as perfusion fluid Ol)

[0439] Table 5. Quantitative composition of perfusion fluid Ol.

[0440] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 93.61 g / L Perfluorodecyl bromide (PFDB) 307-43-7 2.90 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 23.80 g / L (1H, 1H, 2H, 2H- 313997-22-7 3.50 g / L Perfluorooctyl)phosphocholine

[0441] 9048-46-8

[0442] Bovine albumin 40.00 g / L 90604-29-8

[0443] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM

[0444]

[0445] MgCl2• 6H2O 7791-18-6 0.40 mM Component Concentration Units L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0446]

[0447] NaOH 1310-73-2 to determine the pH The following method of obtaining perfusion fluid is shown in Fig. 1.

[0448] The surfactants solution was prepared by weighing 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine and dissolving in ultrapure water. The solution was stirred on a magnetic stirrer at room temperature for about 24 h. The clear solution of the surfactants mixture was then transferred to a 1000 mL volumetric flask and made up to volume with ultrapure water.

[0449] The perfluorinated phase was prepared by weighing 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide and stirring mechanically at room temperature for approximately 0.25 h.

[0450] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. The buffer was stirred on a magnetic stirrer using 350 rpm at 37 °C for approximately 2 h. Once all the ingredients were dissolved, pH adjustment was performed (correction / elevation with 5 M NaOH to a value of 7.4). The solution was then transferred to a 2 L volumetric flask and topped up with an appropriate amount of ultrapure water. A preliminary quality control of the resulting buffer was also performed, including: measurement of pH, osmotic pressure and ion concentration - sodium, potassium, chloride and calcium.

[0451] The primary perfusion fluid was prepared by dispensing appropriate volumes of 40.84 mL of the surfactants solution, 133.33 mL of the buffer, 15.83 mL of the ultrapure water and 10 mL of the perfluorocarbons mixture into a 250 mL beaker using graduated cylinders and automatic pipettes. The mixture was emulsified by mechanical homogenization, and 200 mL of the primary perfusion fluid was obtained (2). APRO25D mechanical homogenizer with a 20 x 115 mm homogenizing tip, PRO Scientific Inc., USA, was used. Process parameters: the motor speed of the homogenizer was approximately 12000 rpm, the process duration was 1 min of homogenization: 1 minute interval / 100 mL emulsion. The homogenization process was carried out for approximately 4 minutes.

[0452] In the next step, microfluidization of the primary perfusion fluid (3) was carried out using a high-pressure homogenizer: Microfluidizer® LM20 processor, Microfluidics (IDEX Health & Science, LLC), Canada. The primary perfusion fluid was pressurized at 2065 bar through an F12Y type diamond chamber cooled by a water bath. The number of times the total volume of emulsion was forced through the microfluidizer chamber was 9, thus obtaining the perfusion fluid. Samples were taken for pH and osmolality testing, as well as ion concentration; if the measurements indicated inadequate pH, it was adjusted by the addition of a solution of alkali (5M NaOH) or acid (IM HC1), to a value of 7.4 at T = 21 - 23° C. The osmolality of the preparation was tested (not to exceed 400 mOsmol / kgJL O), and the ion concentration was tested (concentration in the range: 80 - 200 mmol / L for Na+, 1 - 15 mmol / L for K+, 0.1 - 2.5 mmol / L for Ca2+, 50 - 170 mmol / L for CT).

[0453] The perfusion fluid was subjected to sterilizing filtration (4), carried out under sterile conditions (under a laminar airflow chamber equipped with HEPA filters). The filtration process is designed to remove possible contaminants generated after mixing and microfluidization (metal filings, fragments of device seals) and microbial infections. For sterilization by this method, off-the-shelf filter kits were used, consisting of a funnel with a 0.22 pm pore diameter membrane and a 250 mL receiver, MerckMillipore, USA. Perfusion fluid was placed on the filter and filtered using a vacuum pump into a sterile container.

[0454] The obtained perfusion fluid was then transferred from the intermediate pack to the sterile target pack under the laminar chamber (5). The fluid was bottled into sterile 250 mL or 100 mL plastic bottles fitted with septa stoppers, Nalgene, Thermo Fischer. Each bottle was labelled with batch number, date of manufacture and storage conditions. In addition, samples were also spilled for perfusion fluid quality control tests (5Q): physicochemical tests (measurement of particle size distribution, zeta potential, osmolality, pH, concentration of selected ions, viscosity, oncotic pressure, biological tests (cytotoxicity).

[0455] Example 1.4 Obtaining perfusion fluid_02

[0456] Table 6. Quantitative composition of perfusion fluid_02.

[0457] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 2720 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0458] 9048-46-8

[0459] Bovine albumin 40.00 g / L 90604-29-8

[0460] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM

[0461]

[0462] MgCh • 6H2O 7791-18-6 0.40 mM Component Concentration Units L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0463]

[0464] NaOH 1310-73-2 to determine the pH Perfusion fluid_02 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0465] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0466] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0467] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water.

[0468] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_02 was obtained.

[0469] Example 1.5 Obtaining perfusion fluid_03

[0470] Table 7. Quantitative composition of perfusion fluid_03.

[0471] Component Concentration Perfluorooctyl bromide (PF OB) 423-55-2 280.82 g / L Perfluorodecyl bromide (PFDB) 307-43-7 8.69 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 30.60 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.50 g / L Perfluorooctyl)phosphocholine

[0472] 9048-46-8

[0473] Bovine albumin 40.00 g / L 90604-29-8

[0474] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM Taurine 107-35-7 10.00 mM

[0475] L-tryptophan 73-22-3 2.00 mM

[0476] D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM

[0477]

[0478] KC1 7447-40-7 4.60 mM Component Concentration Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM

[0479] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0480] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0481]

[0482] NaOH 1310-73-2 to determine the pH Perfusion fluid_03 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0483] The surfactants solution was prepared by dissolving 116.6000 g of Pluronik F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0484] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0485] The buffer was prepared by weighing 160.0000 g bovine serum albumin, 0.0697 g L-arginine, 0.5445 g allopurinol, 15.6229 g mannitol, 5.0080 g taurine, 1.6338 g L-tryptophan, 7.9268 g D-glucose • H2O, 14.0256 g NaCl, 1.3717 g KC1, 4.5427 g Na2HPO4, 0.7199 g NaH2PO4, 0.2664 g CaCl2, 0.3253 g MgCl2• 6H2O, 0.2923 g L-glutamine, 3.6880 g glutathione, 0.1009 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water.

[0486] Atotal of 52.51 mL of the surfactants solution, 100 mL of the buffer, 17.49 mL of the ultrapure water and 30 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_03 was obtained.

[0487] Example 1.6 Obtaining perfusion fluid_04

[0488] Table 8. Quantitative composition of perfusion fluid_04.

[0489] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 374.42 g / L Perfluorodecyl bromide (PFDB) 307-43-7 11.58 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 34.00 g / L (1H, 1H, 2H, 2H- 313997-22-7 5.00 g / L Perfluorooctyl)phosphocholine

[0490] 9048-46-8

[0491] Bovine albumin 40.00 g / L 90604-29-8

[0492] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM

[0493]

[0494] Taurine 107-35-7 10.00 mM Component Concentration Units L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60,00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0495]

[0496] NaOH 1310-73-2 to determine the pH Perfusion fluid_04 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0497] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0498] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide and 1000 g of perfluorooctyl bromide.

[0499] The buffer was prepared by weighing 160.0000 g bovine serum albumin, 0.0697 g L-arginine, 0.5445 g allopurinol, 15.6229 g mannitol, 5.0080 g taurine, 1.6338 g L-tryptophan, 7.9268 g D-glucose • H2O, 14.0256 g NaCl, 1.3717 g KC1, 4.5427 g Na2HPO4, 0.7199 g NaH2PO4, 0.2664 g CaCl2, 0.3253 g MgCl2• 6H2O, 0.2923 g L-glutamine, 3.6880 g glutathione, 0.1009 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water.

[0500] A total of 58.34 mL of the surfactants solution, 100 mL of the buffer, 1.66 mL of the ultrapure water and 40 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_04 was obtained.

[0501] Example 1.7 Obtaining perfusion fluid_05

[0502] Table 9. Quantitative composition of perfusion fluid_05.

[0503] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 468.03 g / L Perfluorodecyl bromide (PFDB) 307-43-7 14.48 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 37.40 g / L (1H, 1H, 2H, 2H- 313997-22-7 5.50 g / L Perfluorooctyl)phosphocholine

[0504] 9048-46-8

[0505] Bovine albumin 40.00 g / L

[0506]

[0507] 90604-29-8 Component Concentration Units L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0508]

[0509] NaOH 1310-73-2 to determine the pH

[0510] Perfusion fluid_05 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0511] The surfactants solution was prepared by dissolving 47.6000 g of Pluronik F-68 and 7.0000 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0512] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0513] The buffer was prepared by weighing 160.0000 g bovine serum albumin, 0.0697 g L-arginine, 0.5445 g allopurinol, 15.6229 g mannitol, 5.0080 g taurine, 1.6338 g L-tryptophan, 7.9268 g D-glucose • H2O, 14.0256 g NaCl, 1.3717 g KC1, 4.5427 g Na2HPO4, 0.7199 g NaH2PO4, 0.2664 g CaCl2, 0.3253 g MgCl2• 6H2O, 0.2923 g L-glutamine, 3.6880 g glutathione, 0.1009 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water.

[0514] 15.71 mL of the surfactants solution, 100 mL of the buffer, 34.29 mL of the ultrapure water and 50 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 14 repetitions of pumping the total volume of emulsion through the microfluidizer chamber. After the sterilizng filtration, perfusion fluid_05 was obtained. Example 1.8 Obtaining perfusion fluid_06

[0515] Table 10. Quantitative composition of perfusion fluid_06.

[0516] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 561.63 g / L Perfluorodecyl bromide (PFDB) 307-43-7 17.37 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 40.80 g / L (1H, 1H, 2H, 2H- 313997-22-7 6.00 g / L Perfluorooctyl)phosphocholine

[0517] 9048-46-8

[0518] Bovine albumin 40.00 g / L 90604-29-8

[0519] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0520]

[0521] NaOH 1310-73-2 to determine the pH Perfusion fluid 06 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0522] The surfactants solution was prepared by dissolving 47.6000 g of Pluronic F-68 and 7.0000 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0523] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0524] The buffer was prepared by weighing 160.0000 g bovine serum albumin, 0.0697 g L-arginine, 0.5445 g allopurinol, 15.6229 g mannitol, 5.0080 g taurine, 1.6338 g L-tryptophan, 7.9268 g D-glucose • H2O, 14.0256 g NaCl, 1.3717 g KC1 , 4.5427 g Na2HPO4, 0.7199 g NaH2PO4, 0.2664 g CaCl2, 0.3253 g MgCl2• 6H2O, 0.2923 g L-glutamine, 3.6880 g glutathione, 0.1009 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water.

[0525] Atotal of 17.14 mL of the surfactants solution, 100 mL of the buffer, 22.86 mL of the ultrapure water and 60 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 14 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizng filtration, perfusion fluid_06 was obtained.

[0526] Example 1.9 Obtaining perfusion fluid_07

[0527] Table 11. Quantitative composition of perfusion fluid_07.

[0528] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 655.24 g / L Perfluorodecyl bromide (PFDB) 307-43-7 20.27 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 44.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 6.50 g / L Perfluorooctyl)phosphocholine

[0529] 9048-46-8

[0530] Bovine albumin 40.00 g / L 90604-29-8

[0531] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0532]

[0533] NaOH 1310-73-2 to determine the pH Perfusion fluid 07 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0534] The surfactants solution was prepared by dissolving 47.6000 g Pluronic F-68 and 7.0000 g (1H, 2H, 2H- (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0535] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0536] The buffer was prepared by weighing 160.0000 g bovine serum albumin, 0.0697 g L-arginine, 0.5445 g allopurinol, 15.6229 g mannitol, 5.0080 g taurine, 1.6338 g L-tryptophan, 7.9268 g D-glucose • H2O, 14.0256 g NaCl, 1.3717 g KC1, 4.5427 g Na2HPO4, 0.7199 g NaH2PO4, 0.2664 g CaCl2, 0.3253 g MgCl2• 6H2O, 0.2923 g L-glutamine, 3.6880 g glutathione, 0.1009 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water. 18.57 mL of the surfactants solution, 100 mL of the buffer, 11.43 mL of the ultrapure water and 70 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 14 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizng filtration, perfusion fluid_07 was obtained.

[0537] Example 1.10 Obtaining perfusion fluid_08

[0538] Table 12. Quantitative composition of perfusion fluid_08.

[0539] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 748.84 g / L Perfluorodecyl bromide (PFDB) 307-43-7 23.16 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 47.60 g / L (1H, 1H, 2H, 2H- 313997-22-7 7.00 g / L Perfluorooctyl)phosphocholine

[0540] 9048-46-8

[0541] Bovine albumin 40.00 g / L 90604-29-8

[0542] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 21.40 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0543]

[0544] NaOH 1310-73-2 to determine the pH Perfusion fluid_08 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0545] The surfactants solution was prepared by dissolving 47.6000 g Pluronic F-68 and 7.0000 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0546] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0547] The buffer was prepared by weighing 160.0000 g bovine serum albumin, 0.0697 g L-arginine, 0.5445 g allopurinol, 15.6229 g mannitol, 5.0080 g taurine, 1.6338 g L-tryptophan, 7.9268 g D-glucose • H2O, 14.0256 g NaCl, 1.3717 g KC1, 4.5427 g Na2HPO4, 0.7199 g NaH2PO4, 0.2664 g CaCh, 0.3253 g MgCh • 6H2O, 0.2923 g L-glutamine, 3.6880 g glutathione, 0.1009 g DL-P -hydroxybutyric acid sodium salt and dissolving the ingredients in ultrapure water.

[0548] 20 mL of the surfactants solution, 100 mL of the buffer and 80 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 14 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_08 was obtained.

[0549] Example Lil Obtaining perfusion fluid_09

[0550] Table 13. Quantitative composition of perfusion fluid_09.

[0551] Component Concentration lilhits .

[0552] Perfluorodecalin (PFD) 306-94-5 192.00 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0553] 9048-46-8

[0554] Bovine albumin 40.00

[0555] 90604-29-8 g / L

[0556] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0557] L-tryptophan 73-22-3 2.00 mM

[0558] D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM

[0559] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0560] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0561]

[0562] NaOH 1310-73-2 to determine the pH Perfusion fluid_09 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0563] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0564] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0565] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of perfluorodecalin were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_09 was obtained.

[0566] Example 1.12 Obtaining perfusion fluid lO

[0567] Table 14. Quantitative composition of perfusion fluid lO.

[0568] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 188.40 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0569] 9048-46-8

[0570] Bovine albumin 40.00 g / L 90604-29-8

[0571] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0572]

[0573] NaOH 1310-73-2 to determine the pH Perfusion fluid lO was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0574] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0575] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0576] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of perfluorotributylamine were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 9 repetitions of pumping the total volume of emulsion through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid lO was obtained.

[0577] Example 1.13 Obtaining perfusion fluid ll

[0578] Table 15. Quantitative composition of perfusion fluid ll.

[0579] Component Concentration Perfluoropentane (DDFP) 678-26-2 166.40 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0580] 9048-46-8

[0581] Bovine albumin 40.00

[0582] 90604-29-8 g / L

[0583] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0584] L-tryptophan 73-22-3 2.00 mM

[0585] D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM

[0586] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0587] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0588]

[0589] NaOH 1310-73-2 to determine the pH Perfusion fluid ll was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0590] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0591] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0592] A total of 46.67 mL of the surfactant solution, 133.33 mL of the buffer and 20 mL of perfluoropentane were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid l 1 was obtained.

[0593] Example 1.14 Obtaining perfusion fluid_12

[0594] Table 16. quantitative composition of perfusion fluid_12.

[0595] Component Concentration Units Perfluorohexane 355-42-0 169.10 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0596] 9048-46-8

[0597] Bovine albumin 40.00 g / L 90604-29-8

[0598] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0599]

[0600] NaOH 1310-73-2 to determine the pH Perfusion fluid_12 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0601] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-perfluorooctyl)phosphocholine in ultrapure water.

[0602] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0603] A total of 46.67 mL of the surfactant solution, 133.33 mL of the buffer and 20 mL of perfluorohexane were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 9 repetitions of pumping the total volume of emulsion through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_12 was obtained.

[0604] Example 1.15 Obtaining perfusion fluid_13

[0605] Table 17. Quantitative composition of perfusion fluid_13.

[0606] Component Concentration Units Perfluoroheptane 335-57-9 174.50 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0607] 9048-46-8

[0608] Bovine albumin 40.00 g / L 90604-29-8

[0609] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0610]

[0611] NaOH 1310-73-2 to determine the pH Perfusion fluid_13 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0612] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-perfluorooctyl)phosphocholine in ultrapure water.

[0613] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCh, 0.2440 g MgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0614] A total of 46.67 mL of the surfactant solution, 133.33 mL of the buffer and 20 mL of perfluoroheptane were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid l 3 was obtained.

[0615] Example 1.16 Obtaining perfusion fluid_14

[0616] Table 18. Quantitative composition of perfusion fluid_14.

[0617] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0618] Hydroxy ethylated starch 130 / 0.4 9005-27-0 60.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0619]

[0620] NaOH 1310-73-2 to determine the pH Perfusion fluid_14 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0621] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0622] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide. The buffer was prepared by weighing 22.5000 g hydroxy ethylated starch, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 gNa2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium and dissolving the ingredients in ultrapure water.

[0623] A total of 46.67 mL of the surfactants solution, 133.33 mL of buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_14 was obtained.

[0624] Example 1.17 Obtaining perfusion fluid_15

[0625] Table 19. Quantitative composition of perfusion fluid l 5.

[0626] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0627] Dextran 70 kDa 9004-54-0 50.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0628]

[0629] NaOH 1310-73-2 to determine the pH Perfusion fluid_15 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0630] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water. The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0631] The buffer was prepared by weighing 18.7500 g Dextran 70 kDa, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium and dissolving the ingredients in ultrapure water.

[0632] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_15 was obtained.

[0633] Example 1.18 Obtaining perfusion fluid_16

[0634] Table 20. Quantitative composition of perfusion fluid_16.

[0635] Component Concentration Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0636] Dextran 40 kDa 9004-54-0 40.00 g / L

[0637] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0638] L-tryptophan 73-22-3 2.00 mM

[0639] D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM

[0640] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0641] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0642]

[0643] NaOH 1310-73-2 to determine the pH Perfusion fluid_16 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3. The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0644] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0645] The buffer was prepared by weighing 15.0000 g Dextran 40 kDa, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium and dissolving the ingredients in ultrapure water.

[0646] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbon mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid l 6 was obtained.

[0647] Example 1.19 Obtaining perfusion fluid_17

[0648] Table 21. Quantitative composition of perfusion fluid l 7.

[0649] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L

[0650] (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0651] Poly(ethylene glycol) 35 kDa 25322-68-3 20.00 g / L

[0652] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0653] L-tryptophan 73-22-3 2.00 mM

[0654] D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM

[0655] KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM

[0656] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0657] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0658]

[0659] NaOH 1310-73-2 to determine the pH Perfusion fluid_17 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0660] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0661] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0662] The buffer was prepared by weighing 7.5000 g Poly(ethylene glycol) 35 kDa, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium and dissolving the ingredients in ultrapure water. A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_17 was obtained.

[0663] Example 1.20 Obtaining perfusion fluid_18

[0664] Table 22. Quantitative composition of perfusion fluid l 8.

[0665] Component Concentration OiOiiiii Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0666] Poly(ethylene glycol) 20 kDa 25322-68-3 20.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM

[0667]

[0668] Glutathione 70-18-8 3.00 mM Component Concentration Units DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0669]

[0670] NaOH 1310-73-2 to determine the pH Perfusion fluid_18 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0671] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0672] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0673] The buffer was prepared by weighing 7.5000 g Poly(ethylene glycol) 20 kDa, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium and dissolving the ingredients in ultrapure water. A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid l 8 was obtained.

[0674] Example 1.21 Obtaining perfusion fluid_19

[0675] Table 23. Quantitative composition of perfusion fluid l 9.

[0676] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0677] Succinylated gelatine 68915-24-2 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM

[0678]

[0679] CaCl210043-52-4 0.60 mM Component Concentration Units MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0680]

[0681] NaOH 1310-73-2 to determine the pH Perfusion fluid_19 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0682] The surfactants solution was prepared by dissolving 116.6000 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0683] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0684] The buffer was prepared by weighing 15.0000 g succinylated gelatine, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium and dissolving the ingredients in ultrapure water.

[0685] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid l 9 was obtained.

[0686] Example 1.22 Obtaining perfusion fluid_20

[0687] Table 24. quantitative composition of perfusion fluid_20.

[0688] Component Concentration Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D- 118680-70-9 4.00 g / L Maltopyranoside

[0689] 9048-46-8

[0690] Bovine albumin 40.00 g / L 90604-29-8

[0691] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0692] L-tryptophan 73-22-3 2.00 mM

[0693] D-glucose • H2O 14431-43-7 10.00 mM

[0694]

[0695] NaCl 7647-14-5 60.00 mM Component Concentration Units KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM

[0696] L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM

[0697] DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0698]

[0699] NaOH 1310-73-2 to determine the pH Perfusion fluid_20 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0700] The surfactants solution was prepared by dissolving 11.6600 g of Pluronic F-68 and 1.7140 g of (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside in ultrapure water.

[0701] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0702] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0703] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized, conducting 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_20 was obtained.

[0704] Example 1.23 Obtaining perfusion fluid ll

[0705] Table 25. Quantitative composition of perfusion fluid_21.

[0706] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L l,2-Dioleoyl-sn-glycero-3-phospho-L-serine

[0707] 70614-14-1 4.00 g / L sodium salt (PS 18:1 / 18:1 DOPS-Na)

[0708] 9048-46-8

[0709] Bovine albumin 40.00 g / L 90604-29-8

[0710] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM

[0711]

[0712] Mannitol 69-65-8 50.00 mM Component Concentration Units Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0713]

[0714] NaOH 1310-73-2 to determine the pH Perfusion fluid_21 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0715] The surfactants solution was prepared by dissolving 11.6600 g of Pluronic F-68 and 1.7140 g of l,2-Dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (PS 18:1 / 18:1 DOPS-Na) in ultrapure water.

[0716] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0717] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0718] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_21 was obtained.

[0719] Example 1.24 Obtaining perfusion fluid_22

[0720] Table 26. Quantitative composition of perfusion fluid_22.

[0721] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L sodium salt of l,2-dipalmitoyl-sn-glycero-3- 169051-60-9 4.00 g / L

[0722]

[0723] phosphate (PA 16:0 / 16:0 DPPA-Na) Component Concentration Units 9048-46-8

[0724] Bovine albumin 40.00 g / L 90604-29-8

[0725] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0726]

[0727] NaOH 1310-73-2 to determine the pH

[0728] Perfusion fluid_22 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0729] The surfactants solution was prepared by dissolving 11.6600 g of Pluronik F-68 and 1.7140 g of l,2-dipalmitoyl-sn-glycero-3-phosphate sodium (PA 16:0 / 16:0 DPPA-Na) in ultrapure water. The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0730] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0731] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 13 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_22 was obtained. Example 1.25 Obtaining perfusion fluid_23

[0732] Table 27. Quantitative composition of perfusion fluid_23.

[0733] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L l,2-dimyristoyl-sn-glycero-3-phospho-rac- glycerol sodium salt (PG 14:0 / 14:0 DMPG- 67232-80-8 4.00 g / L Na)

[0734] 9048-46-8

[0735] Bovine albumin 40.00 g / L 90604-29-8

[0736] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10,00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0737]

[0738] NaOH 1310-73-2 to determine the pH Perfusion fluid_23 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0739] The surfactants solution was prepared by dissolving 11.6600 g of Pluronic F-68 and 1.7140 g of l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt (PG 14:0 / 14:0 DMPG-Na) in ultrapure water.

[0740] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0741] The Buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 gCaCl2, 0.2440 gMgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium and dissolving the components in ultrapure water. A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_23 was obtained.

[0742] Example 1.26 Obtaining perfusion fluid_24

[0743] Table 28. quantitative composition of perfusion fluid_24.

[0744] Component Concentration liiiillli Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L N-(carbonyl-methoxypolyethyleneglycol- 2000)- 1 ,2-distearoyl-sn-glycero-3 - 147867-65-0 4.00 g / L phosphoethanolamine sodium salt (PE

[0745] 18:0 / 18:0-PEG 2000)

[0746] 9048-46-8

[0747] Bovine albumin 40.00

[0748] 90604-29-8 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0749]

[0750] NaOH 1310-73-2 to determine the pH Perfusion fluid_24 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0751] The surfactants solution was prepared by dissolving 11.6600 g of Pluronic F-68 and 1.7140 g of N-(carbonyl-methoxypolyethyleneglycol-2000)-l,2-distearoyl-sn-glycero-3-phospho-ethanolamine sodium salt (PE 18:0 / 18 :0-PEG 2000) in ultrapure water.

[0752] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0753] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCh • H2O, 0.2440 g MgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium and dissolving the components in ultrapure water. A total of 46.67 mL of the surfactanst solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_24 was obtained.

[0754] Example 1.27 Obtaining perfusion fluid_25

[0755] Table 29. quantitative composition of perfusion fluid_25.

[0756] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0757] 9048-46-8

[0758] Bovine albumin 67.20 g / L 90604-29-8

[0759] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCh 10043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0760]

[0761] NaOH 1310-73-2 to determine the pH Perfusion fluid_25 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0762] The surfactants solution was prepared by dissolving 11.6600 g of bovine serum albumin and 1.7140 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0763] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide. The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0764] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_25 was obtained.

[0765] Example 1.28 Obtaining perfusion fluid_26

[0766] Table 30. Quantitative composition of perfusion fluid_26.

[0767] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0768] 9048-46-8

[0769] Bovine albumin 40.00 g / L 90604-29-8

[0770] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0771]

[0772] NaOH 1310-73-2 to determine the pH Perfusion fluid_26 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0773] A surfactant solution was prepared by dissolving 1.7140 g of (1H, 1H, 2H, 2H- Perfluorooctyl)phosphocholine in ultrapure water. The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0774] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0775] A total of 46.67 mL of the surfactant solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_26 was obtained.

[0776] Example 1.29 Obtaining perfusion fluid_27

[0777] Table 31. Quantitative composition of perfusion fluid_27.

[0778] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-108 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0779] 9048-46-8

[0780] Bovine albumin 40.00 g / L 90604-29-8

[0781] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0782]

[0783] NaOH 1310-73-2 to determine the pH Perfusion fluid_27 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3. The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-108 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0784] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0785] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0786] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbon mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_27 was obtained.

[0787] Example 1.30 Obtaining perfusion fluid_28

[0788] Table 32. quantitative composition of perfusion fluid_28.

[0789] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-127 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0790] 9048-46-8

[0791] Bovine albumin 40.00 g / L 90604-29-8

[0792] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0793]

[0794] NaOH 1310-73-2 to determine the pH Perfusion fluid_28 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0795] The surfactants solution was prepared by dissolving 11.6600 g of Pluronic F-68 and 17.1400 g of (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0796] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0797] The buffer was prepared by weighing 120.0000 g bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium and dissolving the components in ultrapure water.

[0798] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by conducting 14 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_28 was obtained.

[0799] Example 1.31 Obtaining perfusion fluid_29

[0800] Table 33. Quantitative composition of perfusion fluid_29.

[0801] Component Concentration OiOiiiii Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0802] 9048-46-8

[0803] Bovine albumin 40.00 g / L 90604-29-8

[0804] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM L-isoleucine 73-32-5 1.52 mM L-leucine 61-90-5 3.05 mM L-lysine 56-87-1 2.05 mM L-methionine 63-68-3 1.34 mM L-threonine 72-19-5 1.68 mM L-valine 72-18-4 2.56 mM L-histidine 71-00-1 0.64 mM L-alanine 56-41-7 5.61 mM

[0805]

[0806] L-glycine 56-40-6 6.66 mM Component Concentration Units L-aspartic acid 56-84-8 1.88 mM L-proline 147-85-3 2.17 mM L-serine 56-45-1 0.95 mM L-tyrosine 60-18-4 0.06 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0807]

[0808] NaOH 1310-73-2 to determine the pH Perfusion fluid_29 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0809] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0810] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0811] The buffer was prepared by weighing 15.0000 g bovine serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.0500 g L-isoleucine, 0.1000 g L-leucine, 0.0750 g L-lysine, 0.0500 g L-methionine, 0.0500 g L-threonine, 0.0750 g L-valine, 0.0250 g L-histidine, 0.1250 g L-alanine, 0.1250 g L-glycine, 0.0625 g L-aspartic acid, 0.0625 g L-proline, 0.0250 g L-serine, 0.0025 g L-tyrosine, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 gNa2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0812] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbons mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running nine repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid_29 was obtained. Example 1.32 Obtaining perfusion fluid_30

[0813] Table 34. quantitative composition of perfusion fluid_30.

[0814] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0815] 9048-46-8

[0816] Bovine albumin 40.00 g / L 90604-29-8

[0817] L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM L-isoleucine 73-32-5 4.19 mM L-leucine 61-90-5 6.86 mM L-lysine 56-87-1 4.79 mM L-methionine 63-68-3 3.35 mM L-threonine 72-19-5 3.78 mM L-valine 72-18-4 5.55 mM L-histidine 71-00-1 2.26 mM L-alanine 56-41-7 12.35 mM L-glycine 56-40-6 17.32 mM L-aspartic acid 56-84-8 4.51 mM L-proline 147-85-3 5.21 mM L-serine 56-45-1 2.38 mM L-tyrosine 60-18-4 0.28 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCh • 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0818]

[0819] NaOH 1310-73-2 to determine the pH Perfusion fluid_30 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0820] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water. The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0821] The buffer was prepared by weighing 15.0000 g bovine serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.1375 g L-isoleucine, 0.2250 g L-leucine, 0.1750 g L-lysine, 0.1250 g L-methionine, 0.1125 g L-threonine, 0.1625 g L-valine, 0.0875 g L-histidine, 0.2750 g L-alanine, 0.3250 g L-glycine, 0.1500 g L-aspartic acid, 0.1500 g L-proline, 0.0625 g L-serine, 0.0125 g L-tyrosine, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0822] A total of 46.67 mL of the surfactants solution, 133.33 mL of the buffer and 20 mL of the perfluorocarbon mixture were combined to obtain 200 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 30 was obtained.

[0823] Example 1.33 Obtaining perfusion fluid_31

[0824] Table 35. quantitative composition of perfusion fluid_31.

[0825] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0826] Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0827]

[0828] NaOH 1310-73-2 to determine the pH Perfusion fluid_31 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0829] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0830] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0831] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0832] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid _31 was obtained.

[0833] Example 1.34 Obtaining perfusion fluid_32

[0834] Table 36. quantitative composition of perfusion fluid_32.

[0835] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0836] Recombinant human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM

[0837]

[0838] Glutathione 70-18-8 3.00 mM Component Concentration Units DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0839]

[0840] NaOH 1310-73-2 to determine the pH Perfusion fluid_32 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0841] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0842] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0843] The buffer was prepared by weighing 15.0000 g recombinant human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0844] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 32 was obtained.

[0845] Example 1.35 Obtaining perfusion fluid_33

[0846] Table 37. quantitative composition of perfusion fluid_33.

[0847] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 94.15 g / L Perfluorodecalin (PFD) 306-94-5 97.3 g / L Pluronic F-68 (Kolliphor P188) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0848] Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO4 7558-79-4 8.00 mM

[0849]

[0850] NaH2PO47558-80-7 1.50 mM Component Concentration Units CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0851]

[0852] NaOH 1310-73-2 to determine the pH Perfusion fluid_33 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0853] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0854] The perfluorinated phase was prepared by mixing 9 mL of PFTBA with 9 mL of PFD.

[0855] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0856] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 33 was obtained.

[0857] Example 1.36 Obtaining perfusion fluid_34

[0858] Table 38. quantitative composition of perfusion fluid_34.

[0859] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 37.66 g / L Perfluorodecalin (PFD) 306-94-5 155.68 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0860] Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM

[0861]

[0862] KC1 7447-40-7 4.60 mM Component Concentration Units Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0863]

[0864] NaOH 1310-73-2 to determine the pH Perfusion fluid_34 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0865] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0866] The perfluorinated phase was prepared by mixing 3.6 mL of PFTBA with 14.4 mL of PFD. The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0867] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 34 was obtained.

[0868] Example 1.37 Obtaining perfusion fluid_35

[0869] Table 39. quantitative composition of perfusion fluid_35.

[0870] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 150.64 g / L Perfluorodecalin (PFD) 306-94-5 38.92 g / L Pluronic F-68 (Kolliphor P188) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0871] Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM

[0872]

[0873] D-glucose • H2O 14431-43-7 10.00 mM Component Concentration Units NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0874]

[0875] NaOH 1310-73-2 to determine the pH Perfusion fluid_35 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0876] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0877] The perfluorinated phase was prepared by mixing 14.4 mL of PFTBA with 3.6 mL of PFD. The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0878] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 35 was obtained.

[0879] Example 1.38 Obtaining perfusion fluid_36

[0880] Table 40. quantitative composition of perfusion fluid_36.

[0881] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 188.3 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0882] Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM

[0883]

[0884] D-glucose • H2O 14431-43-7 10.00 mM Component Concentration Units NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0885]

[0886] NaOH 1310-73-2 to determine the pH Perfusion fluid_36 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0887] The surfactant solution was prepared by dissolving 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0888] The perfluorinated phase was prepared by measuring out 18 mL of PFTBA.

[0889] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0890] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 36 was obtained.

[0891] Example 1.39 Obtaining perfusion fluid_37

[0892] Table 41. quantitative composition of perfusion fluid_37.

[0893] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 188.3 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0894] Human serum albumin 70024-90-7 47.20 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM

[0895]

[0896] D-glucose • H2O 14431-43-7 10.00 mM Component Concentration Units NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0897]

[0898] NaOH 1310-73-2 to determine the pH Perfusion fluid_37 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0899] The surfactant solution was prepared by dissolving 116.6000 g Human serum albumin and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0900] The perfluorinated phase was prepared by measuring out 18 mL of PFTBA.

[0901] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 g NaCl, 0.1286 g KC1, 0.4259 g Na2HPO4, 0.0675 g NaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0902] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 37 was obtained.

[0903] Example 1.40 Obtaining perfusion fluid_38

[0904] Table 42. quantitative composition of perfusion fluid_38.

[0905] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor P188) 9003-11-6 27.20 g / L Sphingomyelin derived from egg yolk 85187-10-6 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM

[0906]

[0907] L-tryptophan 73-22-3 2.00 mM Component Concentration Units D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0908]

[0909] NaOH 1310-73-2 to determine the pH Perfusion fluid_38 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0910] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g Sphingomyelin derived from egg yolk in ultrapure water.

[0911] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0912] The buffer was prepared by weighing 120.0000 g Bovine serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 38 was obtained.

[0913] Example 1.41 Obtaining perfusion fluid_39

[0914] Table 43. quantitative composition of perfusion fluid_39.

[0915] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 188.3 g / L Pluronik F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 1H, 2H, 2H- 313997-22-7 4.00 g / L Perfluorooctyl)phosphocholine

[0916] Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM

[0917]

[0918] Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0919]

[0920] NaOH 1310-73-2 to determine the pH Perfusion fluid_39 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0921] The surfactants solution was prepared by dissolving 116.6000 g Pluronik F-68 and 17.1400 g (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0922] The perfluorinated phase was prepared by measuring out 18 mL of PFTBA.

[0923] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0065 g L-arginine, 0.0510 g allopurinol, 3.4157 g mannitol, 0.4695 g taurine, 0.1532 g L-tryptophan, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 g Na2HPO4, 0.0675 gNaH2PO4, 0.0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.0274 g L-glutamine, 0.3457 g glutathione, 0.0095 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0924] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 39 was obtained.

[0925] Example 1.42 Obtaining perfusion fluid_40

[0926] Table 44. quantitative composition of perfusion fluid_40.

[0927] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 149.7 g / L Perfluorodecyl bromide (PFDB) 307-43-7 4.63 g / L Perfluorotributyl amine (PFTBA) 311-89-7 37.66 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM

[0928]

[0929] Allopurinol 315-30-0 1.00 mM Component Concentration Units Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0930]

[0931] NaOH 1310-73-2 to determine the pH Perfusion fluid_40 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0932] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0933] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide. Next, 14.4 mL of PFOB and PFDB were mixed with 3.6 mL ofPFTBA.

[0934] The buffer was prepared by weighing 120.0000 g Human serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 40 was obtained.

[0935] Example 1.43 Obtaining perfusion fluid_41

[0936] Table 45. quantitative composition of perfusion fluid_41.

[0937] Component Concentration Units Perfluorooctyl bromide (PFOB) 423-55-2 93.61 g / L Perfluorodecyl bromide (PFDB) 307-43-7 2.90 g / L Perfluorotributyl amine (PFTBA) 311-89-7 94.15 g / L

[0938]

[0939] Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L Component Concentration Units (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0940]

[0941] NaOH 1310-73-2 to determine the pH Perfusion fluid_41 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0942] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0943] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide. Next, 9 mL of PFOB and PFDB were mixed with 9 mL of PFTBA.

[0944] The buffer was prepared by weighing 120.0000 g Human serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 gCaCl2, 0.2440 gMgCh • 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P-hydroxybutyric acid sodium salt and dissolving the components in ultrapure water.

[0945] A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 41 was obtained. Example 1.44 Obtaining perfusion fluid_42

[0946] Table 46. quantitative composition of perfusion fluid_42.

[0947] Component Concentration Units Perfluorooctyl bromide (PFOB) 423-55-2 37.44 g / L Perfluorodecyl bromide (PFDB) 307-43-7 1.16 g / L Perfluorotributyl amine (PFTBA) 311-89-7 150.64 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0948]

[0949] NaOH 1310-73-2 to determine the pH Perfusion fluid_42 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0950] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0951] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide. Next, 3.6 mL of PFOB and PFDB were mixed with 14.4 mL ofPFTBA.

[0952] The buffer was prepared by weighing 120.0000 g Human serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 42 was obtained.

[0953] Example 1.45 Obtaining perfusion fluid_43

[0954] Table 47. quantitative composition of perfusion fluid_43.

[0955] Component Concentration Units Perfluorooctyl bromide (PFOB) 423-55-2 154.40 g / L Perfluorotributyl amine (PFTBA) 311-89-7 37.66 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0956]

[0957] NaOH 1310-73-2 to determine the pH Perfusion fluid_43 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0958] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0959] The perfluorinated phase was prepared by mixing 14.4 mL of PFOB with 3.6 mL of PFTBA. The buffer was prepared by weighing 120.0000 g Human serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0.1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 43 was obtained.

[0960] Example 1.46 Obtaining perfusion fluid_44

[0961] Table 48. quantitative composition of perfusion fluid_44.

[0962] Component Concentration Units Perfluorooctyl bromide (PFOB) 423-55-2 96.50 g / L Perfluorotributyl amine (PFTBA) 311-89-7 94.15 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0963]

[0964] NaOH 1310-73-2 to determine the pH Perfusion fluid_44 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0965] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0966] The perfluorinated phase was prepared by mixing 9 mL of PFOB with 9 mL of PFTBA.

[0967] The buffer was prepared by weighing 120.0000 g Human serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0,1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 44 was obtained.

[0968] Example 1.47 Obtaining perfusion fluid_45

[0969] Table 49. quantitative composition of perfusion fluid_45.

[0970] Component Concentration Units Perfluorooctyl bromide (PFOB) 423-55-2 38.60 g / L Perfluorotributyl amine (PFTBA) 311-89-7 150.64 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L L-arginine 74-79-3 0.10 mM Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM Taurine 107-35-7 10.00 mM L-tryptophan 73-22-3 2.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM L-glutamine 56-85-9 0.50 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0971]

[0972] NaOH 1310-73-2 to determine the pH Perfusion fluid_45 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0973] The surfactants solution was prepared by dissolving 11.6600 g Pluronic F-68 and 1.7140 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0974] The perfluorinated phase was prepared by mixing 3.6 mL of PFOB with 14.4 mL of PFTBA. The buffer was prepared by weighing 120.0000 g Human serum albumin, 0.0523 g L-arginine, 0.4083 g allopurinol, 27.3255 g mannitol, 3.7560 g taurine, 1.2254 g L-tryptophan, 5.9451 g D-glucose • H2O, 10.5192 g NaCl, 1.0288 g KC1, 3.4070 g Na2HPO4, 0.5399 g NaH2PO4, 0,1998 g CaCl2, 0.2440 g MgCl2• 6H2O, 0.2192 g L-glutamine, 2.7660 g glutathione, 0.0757 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 45 was obtained.

[0975] Example 1.48 Obtaining perfusion fluid_46

[0976] Table 50. quantitative composition of perfusion fluid_46.

[0977] Component Concentration Units Perfluorotributyl amine (PFTBA) 311-89-7 188.30 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 40.00 g / L Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0978]

[0979] NaOH 1310-73-2 to determine the pH Perfusion fluid_46 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0980] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0981] The perfluorinated phase was prepared by measuring out 18 mL of PFTBA.

[0982] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0510 g allopurinol, 3.4157g mannitol, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 gKCl, 0.4259 gNa2HPO4, 0.0675 g NaH2PO4, 0,0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.3457 g glutathione, 0.0095 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 46 was obtained. Example 1.49 Obtaining perfusion fluid_47

[0983] Table 51. quantitative composition of perfusion fluid_47.

[0984] Component Concentration Units Perfluorooctyl bromide (PF OB) 423-55-2 187.21 g / L Perfluorodecyl bromide (PFDB) 307-43-7 5.79 g / L Pluronic F-68 (Kolliphor Pl 88) 9003-11-6 27.20 g / L (1H, 2H, 2H-Perfluorooctyl)phosphocholine 313997-22-7 4.00 g / L Human serum albumin 70024-90-7 67.20 g / L Allopurinol 315-30-0 1.00 mM Mannitol 69-65-8 50.00 mM D-glucose • H2O 14431-43-7 10.00 mM NaCl 7647-14-5 60.00 mM KC1 7447-40-7 4.60 mM Na2HPO47558-79-4 8.00 mM NaH2PO47558-80-7 1.50 mM CaCl210043-52-4 0.60 mM MgCl2• 6H2O 7791-18-6 0.40 mM Glutathione 70-18-8 3.00 mM DL-P-hydroxybutyric acid sodium salt 150-83-4 0.20 mM

[0985]

[0986] NaOH 1310-73-2 to determine the pH Perfusion fluid_47 was obtained according to the method for obtaining perfusion fluid Ol, described in Example 1.3.

[0987] The surfactants solution was prepared by dissolving 116.6000 g Pluronic F-68 and 17.1400 g (1H, 2H, 2H-Perfluorooctyl)phosphocholine in ultrapure water.

[0988] The perfluorinated phase was prepared by dissolving 30.9278 g of perfluorodecyl bromide in 1000 g of perfluorooctyl bromide.

[0989] The buffer was prepared by weighing 15.0000 g Human serum albumin, 0.0510 g allopurinol, 3.4157 g mannitol, 0.7431 g D-glucose • H2O, 1.3149 gNaCl, 0.1286 g KC1, 0.4259 gNa2HPO4, 0.0675 g NaH2PO4, 0,0250 g CaCl2, 0.0305 g MgCl2• 6H2O, 0.3457 g glutathione, 0.0095 g DL-P -hydroxybutyric acid sodium salt and dissolving the components in ultrapure water. A total of 42 mL of the surfactants solution, 120 mL of the buffer and 18 mL of the perfluorocarbon mixture were combined to obtain 180 mL of the primary perfusion fluid, which was homogenized and microfluidized by running 9 repetitions of pumping the total emulsion volume through the microfluidizer chamber. After the sterilizing filtration, perfusion fluid 47 was obtained. Example II:

[0990] Methods for quality control of perfusion fluid

[0991] Example ILL Determination of particle size distribution of perfusion fluid

[0992] Emulsion particle size distributions were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments. Ltd . Worcestershire. UK). A series of dilutions were performed for each sample: 5-fold and 50-fold with ultrapure water (MilliQ). Measurements were made at 25°C and at a scattering angle of 173°. Results were presented by two parameters: mean particle size and polydispersity index (Pdl). The mean particle size (reported as mean diameter in nanometres) is a value calculated by the instrument from the particle intensity signal, according to the ISO standard provided by the manufacturer (ISO 13321 :1996E and 22412). The polydispersity index is a dimensionless value expressing the width of the particle size distribution of the emulsion, i.e. the homogeneity of the analysed sample, calculated according to ISO 13321 :1996E and 22412. All measurements were carried out in triplicate. Values were given as the mean value of the three measurements and the standard deviation was calculated.

[0993] Example II.2. Determination of the zeta potential of particles in a perfusion fluid Zeta potential measurements were performed using Malvern's Zetasizer Nano ZS and DTS 1070 zeta potential cuvettes. The resulting o / w emulsion was diluted 10-fold, with MilliQ ultrapure water. A minimum of 3 measurements were made, taking approximately 2 ml of the diluted emulsion each time. The result was given by averaging all the zeta potential values obtained, which were averaged, and the standard deviation was calculated, in units: mV.

[0994] Example II.3. Determination of osmotic pressure of perfusion fluid

[0995] Osmolality measurements were performed using Osmometer 800CLG, Trident Med and OSMO-KRIO measuring vessels with crystallisation germs. Measurements were performed in a minimum of 3 replicates, taking 100 pl of the obtained emulsion each time. The result was given by averaging all the osmolality values obtained and calculating the standard deviation, in units: mOsm / kgJLO.

[0996] Example II.4. Determination of oncotic pressure of perfusion fluid

[0997] Oncotic pressure measurements were performed using a BMT 923 Oncometer with a cellulose triacetate membrane with a cut-off mass of 20,000 daltons, BMT MESSTECHNIK GMBH, Germany. Measurements were performed in a minimum of 3 replicates, taking 100 pl of the fluid obtained each time. The result was given by averaging all osmolality values obtained and calculating the standard deviation, in units: mmHg. Example II.5. Determination of the concentration of selected ions in perfusion fluid Concentrations of sodium, potassium, calcium and chloride ions were measured using an EC90 ion analyser with interchangeable iCa measuring cartridges2+, Erba Polska Sp. z o.o.. A one-and two-point calibration of the instrument was performed prior to the measurements. Measurements were performed in a minimum of 3 repetitions, with the device taking 35 pl of the fluid obtained each time. The result was given by averaging all the ion concentration values obtained and calculating the standard deviation, in units: mmol / L.

[0998] Example II.6. Determination of pH of perfusion fluid

[0999] pH measurements were made using a pHmeter, Mettler Toledo, Italy, and calibration solutions of pH: 2.00, 4.00, 7.00, 9.21, 11.00. The instrument was calibrated before measurement. Measurements were performed in a minimum of 3 repetitions, taking 3 mL of the fluid obtained each time. The result was given by averaging all the pH values obtained and calculating the standard deviation, in dimensionless units.

[1000] Example II.7. Assessment of cytotoxicity of perfusion fluid

[1001] Tests on extracts from the obtained perfusion fluid, were performed in vitro on mouse fibroblast cell cultures (L929), using the Cell Proliferation Kit II (XTT) cytotoxicity assay. The test was performed according to ISO 10993-5 (standard describing test methods for the cytotoxicity evaluation of medical devices).

[1002] The first step of the work was to prepare mouse fibroblast cultures. In a 96-well plate, 104cells per well were seeded and 100 pL of culture medium (DMEM with 10 % (v / v) admixture of fetal bovine serum (FBS) and 1 % (v / v) addition of a mixture of penicillin and streptomycin) was added. The cells prepared in this way were kept in an incubator for 24 h. After 24 h under sterile conditions, 50 % (v / v) solutions of the resulting fluids in culture medium were prepared and then transferred to the cell plates, previously removing the old culture medium from the wells. In addition to the test materials, a negative control for cytotoxicity (NC) was prepared in the form of culture medium incubated under the same conditions as the perfusion fluid samples tested, and a positive control for cytotoxicity (PC) was prepared in the form of culture medium with 0.2 % (v / v) Triton X-100. After 24 h incubation with the cells, the solutions were removed from the cells and each well was washed three times with phosphate-buff ered saline (PBS). The XTT assay was then performed according to the manufacturer's protocol. The results obtained were expressed as a percentage relative to the value of the corresponding negative control. Example II.8. Determination of viscosity of perfusion fluid

[1003] Viscosity measurements were performed using a Brookfield DV-III Ultra rheometer with a CPE-40 conical spindle (shear rate range: 0-1500 1 / s) and equipped with a thermostated sheath, BROOKFIELD ENGINEERING LABORATORIES, INC, USA. Measurements were performed in a minimum of 3 replicates, taking 500 pl of the fluid obtained each time. Samples were thermostated at 20 - 23 °C. The result was given by averaging all the viscosity values obtained and calculating the standard deviation, in units of: mPa • s

[1004] Example II.9. Assessment of stability / sustainability of perfusion fluid

[1005] Fluid stability / persistence was assessed by measuring emulsion particle size distribution by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments. Ltd. Worcestershire. UK), according to Example 1. Fluid samples were stored under reduced temperature conditions of 4 - 6° C.

[1006] Table 35. Quality control results of organ perfusion fluids, particle size distribution, zeta potential, viscosity and pH.

[1007] Name of Average

[1008] Average Pdl Zeta

[1009] the diameter Viscosity diameter Pdl [-] after 7 potentia

[1010] perfusion after 7 days [mPa-s] pH [-]

[1011] [nm] days [-] 1 [mV]

[1012] fluid [nm]

[1013] Perfusion 105.31 ± 0.202 ± 0.083 ± -2.57 ± 2.264 ± 7.24 ±

[1014] 178.70 ± 1.61

[1015] fluid 01 1.31 0.005 0.016 0.86 0.019 0.01 Perfusion 121.29 ± 0.121 ± 0.049 ± -1.96 ± 2.975 ± 7.23 ±

[1016] 200.60 ± 1.69

[1017] fluid 02 1.53 0.008 0.009 0.43 0.033 0.00 Perfusion 116.73 ± 0.111 ± 0.027 ± -2.33 ± 4.201 ± 7.26 ±

[1018] 210.28 ± 2.54

[1019] fluid 03 1.42 0.013 0.014 0.86 0.044 0.01 Perfusion 137.42 ± 0.069 ± 0.038 ± -2.08 ± 6.754 ± 7.26 ±

[1020] 218.74 ± 2.05

[1021] fluid 04 1.39 0.016 0.019 0.34 0.037 0.00 Perfusion 124.79 ± 0.078 ± 0.042 ± -3.28 ± 8.470 ± 7.24 ±

[1022] 215.94 ± 2.47

[1023] fluid 05 1.57 0.017 0.015 0.34 0.040 0.01 Perfusion 116.16 ± 0.062 ± 0.037 ± -2.67 ± 19.000 ± 7.24 ±

[1024] 214.28 ± 2.15

[1025] fluid 06 1.12 0.011 0.019 0.45 0.000 0.01 Perfusion 135.77 ± 0.030 ± 0.042 ± -1.70 ± 19.000- 7.27 ±

[1026] 220.16 ± 2.03

[1027] fluid 07 0.96 0.008 0.019 0.43 30.000 0.00 Perfusion 131.71 ± 0.032 ± 0.032 ± -2.70 ± 30.000- 7.29 ±

[1028] 222.36 ± 2.59

[1029] fluid 08 1.67 0.011 0.016 0.51 40.000 0.01 Perfusion 136.39 ± 0.166 ± 0.081 ± 3.03 ± 3.072 ± 7.24 ±

[1030] 239.14 ± 3.53

[1031] fluid 09 1.13 0.014 0.017 0.91 0.037 0.02 Perfusion 0.199 ± 0.197 ± 3.69 ± 2.714 ± 7.25 ±

[1032] 95.72 ± 0.71 96.23 ± 1.33

[1033] fluid 10 0.009 0.004 0.39 0.020 0.00 Perfusion 388.14 ± 0.138 ± 526.54 ± 0.081 ± -4.31 ± 2.700- 7.27 ± fluid 11 7.08 0.020 12.73 0.028 1.36 3.000 0.01 Perfusion 261.42 ± 0.053 ± 457.32 ± 0.117 ± -2.06 ± 2.511 ± 7.20 ± fluid 12 6.69 0.039 11.50 0.032 0.27 0.163 0.01 Perfusion 184.79 ± 0.035 ± 0.073 ± -2.31 ± 2.582 ± 7.22 ±

[1034] 277.92 ± 4.03

[1035] fluid 13 1.50 0.020 0.035 0.75 0.005 0.01 Perfusion 0.175 ± 0.062 ± -1.65 ± 8.170 ± 7.20 ±

[1036] 94.84 ± 1.01 219.87 ± 3.33

[1037] fluid 14 0.010 0.017 0.23 0.072 0.00 Perfusion 112.29 ± 0.098 ± 0.032 ± -2.76 ± 8.453 ± 7.29 ±

[1038] 216.37 ± 2.82

[1039] fluid 15 1.02 0.009 0.019 0.14 0.090 0.01 Perfusion 124.50 ± 0.066 ± 0.064 ± -2.31 ± 6.766 ± 7.21 ±

[1040] 220.90 ± 1.76

[1041] fluid 16 1.03 0.011 0.020 0.83 0.108 0.01 Perfusion 114.52 ± 0.035 ± 0.019 ± -1.64 ± 7.626 ± 7.31 ±

[1042] 222.17 ± 4.22

[1043]

[1044] fluid 17 1.08 0.009 0.012 0.16 0.118 0.00 Name of Average

[1045] Average Pdl Zeta

[1046] the diameter Viscosity diameter Pdl [-] after 7 potentia

[1047] perfusion after 7 days [mPa-s] pH [-]

[1048] [nm] days [-] 1 [mV]

[1049] fluid [nm]

[1050] Perfusion 109.62 ± 0.050 ± 0.033 ± -2.33 ± 6.392 ± 7.31 ±

[1051] 223.41 ± 2.63

[1052] fluid 18 1.10 0.016 0.021 0.70 0.136 0.00 Perfusion 122.63 ± 0.066 ± 0.034 ± -1.66 ± 5.501 ± 6.08 ±

[1053] 215.71 ± 2.64

[1054] fluid 19 1.72 0.013 0.021 0.39 0.072 0.01 Perfusion 116.39 ± 0.139 ± 0.055 ± -1.55 ± 2.830 ± 7.20 ±

[1055] 204.04 ± 2.18

[1056] fluid 20 1.02 0.011 0.015 0.57 0.026 0.01 Perfusion 109.16 ± 0.189 ± 0.164 ± -22.41 ± 3.343 ± 7.23 ±

[1057] 333.98 ± 5.63

[1058] fluid 21 0.82 0.010 0.016 1.53 0.039 0.01 Perfusion 262.02 ± 0.391 ± 0.232 ± -16.62 ± 2.700- 7.16 ±

[1059] 348.86 ± 3.39

[1060] fluid 22 3.62 0.025 0.019 1.08 3.000 0.00 Perfusion 126.54 ± 0.117 ± 0.144 ± -23.03 ± 5.707 ± 7.22 ±

[1061] 301.59 ± 4.93

[1062] fluid 23 1.29 0.012 0.020 1.07 0.228 0.00 Perfusion 110.82 ± 0.165 ± 0.061 ± -14.78 ± 3.519 ± 7.24 ±

[1063] 242.79 ± 2.48

[1064] fluid 24 0.96 0.011 0.023 1.10 0.006 0.00 Perfusion 0.279 ± 0.242 ± -22.70 ± 2.327 ± 7.15 ±

[1065] 74.27 ± 1.89 227.77 ± 2.18

[1066] fluid 25 0.006 0.014 1.99 0.066 0.00 Perfusion 95.05 ± 0.203 ± 0.172 ± -25.46 ± 1.803 ± 7.21 ±

[1067] 217.26 ± 1.80

[1068] fluid 26 0.690 0.011 0.013 1.48 0.032 0.00 Perfusion 126.50 ± 0.172 ± 0.043 ± -1.38 ± 3.123 ± 7.21 ±

[1069] 202.72 ± 2.31

[1070] fluid 27 1.33 0.009 0.019 0.34 0.118 0.00 Perfusion 128.87 ± 0.183 ± 0.047 ± -1.46 ± 3.458 ± 7.22 ±

[1071] 212.90 ± 2.26

[1072] fluid 28 0.75 0.007 0.016 0.48 0.050 0.00 Perfusion 121.42 ± 0.118 ± 0.049 ± -2.47 ± 2.898 ± 7.26 ±

[1073] 206.84 ± 1.94

[1074] fluid 29 1.33 0.011 0.011 0.87 0.049 0.01 Perfusion 119.06 ± 0.118 ± 0.046 ± -1.49 ± 3.080 ± 7.24 ±

[1075] 206.78 ±1.59

[1076] fluid 30 0.96 0.007 0.013 0.44 0.052 0.01 Perfusion 113.01 ± 0.150 ± 0.060 ± -1.75 ± 2.581 ± 7.46 ±

[1077] 204.70 ± 2.13

[1078] fluid 31 1.14 0.010 0.010 0.23 0.008 0.00 Perfusion 0.180 ± 0.030 ± -6.19 ± 3.648 ± 7.33 ±

[1079] 89.37 ± 1.88 174.57 ± 1.30

[1080] fluid 32 0.020 0.020 0.38 0.010 0.00 Perfusion 0.330 ± 0.300 ± -3.88 ± 3.107 ± 7.47 ±

[1081] 81.00 ± 0.49 82.96 ± 0.83

[1082] fluid 33 0.000 0.010 0.33 0.028 0.00 Perfusion 117.49 ± 0.290 ± 0.340 ± -1.65 ± 2.800 ± 7.47 ±

[1083] 155.02 ± 2.38

[1084] fluid 34 1.32 0.010 0.030 0.54 0.065 0.00 Perfusion 0.260 ± 0.230 ± -4.27 ± 3.315 ± 7.47 ±

[1085] 85.64 ± 0.83 86.92 ± 0.53

[1086] fluid 35 0.010 0.010 1.05 0.049 0.00 Perfusion 106.20 ± 0.170 ± 0.200 ± -27.09 ± 2.757 ± 7.48 ±

[1087] 119.64 ± 1.40

[1088] fluid 36 1.99 0.010 0.010 0.79 0.053 0.01 Perfusion 0.270 ± 0.290 ± -25.88 ± 2.516 ± 7.38 ±

[1089] 87.82 ± 1.37 93.19 ± 1.19

[1090] fluid 37 0.010 0.010 0.72 0.051 0.00 Perfusion 0.270 ± 0.050 ± -14.04 ± 2.811 ± 7.47 ±

[1091] 93.63 ± 1.37 237.99 ± 2.67

[1092] fluid 38 0.010 0.020 0.64 0.032 0.00 Perfusion 0.190 ± 0.180 ± -3.95 ± 3.184 ± 7.45 ±

[1093] 91.46 ± 1.04 92.59 ± 0.93

[1094] fluid 39 0.010 0.010 0.28 0.015 0.00 Perfusion 113.31 ± 0.190 ± 0.180 ± -2.54 ± 2.790 ± 7.38 ±

[1095] 153.22 ± 1.76

[1096] fluid 40 0.68 0.010 0.020 0.33 0.063 0.00 Perfusion 0.270 ± 0.260 ± -3.99 ± 2.821 ± 7.39 ±

[1097] 83.43 ± 0.54 85.80 ± 0.83

[1098] fluid 41 0.010 0.010 0.50 0.024 0.00 Perfusion 0.250 ± 0.230 ± -3.05 ± 2.920 ± 7.40 ±

[1099] 82.21 ± 1.62 83.87 ± 0.88

[1100] fluid 42 0.010 0.010 0.42 0.057 0.00 Perfusion 0.220 ± 0.190 ± -2.53 ± 2.799 ± 7.39 ±

[1101] 97.66 ± 1.17 162.72 ± 1.50

[1102] fluid 43 0.010 0.010 0.57 0.014 0.00 Perfusion 0.270 ± 0.250 ± -3.48 ± 2.912 ± 7.37 ±

[1103] 83.01 ± 0.97 86.46 ± 0.92

[1104]

[1105] fluid 44 0.010 0.010 0.46 0.020 0.01 Name of Average

[1106] Average Pdl Zeta

[1107] the diameter Viscosity diameter Pdl [-] after 7 potentia

[1108] perfusion after 7 days [mPa-s] pH [-]

[1109] [nm] days [-] 1 [mV]

[1110] fluid [nm]

[1111] Perfusion 0.240 ± 0.220 ± -3.22 ± 2.911 ± 7.38 ±

[1112] 82.55 ± 0.29 84.77 ± 0.82

[1113] fluid 45 0.010 0.010 0.48 0.017 0.00 Perfusion 0.205 ± 0.198 ± -4.21 ± 3.043 ± 7.37 ±

[1114] 88.98 ± 1.28 88.29 ± 0.73

[1115] fluid 46 0.011 0.01 0.59 0.025 0.00 Perfusion 0.287 ± 0.042 ± -2.96 ± 2.761 ± 7.40 ±

[1116] 83.94 ± 3.18 170.12 ± 1.02

[1117] fluid 47 0.016 0.018 0.46 0.019 0.00 Perfusion 100.11 ± 0.119 ± 0.118 ± -2.24 ± 3.317 ± 8.48 ±

[1118] 101.49 ± 0.83

[1119] fluid 48 0.83 0.016 0.012 0.27 0.019 0.00 Perfusion

[1120] fluid_02_

[1121] 163.81 ± 0.054 ± 0.056 ± -2.61 ± 2.880 ± 7.43 ± II 220.52 ± 3.75

[1122] 1.43 0.012 0.019 0.52 0.040 0.01 production

[1123] method

[1124] Perfusion

[1125] fluid_04_

[1126] 190.70 ± 0.068 ± 0.067 ± -4.86 ± 5.330 ± 7.32 ± II 213.46 ±2.43

[1127] 1.50 0.020 0.019 0.63 0.050 0.00 production

[1128]

[1129] method

[1130] Table 36. Results of quality control of organ perfusion fluids, osmotic pressure, oncotic pressure, sodium, potassium, calcium and chloride ion concentrations and cytotoxicity. Name of Osmotic

[1131] Oncotic Cell the pressure Ca

[1132] pressure Na+[mM] K [mM]2+Cl viability perfusion [mOsm / kgfkO [mM] [mM]

[1133] [mmHg] [%] fluid 1

[1134] Perfusion 94.62 ± 5.16 ± 0.19 ± 67.21 ±

[1135] 281 ± 1 34.9 ± 3.8 83 ± 4 fluid 01 0.78 0.01 0.00 0.59 Perfusion 101.08 ± 5.77 ± 0.22 ± 72.53 ±

[1136] 317 ± 4 28.8 ± 2.1 89 ± 12 fluid 02 0.67 0.04 0.01 0.42 Perfusion 107.97 ± 6.17 ± 0.42 ± 81.31 ±

[1137] 300 ± 4 26.9 ± 1.8 83 ± 20 fluid 03 0.85 0.04 0.01 0.66 Perfusion 114.34 ± 6.56 ± 0.44 ± 86.89 ±

[1138] 328 ± 2 26.0 ± 1.0 83 ± 15 fluid 04 0.30 0.04 0.00 0.44 Perfusion 123.47 ± 6.97 ± 0.46 ± 96.16 ±

[1139] 355 ± 2 18.3 ± 0.6 83 ± 2 fluid 05 0.32 0.04 0.00 0.11 Perfusion 136.32 ± 7.57 ± 0.50 ± 106.72 ±

[1140] 395 ± 13 33.7 ± 5.3 90 ± 3 fluid 06 0.50 0.16 0.00 0.52 Perfusion 106. 85 ± 5.86 ± 0.37 ± 81.97 ±

[1141] 417 ± 10 35.4 ± 1.9 88 ± 7 fluid 07 1.70 0.11 0.01 1.02 Perfusion 113.31 ± 6.20 ± 0.32 ± 86.80 ±

[1142] 478 ± 17 22.8 ± 0.4 92 ± 4 fluid 08 1.13 0.08 0.01 1.39 Perfusion 98.83 ± 5.62 ± 0.20 ± 74.29 ±

[1143] 303 ± 4 33.8 ± 5.1 117 ± 3 fluid 09 2.75 0.19 0.02 3.41 Perfusion 93.56 ± 5.24 ± 0.19 ± 67.99 ±

[1144] 277 ± 4 23.2 ± 0.6 83 ± 7 fluid 10 0.95 0.02 0.01 0.72 Perfusion 105.68 ± 5.93 ± 0.24 ± 80.06 ±

[1145] 334 ± 11 43.2 ± 4.6 123 ± 19 fluid 11 2.27 0.03 0.02 0.53 Perfusion 25.3 ± 99.11 ± 5.52 ± 0.19 ± 73.09 ±

[1146] 301 ± 1 86 ± 13 fluid 12 14.2 1.28 0.06 0.01 0.75 Perfusion 99.64 ± 5.53 ± 0.20 ± 72.29 ±

[1147] 301 ± 1 24.7 ± 1.3 92 ± 10 fluid 13 0.36 0.01 0.01 0.21 Perfusion 94.49 ± 5.85 ± 0.42 ± 74.67 ±

[1148] 322 ± 4 33.1±1.0 113 ± 14

[1149]

[1150] fluid 14 1.58 0.08 0.01 1.42 Name of Osmotic

[1151] Oncotic Cell the pressure Ca2+Cl pressure Na+[mM] K [mM] viability perfusion [mOsm / kgEhO [mM] [mM]

[1152] [mmHg] [%] fluid 1

[1153] Perfusion 90.62 ± 5.48 ± 0.43 ± 76.95 ±

[1154] 310 ± 3 35.4 ± 1.3 98 ± 9 fluid 15 4.22 0.22 0.04 3.15 Perfusion 90.95 ± 5.57 ± 0.45 ± 71.94 ±

[1155] 297 ± 2 33.9 ± 1.3 91 ± 13 fluid 16 1.22 0.03 0.01 0.62 Perfusion 93.42 ± 5.81 ± 0.44 ± 70.69 ±

[1156] 302 ± 1 21.6 ± 0.9 104 ± 5 fluid 17 3.77 0.22 0.04 2.95 Perfusion 89.12 ± 5.55 ± 0.41 ± 73.87 ±

[1157] 308 ± 3 19.3 ± 0.6 105 ± 7 fluid 18 3.45 0.16 0.03 2.95 Perfusion 145.78 ± 4.35 ± 0.57 ± 103.68 ±

[1158] 339 ± 7 19.8 ± 1.0 115 ± 11 fluid 19 1.19 0.04 0.01 0.61 Perfusion 99.62 ± 5.50 ± 0.21 ± 70.82 ±

[1159] 295 ± 2 20.3 ± 0.7 97 ± 6 fluid 20 0.54 0.05 0.01 0.85 Perfusion 101.76 ± 5.42 ± 0.10 ± 71.92 ±

[1160] 301 ± 2 31.4 ± 2.8 74 ± 6 fluid 21 0.21 0.02 0.00 0.64 Perfusion 100.25 ± 5.53 ± 0.10 ± 73.96 ±

[1161] 305 ± 3 24.2 ± 1.3 113 ± 12 fluid 22 0.95 0.02 0.00 0.83 Perfusion 103.77 ± 5.37 ± 0.10 ± 72.97 ±

[1162] 305 ± 1 24.5 ± 0.9 71 ± 6 fluid 23 0.33 0.01 0.00 0.63 Perfusion 103.28 ± 5.65 ± 0.11 ± 74.64 ±

[1163] 312 ± 3 25.1 ± 1.5 81 ± 13 fluid 24 0.35 0.02 0.01 0.19 Perfusion 103.22 ± 5.03 ± 0.11 ± 70.50 ±

[1164] 282 ± 2 25.8 ± 1.4 77 ± 10 fluid 25 0.25 0.02 0.00 0.29 Perfusion 96.39 ± 4.97 ± 0.18 ± 69.26 ±

[1165] 274 ± 2 15.9 ± 0.2 83 ± 12 fluid 26 0.27 0.01 0.01 0.26 Perfusion 101.05 ± 5.91 ± 0.20 ± 73.19 ±

[1166] 305 ± 0 25.3 ± 0.6 75 ± 12 fluid 27 0.44 0.03 0.00 0.35 Perfusion 102.07 ± 5.40 ± 0.20 ± 74.05 ±

[1167] 309 ± 1 33.0 ± 1.7 93 ± 12 fluid 28 0.24 0.02 0.01 0.07 Perfusion 102.84 ± 5.68 ± 0.22 ± 74.61 ±

[1168] 342 ± 2 20.8 ± 2.3 107 ± 14 fluid 29 0.33 0.03 0.00 0.44 Perfusion 104.26 ± 5.83 ± 0.21 ± 75.75 ±

[1169] 406 ± 7 18.5 ± 0.8 100 ± 15 fluid 30 0.48 0.03 0.01 0.73 Perfusion 104.83 ± 5.77 ± 0.25 ± 76.90 ±

[1170] 300 ± 1 23.2 ± 1.4 116 ± 21* fluid 31 0.21 0.03 0.00 0.22 Perfusion 90.37 ± 4.53 ± 0.13 ± 61.90 ±

[1171] 266 ± 4 20.0 ± 2.2 91 ± 10* fluid 32 0.12 0.00 0.00 0.08 Perfusion 108.87 ± 5.90 ± 0.26 ± 80.43 ±

[1172] 307 ± 4 17.7 ± 1.2 115 ± 9* fluid 33 0.05 0.00 0.00 0.17 Perfusion 108.73 ± 5.89 ± 0.26 ± 80.30 ±

[1173] 309 ± 1 20.0 ± 0.5 111 ± 8* fluid 34 0.17 0.01 0.01 0.14 Perfusion 108.63 ± 5.88 ± 0.26 ± 80.20 ±

[1174] 305 ± 3 20.7 ± 0.8 98 ± 4* fluid 35 0.05 0.01 0.00 0.14 Perfusion 99.97 ± 5.11 ± 0.21 ± 72.73 ±

[1175] 269 ± 3 9.9 ± 0.0 104 ± 12* fluid 36 0.12 0.02 0.00 0.21 Perfusion 97.17 ± 4.68 ± 0.17 ± 68.80 ±

[1176] 274 ± 0 20.8 ± 0.2 78 ± 11* fluid 37 0.05 0.01 0.00 0.08 Perfusion 106.93 ± 5.91 ± 0.20 ± 78.30 ±

[1177] 309 ± 2 21.8 ± 1.1 152 ± 11* fluid 38 0.12 0.01 0.00 0.29 Perfusion 107.33 ± 5.91 ± 0.26 ± 79.50 ±

[1178] 310 ± 1 19.6 ± 0.3 99 ± 7* fluid 39 0.12 0.01 0.00 0.08 Perfusion 101.87 ± 7.67 ± 0.34 ± 77.07 ±

[1179] 299 ± 2 21.9 ± 0.1 145 ± 7* fluid 40 0.17 0.02 0.00 0.47 Perfusion 102.67 ± 6.00 ± 0.35 ± 76.70 ±

[1180] 298 ± 1 20.6 ± 0.6 151 ± 5*

[1181]

[1182] fluid 41 0.09 0.00 0.00 0.24 Name of Osmotic

[1183] Oncotic Cell the pressure Ca2+Cl pressure Na+[mM] K [mM] viability perfusion [mOsm / kgEhO [mM] [mM]

[1184] [mmHg] [%] fluid 1

[1185] Perfusion 102.13 ± 6.17 ± 0.35 ± 76.70 ±

[1186] 297 ± 2 20.4 ± 0.7 149 ± 10* fluid 42 0.17 0.01 0.00 0.37 Perfusion 103.10 ± 5.81 ± 0.35 ± 76.90 ±

[1187] 298 ± 3 21.6 ± 0.0 157 ± 11* fluid 43 0.08 0.00 0.00 0.37 Perfusion 102.07 ± 7.21 ± 0.35 ± 78.13 ±

[1188] 295 ± 2 21.5 ± 0.2 143 ± 10* fluid 44 0.24 0.02 0.00 0.63 Perfusion 103.80 ± 5.69 ± 0.35 ± 77.95 ±

[1189] 298 ± 2 22.5 ± 0.1 149 ± 6* fluid 45 0.00 0.01 0.00 0.15 Perfusion 82.70 ± 4.60 ± 0.60 ± 66.60 ±

[1190] 283 ± 0 21.7 ± 0.1 88 ± 3 fluid 46 0.00 0.00 0.00 0.00 Perfusion 82.70 ± 4.60 ± 0.60 ± 66.60 ±

[1191] 292 ± 0 22.6 ± 1.3 102 ± 5 fluid 47 0.00 0.00 0.00 0.00 Perfusion

[1192] fluid_02_

[1193] 105.20 ± 5.63 ± 0.22 ± 75.77 ±

[1194] II 320 ± 1 17.8 ± 0.9 91 ± 9

[1195] 0.20 0.03 0.01 0.58 production

[1196] method

[1197] Perfusion

[1198] fluid_04_

[1199] 21.27 ± 118.90 ± 6.73 ± 0.26 ± 91.00 ±

[1200] II 343 ± 4 97 ± 13

[1201] 3.45 4.27 0.06 0.00 0.69

[1202] production

[1203]

[1204] method

[1205] * Cell viability for undiluted perfusion fluid formulations (without culture medium), calculated relative to the negative control using Belzer MPS.

[1206] Table 37. Stability results of selected perfusion fluids prepared according to production method I: mean particle diameter.

[1207] Name of the Mean diameter [nm]

[1208] perfusion fluid Day 0 Day 7 Month 3 Months 6 Months Perfusion

[1209] 113.01 ± 1.14 204.70 ± 2.13 228.30 ± 1.49 259.27 ± 3.25 273.00 ± 1.97 fluid 31

[1210] Perfusion

[1211] 89.37 ± 1.88 174.57 ± 1.30 200.28 ± 1.26 229.16 ± 2.36 235.03 ± 3.12 fluid 32

[1212] Perfusion

[1213] 81.00 ± 0.49 82.96 ± 0.83 87.37 ± 0.42 100.78 ± 0.60 106.58 ± 0.92 fluid 33

[1214] Perfusion

[1215] 117.49 ± 1.32 155.02 ± 2.38 137.56 ± 3.57 147.19 ± 30.11 136.44 ± 1.01 fluid 34

[1216] Perfusion

[1217] 85.64 ± 0.83 86.92 ± 0.53 90.37 ± 0.64 101.50 ± 1.11 105.00 ± 0.54 fluid 35

[1218] Perfusion

[1219] 106.20 ± 1.99 119.64 ± 1.40 128.81 ± 1.01 149.44 ± 2.10 156.69 ± 1.36 fluid 36

[1220] Perfusion

[1221] 87.82 ± 1.37 93.19 ± 1.19 97.53 ± 0.57 109.87 ± 0.79 111.02 ± 0.76 fluid 37

[1222] Perfusion

[1223] 94.46 ± 2.22 213.11 ± 3.19 235.42 ± 1.45 259.24 ± 2.45 274.79 ± 2.17 fluid 2

[1224] Perfusion

[1225] 91.46 ± 1.04 92.59 ± 0.93 94.25 ± 0.61 102.20 ± 1.30 101.80 ± 1.17

[1226]

[1227] fluid 39

[1228] Table 38. Stability results of selected perfusion fluids prepared according to production method I: polydispersity index.

[1229] Name of the Pdl [-]

[1230] perfusion fluid

[1231]

[1232] Day 0 Day 7 Month 3 Months 6 Months Perfusion fluid_31 0.145 ± 0.014 0.056 ± 0.014 0.045 ± 0.02 0.093 ± 0.023 0.135 ± 0.019 Perfusion fluid_32 0.175 ± 0.018 0.033 ± 0.015 0.046 ± 0.02 0.053 ± 0.021 0.057 ± 0.032 Perfusion fluid_33 0.329 ± 0.005 0.297 ± 0.008 0.267 ± 0.007 0.231 ± 0.005 0.223 ± 0.016 Perfusion fluid_34 0.292 ± 0.007 0.339 ± 0.027 0.417 ± 0.044 0.227 ± 0.046 0.252 ± 0.008 Perfusion fluid_35 0.259 ± 0.009 0.235 ± 0.009 0.212 ± 0.011 0.19 ± 0.005 0.184 ± 0.006 Perfusion fluid_36 0.169 ± 0.013 0.197 ± 0.012 0.218 ± 0.01 0.245 ± 0.009 0.259 ± 0.005 Perfusion fluid_37 0.267 ± 0.005 0.286 ± 0.008 0.291 ± 0.011 0.304 ± 0.008 0.315 ± 0.021 Perfusion fluid_2 0.231 ± 0.013 0.029 ± 0.018 0.046 ± 0.019 0.063 ± 0.026 0.082 ± 0.021

[1233]

[1234] Perfusion fluid_39 0.190 ± 0.007 0.178 ± 0.014 0.168 ± 0.014 0.164 ± 0.019 0.138 ± 0.022

[1235] Table 39. Stability results of perfusion fluid_02_II production method, emulsion particle size distribution.

[1236] Storage time at 4 C° Average diameter [nm] Pdl [-]

[1237] 2 days 163.81 ± 1.43 0.054 ± 0.012 7 days 220.52 ± 3.75 0.056 ± 0.019 month 239.29 ± 0.79 0.041 ± 0.012 2 months 244.50 ± 2.25 0.106 ± 0.020 3 months 252.23 ± 1.44 0.080 ± 0.018 4 months 281.61 ± 2.67 0.097 ± 0.026 8 months 317.09 ± 3.04 0.135 ± 0.016

[1238]

[1239] Table 40. Stability results of perfusion fluid_04_II production method, emulsion particle size distribution.

[1240] Storage time at 4 C° Average diameter [nm] Pdl [-]

[1241] 3 days 190.70 ± 1.50 0.068 ± 0.020 7 days 213.46 ± 2.43 0.067 ± 0.019 month 238.63 ± 1.36 0.091 ± 0.045 8 months 360.78 ± 22.79 0.260 ± 0.045

[1242]

[1243] Example III.

[1244] Perfusion of isolated organs ex vivo.

[1245] Organ transplantation is a modality used in the end-stage failure of an organ. The gold standard for ex vivo organ storage is pumping (perfusion) through the vascular bed suitable fluid at a low temperature, which reduces the metabolic rate and alleviates the effects of ischaemia. In order to demonstrate the efficacy of the perfusion fluid according to the invention, a number of the perfusion experiments were carried out on isolated organs (kidneys, liver and heart). Example III. 1. Perfusion of porcine kidney ex vivo

[1246] Efficacy studies of the perfusion fluids were conducted on isolated porcine kidneys (domestic Polish White Hog, females weighing 40-50kg) in the donation after cardiac death (DCD) model subjected to a 7-40 minutes warm ischaemic time (DCD model). This model involves organ retrieval after arrest of the blood flow in the organ. Maximum ischaemic time of the organ at physiological temperature (37 degrees C.) is 40 minutes. After retrieval, organs underwent preservation. The perfusion process was carried out using a prototype organ perfusion device that is the subject of an independent patent, application number P.449924.

[1247] Example III.l.l. Pre-operative procedure

[1248] The pigs were being acclimatised at the Jan Kielanowski Institute of Animal Physiology and Nutrition in Jablonna for at least 2 weeks. Eight hours before the operative procedure, food and drink were withdrawn.

[1249] Anaesthesia was started by intramuscular premedication: medetomidine lmg / m2, midazolam 5mg / m2, ketamine 8mg / kg body weight, methadone 0.2mg / kg body weight. The animal was then transported to the operating theatre. 1.1 x 32 mm or 0.9 x 25 mm intravenous cannula was inserted into the marginal ear vein or the saphenous vein or the caudal vein. Drugs for induction of anaesthesia - propofol 1-2 mg / kg and lidocaine Img / kg - were administered through the cannula.

[1250] The animal was placed in the sternal position on the operating table. Preoxygenation of 31 / min with a mask was started, then monitoring of vital functions (spO2, PR, NIBP: SYS / DIA, MAP, temperature) was connected.

[1251] The isoflurane concentration was set at 2-2.5% and the oxygen flow rate at 21 / min and the pig was carefully being observed. Normal anaesthesia was induced by reducing the heart rate below 150 beats / minute and blood pressure (systolic value below 100 mmHg) no movement of the pig was observed. The incision site was anaesthetised intrathecally with 2% lidocaine; the total dose of lidocaine used mustn’t have been more than 8 mg / kg. The concentration of isoflurane was gradually reduced to 1-1.5%.

[1252] Fluid therapy with Ringer’s lactate or acetate solution was then initiated. A suitable fluid dose was calculated from the following formulas:

[1253] - Fluid deficiency (1): body weight (kg) x [% dehydration / 100)

[1254] - daily fluid requirements: body weight (kg)0.75 x 70, average 40-60ml / kg / day

[1255] - running losses: average Iml / kg / h x body weight (kg)

[1256] Under sterile conditions, the skin was incised caudally from the angle of the mandible, then the salivary gland was dissected bluntly. The external jugular vein was exposed under the salivary gland. The vessel was dissected and ligated intracranially from the planned site of the access using Silk 3-0 suture. For the catheter insertion, the vessel was pinched, e.g. with a thin peanut. A small incision was made in the vessel with vascular scissors and the catheter was inserted in the distocardial side. Using Silk 3-0 thread, a loop was placed over the vessel to fixate the catheter to the vein.

[1257] The free side of the catheter was threaded onto a suitably large cutting needle and brought to the skin surface dorsal to the incision line. The incision line was closed with a mattress suture.

[1258] 20mg / kg of cefuroxime was administered 30 minutes before the start of surgery and every 90 minutes during surgery intravenously. Shotapen intramuscularly at a dose of 1 ml per 25kg and maropitant subcutaneously at a dose of Img / kg were also administered. Intramuscular metamizole at a dose of 50 mg / kg and intravenous bolus fentanyl at a dose of 1.5-3 ug / kg were administered as analgesic protection, followed by a CRI of fentanyl in 0.9% NaCl throughout the operation at a dose of 1.5 ug / kg / h. Veterinary ophthalmic ointment was applied to the eyes to prevent dryness during anaesthesia.

[1259] Example DI.1.2. Operating procedure

[1260] After disinfecting the surgical field and spreading the drape, a 25 cm long median incision was made and a retractor was inserted. The large and small bowel were covered with a drape and placed on the opposite side of the organ for better access during the retrieval of the organ . The ureter and the kidney were freed from any adjacent tissue using coagulation.

[1261] The renal vein and renal artery were dissected using coagulation until their exit from the inferior vena cava and aorta, respectively, was freed.

[1262] After complete dissection of the kidney, the ureter was tied (silk thread, 3-0) and cut distally. An ice bowl and sterile organ bag were prepared.

[1263] 100 lU / kg body weight of intravenous heparin was administered, the condition was then observed for 5 minutes. The renal artery close to the aorta and the renal vein close to the vena cava were then occluded with vascular clips for up to 30 minutes. Subsequently, the organ was harvested and a cannula was immediately inserted into the renal artery to start flushing the vascular bed. 500 ml of cold Belzer UW solution (or equivalent e.g. Store Protect) with 5000 U / l heparin was used to flush out the blood. The renal vein and the ureter were also cannulated. The kidney was sterile packaged and stored in the refrigerator until the perfusion was initiated. After retrieval, the animal was euthanized by intravenous administration of pentobarbital at a dose of 140 mg / kg body weight.

[1264] Example in.1.3.1. Normothermic perfusion of isolated kidney 260122N2 - DCD model WIT: lOmin, CIT: 60h

[1265] Kidney from a female weighing approximately 40 kg. Organ harvesting followed a 10-minute warm ischaemia, the kidney was stored for 60h in hypothermia: on ice or in a refrigerator). The weight of the kidney before the perfusion was 100g and after the perfusion 130g. Perfusion was carried out with perfusion fluid_04_II production method. The normothermic perfusion plan included:

[1266] (a) initial hypothermic perfusion for 2.5 - 4 hours

[1267] (b) controlled, linear temperature rise from 5°C to 37 °C for 90 minutes

[1268] (c) normothermic perfusion at 37°C for 24 hours (d) controlled linear cooling of the organ from 37°C to 5 °C for 90 minutes

[1269] (e) Langendorff model reperfusion at 37°C and MAP (mean arterial pressure) 90 - 100 mmHg using Krebs-Henseleit solution enriched with albumin to simulate reperfusion in the recipient's body.

[1270] Urine production started at 6thhour of the perfusion and was small, however sufficient to allow biochemical tests to be made.

[1271] LDH activity in the fluid (see Fig. 5.) fluctuates strongly during the perfusion. The level of the parameter increases strongly during the first 9 hours of the process, it is caused by the increase of the perfusion fluid’s temperature to 37°C. This leads to the increase of metabolic activity of the organ. Then the LDH activity drops to a lower level, around 300 U / l. Increase is observed again around the 15th hour. From the 17th hour a progressive decrease in enzyme activity is observed, with stabilisation occurring in the last three hours of the process.

[1272] The urinary LDH activity curve (See Fig. 6.) changes intensely during the perfusion. At the 6th hour of the process, the level of the marker is very high, however, it steadily decreases, reaching a minimum at the 9th hour of the process. From then on, the level starts to rise again, reaching value about 1,100 U / l. In the following hours, a decrease in LDH activity and a temporary stabilisation at a low level is observed. During the last hours of perfusion, the activity increases and exceeds the level of 1000 U / l. However, at the end of the process, the level of the parameter decreases to a below-average value.

[1273] The ASAT activity curve (See Fig. 7.) has an increasing trend. During the first eight hours of the perfusion, ASAT remains at a low activity level, slightly increasing. At the 9th hour of the process, there is an intense increase in the level of the parameter. From the 11th to the 16th hour of the process, a progressive increase is observed. Then the upward trend turns into a downward one. The decrease continues to the 26th hour, with a slight deviation in the form of the peak at the 24th hour of the process. By the end of the perfusion, a stabilisation of the parameter level is observed.

[1274] The urinary ASAT activity (see Fig. 8.) is very high at the beginning of the process and reaches a value of approximately 66 U / l, the highest value during the entire perfusion. Until 8 hours into the process, there is a decrease in activity. From the 9th hour, an increase in the level of the parameter is observed, this condition maintained until the very end of the process. There are local decreases in the level, however, when looking at the entire experiment the trend is upwards.

[1275] Curve of ALAT activity in the perfusion fluid (See Fig. 9.) is irregular. It can be seen that the level of the parameter alternately increases and decreases during the first part of the process, until around the 14th hour it remains just above the limit of quantification. In the second half of the experiment, greater fluctuations of the parameter are noticeable, but the trend of alternating increases and decreases is maintained.

[1276] Urinary ALAT activity (see Fig. 10.) remains at a level just above the parameter's limit of quantification for most of the process. An upward and downward trend is noticeable, the parameter has a very irregular course. The highest activity value is observed at 24thhour of the process (102 U / l).

[1277] The value of GGT activity in the perfusion fluid (see Fig. 11.) remains below the limit of quantification for the first 5 hours of the process. Thereafter, an increase in the level of the parameter is observed, this phenomenon is probably related to raising the temperature of the fluid to 37°C, with a maximum of 67 U / l at around 18hr of the perfusion. From then, the curve tends to decrease, until the end of the last four hours of the process, GGT activity stabilises and remains at around 25 U / l level.

[1278] Urinary GGT activity (see Fig. 12.) is high at the 6th hour of the process, at 402 U / l level, however, it starts to decrease intensely in the following hours of the perfusion. From the 9th hour onwards, the activity remains at a low level, nevertheless, it is still higher than the limit of quantification. This trend is observed until the end of the experiment.

[1279] The creatinine concentration in the perfusion fluid (See Fig. 13.) increases from the beginning of the process until around the 13th hour of the perfusion, when the value reaches a maximum of 36 U / l. This is due to the rise in the temperature of the perfusion fluid to 37°C and consequently, acceleration of the metabolic processes in the organ. From the half the time of the perfusion, the level of the parameter begins to decrease, which indicates that the creatinine in the urine is being excreted. This is a confirmation that the kidney works and filters the perfusion fluid correctly.

[1280] Creatinine concentration in the urine (see Fig. 14.) at the beginning of its production is about 20 mg / dl, when at the 10th hour of the process it is already 35 mg / dl. In the following hours, a decrease in the concentration of the parameter is observed, but the decrease is smaller than that observed for creatinine concentration in the perfusion fluid. This is a confirmation of the correct functioning of the kidney, which correctly filters the perfusion fluid and excretes excess metabolites.

[1281] In summary, porcine kidney subjected to the ischaemic injury was successfully perfused. The kidney was initially subjected to ischaemia at physiological temperature (37°C), followed by further long-term simple hypothermia for a few dozen of hours. Based on the aforementioned conditions, kidney was damaged severely before the perfusion. The data obtained from the perfusion process allowed to consider the overall condition of the organ after perfusion as fit for transplantation. The kidney started working during the reperfusion process by producing urine and filtering creatinine.

[1282] Example IIL1.3.2. Normothermic perfusion of isolated kidney 070223N2 - DCD model WIT: 30min, CIT: 32h

[1283] Kidney from a female weighing approximately 40 kg. Organ retrieval followed a 30-minute warm ischaemia, the kidney was stored for 32 hours under cold conditions: on ice or in a refrigerator. Kidney weight before perfusion was 105g and 182g after the perfusion. Perfusion was carried out using perfusion fluid_04_II production method. The normothermic perfusion scheme included:

[1284] (a) initial hypothermic perfusion for 2.5 -4 hours

[1285] (b) controlled, linear rise in temperature from 5°C to 37 °C Cover 90 minutes

[1286] (c) normothermic perfusion at 37°C for 24 hours

[1287] (d) controlled in-line cooling of the organ from 37°C to 5 °C for 90 minutes

[1288] (e) Langendorff model reperfusion at 37°C and MAP (mean arterial pressure) 90 - 100 mmHg using Krebs-Henseleit solution enriched with albumin to simulate reperfusion in the recipient's body.

[1289] The kidney started to produce urine at the 3rd hour of the process. (See Fig. 15.). Fig. 15. urine flow was irregular due to the sensitivity of the flowmeter to any disturbance. The activity of LDH in the fluid (see Fig. 16.) gradually increases during perfusion, starting at values below the limit of quantification at the start of the process and reaching very high values of approximately 2600 U / l at the end of the experiment. The increase is related to the heating of the perfusion fluid up to 37°C.

[1290] ASAT activity in the fluid (see Fig. 17.) gradually increases during perfusion, starting at values below the limit of quantification at the start of the process and reaching values in the order of 600 U / l at the end of the experiment. The increase in the level of the parameter is related to the heating of the perfusion fluid to 37°C.

[1291] The creatinine concentration in the perfusion fluid (See Fig. 18.) decreases for the first five hours. Thereafter, the level of the parameter begins to rise slightly, reaching a value of over 20 mg / dl around the 7th hour of the process. Then, from 9thhour, it starts to oscillate around a value of 15 mg / dl, and from the 19th hour of the process it decreases steadily.

[1292] Creatinine concentration in the urine (See Fig. 19.) increases with the beginning of the urine production. Creatinine concentration in the urine decreases significantly and reaches its lowest value around the 30th hour of perfusion. The values measured at the very end of perfusion are significantly lower than those at the beginning of urine production. This condition confirms correct both functioning of the organ and creatinine filtration.

[1293] The activity of LDH in the urine (see Fig. 20.) is approximately constant during almost the entire experiment. Only at the end of the perfusion, around 28thhour, the activity increases strongly, and reaches values above 1,000 U / l.

[1294] Urinary AST activity (see Fig. 21.) increases strongly during the perfusion. Initial values are located quite close to the limit of quantification. However, it changes with the perfusion duration and at the end of perfusion values reach levels of approximately 450 U / l.

[1295] Creatinine clearance (See Fig. 22.) is a parameter necessary to assess the filtration capacity of the glomeruli. The amount of creatinine present in the perfusion fluid and urine is compared. The parameter increases during the process. However, the clearance value decreases at the end of the experiment. This may indicate a lower ability of the kidney to filter metabolites at the end of perfusion, this phenomenon may occur due to the low temperature.

[1296] Concentration of glucose in the perfusion fluid (see Fig. 23, blue line) successively decreases during the course of perfusion. This phenomenon occurs due to the increasing organ's demand for this component which is related to the accelerating metabolic processes. This demand increases specifically between the 4th and 30th hour of the process, where the temperature of the fluid is being raised from 4°C to 37°C. The urinary glucose concentration (orange line) at the start of the urine production is about 4 mmol / L, it falls to about 2 mmol / L by the 5th hour of the process. In the last hours of the perfusion t, glucose concentration rises slightly, which is due to the fact that glucose is externally dosed once the 4 mmol / L limit is reached.

[1297] In summary, porcine kidney subjected to significant ischaemic injury was successfully perfused. The kidney was initially subjected to ischaemia at physiological temperature (37°C), followed by further long-term simple hypothermia for a few dozen of hours. Based on the aforementioned conditions, the damage to the kidney before perfusion can be considered severe. The data obtained from the perfusion process allow the overall condition of the organ after perfusion to be determined as fit for transplantation. The kidney started working during the reperfusion process by producing urine and filtering creatinine. Example IIL1.3.3. Normothermic perfusion of isolated kidney 270223N1 - DCD model WIT: 30min, CIT: 16h

[1298] Kidney from a female weighing approximately 40 kg. Organ harvesting followed a 30-minute warm ischaemia, the kidney was stored 16h under cold conditions: on ice or in a refrigerator. The weight of the kidney before perfusion was assessed at 128g and after perfusion at 230g. Perfusion was performed with perfusion fluid_04_II production method. The normothermic perfusion plan included:

[1299] (a) initial hypothermic perfusion for 2.5 - 4 hours

[1300] (b) controlled, linear rise of the temperature from 5°C to 37°C over 90 minutes

[1301] (c) normothermic perfusion at 37°C for 24 hours

[1302] (d) controlled linear cooling of the organ from 37°C to 5°C for 90 minutes

[1303] (e) Langendorff model reperfusion at 37°C and MAP (mean arterial pressure) 90 - 100 mmHg using Krebs-Henseleit solution enriched with albumin to simulate reperfusion in the recipient's body.

[1304] The kidney started to produce urine around the 6th hour of the perfusion process (See Fig. 24.). The urine flow increases as a function of the duration of perfusion until the cooling of the organ and consequently the decrease of the metabolic activity of the organ.

[1305] LDH activity in the fluid (see Fig. 25.) gradually increases during perfusion, starting at a value around the limit of quantification at the start of the process and reaching high values of approximately 1,200 U / l at the end of the experiment. The increase in the level of the parameter is related to the heating of the perfusion fluid to 37°C and the increase in metabolic activity of the organ.

[1306] The activity of ASAT in the fluid (see Fig. 26.) gradually increases during perfusion, starting below the limit of detection at the start of the process and reaching values of 150 U / l around the 15th hour of the process. After the 15th hour of the process, the level of the parameter stabilises at the same level. Peak of activity value is observed at the 28th hour of the process, but the parameter level returns to the previously established value.

[1307] GGT activity (see Fig. 27.) maintain limit of detection up to 5th hour of perfusion. From the 6th hour the values start to increase reaching levels of approximately 5 U / l. Around the 9th hour of the trial, a downward trend is observed. At the 20th hour, the values stabilise at around 5 U / l until the end of the experiment. The level of the damage marker is not high for the entire process.

[1308] The concentration of creatinine in the fluid (See Fig. 28.) for the first 5 hours of the process is at a similar level. After 5 hours, a systematic decrease is observed, indicating the beginning of urine production and creatinine excretion. After the 20th hour of the process, an increase in the concentration of the parameter is observed.

[1309] The concentration of creatinine in the urine (See Fig. 29.) at the beginning of its production maintains level of about 10 mg / dl, with a slight decrease around the 8th hour of perfusion. Around the 13th hour of the process, a slight decrease in the parameter is noticeable; however, at the 19th hour of the process, an increasing creatinine concentration ...

Claims

1. Claims1. A perfusion fluid in the form of an oil-in-water PFC nanoemulsion including:3.the dispersed phase, which includes:4.- an oxygen carrier, preferably in an amount of 100.00 - 800.00 g / L, more preferably in an amount of 100 - 400 g / L, which is one or more compounds selected from:5.perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane, the water phase, which includes:6.- a surface active agent which is the main surfactant or at least one main surfactant and at least one co-surfactant, wherein:7.a main surfactant, preferably in an amount of 23.80 - 40.08 g / L, more preferably in an amount of 23.80 - 34.00 g / L, which is Pluronic F-68 (Kolliphor P188) or Pluronic F- 108 or Pluronic F- 127, or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof, diblock / triblock / multiblock Pluronics, such as, for example Pluronics (condensation polymers of polypropylene glycols with polyoxyethylene glycols) and derivatives thereof, perfluorinated copolymers of the hydrocarbon-fluorocarbon type, gelatine and derivatives thereof,8.a co-surfactant, preferably in an amount of 0.35 - 0.60 g / L, more preferably in an amount of 0.35 - 0.50 g / L, which is: an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)- P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3- phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl- methoxy polyethyl eneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, perfluorinated phospholipids (e.g. phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids;9.- an oncotic agent, preferably in an amount of 20.00 - 40.00 g / L, more preferably in an amount of 40.00 g / L, which is one oncotic agent selected from the group comprising: of bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch or recombinant human serum albumin or derivatives thereof, Dextran 30 kDa, Dextran 50 kDa or derivatives thereof, gelatine derivatives, cyclodextrins, preferably bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch,10.- a main osmotic agent, preferably in an amount of 10.00 - 100.00 mM, most preferably in an amount of 21.40 - 50.00 nM, which is mannitol or raffinose or sucrose or glucose or citrate chelates or salts of lactobionic acid or gluconic acid, preferably mannitol, most preferably mannitol in an amount of 21.40 - 50.00 nM,11.- the main components of the buffer are one or more compounds selected from the12.- sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O preferably in an amount of 5.00 - 20.00 mM, most preferably in an amount of 10.00 mM;13.- sodium salt of DL-P-hydroxybutyric acid, preferably in an amount of 0.10 - 0.50 mM, most preferably in an amount of 0.20 mM;14.- antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N- acetylcysteine or catalase or peroxidase, preferably glutathione, preferably in an amount of 1.50 - 6.00 mM, most preferably in an amount of 3.00 mM, and15.- pharmacologically active substances - allopurinol or adenine, adenosine or phosphates or hormones: insulin, steroids i.e. dexamethasone, preferably allopurinol, preferably in an amount of 0.50 - 3.00 mM, most preferably in an amount of 1.00 mM;16.- buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more of NaCl, preferably in an amount of 20,00 - 100.00 mM, most preferably in an amount of 60.00 mM, KC1, preferably in an amount of 2.00 - 10.00 mM, most preferably in an amount of 4.60 mM, CaCL, preferably in an amount of 0.30 - 2.00 mM, most preferably in an amount of 0.60 mM, MgCh • 6H2O, preferably in an amount of 0.20 - 2.00 mM, most preferably in an amount of 0.40 mM; - buffering components maintaining the pH in the range 7.1 - 7.6, preferably one or more compounds selected from the group Na2HPO4 and NaH2PO4, buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine-HCl and L-histidine, preferably these are Na2HPO4 and / or preferably Na2HPO4, most preferably these are: Na2HPO4 in an amount of 6.00 - 15.00 mM, most preferably in an amount of 8.00 mM and NaH2PO4, preferably in an amount of 0.50 - 3.00 mM, most preferably in an amount of 1.5 mM;17.wherein, preferably, the perfusion fluid exhibits osmotic pressure in the range of: 280 - 400 mOsm / kgJLO, pH in the range of 7.1 - 7.6, oncotic pressure in the range of 15 - 36 mmHg, concentration of the main ions: sodium in the range of 80 - 200 mM, potassium in the range of 1 - 15 mM, calcium in the range of 0.1 - 2.5 mM and chloride in the range of 50 - 170 mM; furthermore, the average diameter of the dispersed phase nanoparticles does not exceed 300 nm, preferably is below 220 nm, most preferably is below 130 nm.

2. The perfusion fluid according to claim 1, wherein the aqueous phase further comprises amino acids, including one or more amino acids selected from the group: taurine, L-arginine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine.

3. The perfusion fluid according to claim 1 or 2, wherein the aqueous phase further comprises amino acids including preferably one or more amino acids selected from the group of: taurine preferably in an amount of 5.00 - 15.00 mM, most preferably in an amount of 10.00 mM; L-arginine preferably in an amount of 0.05 - 0.20 mM, most preferably in an amount of 0.10 mM; L-tryptophan preferably in an amount of 1,00 - 3.00 mM, most preferably 2.00 mM; L-glutamine preferably in an amount of 0.10 - 1.00 mM, most preferably in an amount of 0.50 mM; L-isoleucine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.52 - 4.19 mM; L-leucine preferably in an amount of 1.00 - 10.00 mM, most preferably in an amount of 3.05 - 6.86 mM; L-lysine preferably in an amount of 1.00 - 10.00 mM, most preferably in an amount of 2.05 - 4.79 mM; L-methionine preferably in an amount of 0.50 -10.00 mM, most preferably in an amount of 1.34 - 3.35 mM; L-threonine preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1,69 - 3.78 mM; L-v alinepreferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 2.56 - 5.55; L-histidine preferably in an amount of 0.10 - 10.00 mM, most preferably in an amount of 0.64 -2,26 mM; L-alanine preferably in an amount of 1.00 - 20.00 mM, most preferably in an amount of 5.61 - 12.35 mM; L-glycine preferably in an amount of 1.00 - 20.00 mM, most preferably in an amount of 6.66 - 17.32 mM; L-aspartic acid preferably in an amount of 0.50 - 10.00 mM, most preferably in an amount of 1.88 - 4.51 mM; L-proline preferably in an amount of 0.50 -10.00 mM, most preferably in an amount of 2.17 - 5,21 mM; L-serine preferably in an amount of 0.10 - 10.00 mM, most preferably in an amount of 0.95 - 2.38 mM; L-tyrosine preferably in an amount of 0.01 - 1.00 mM, most preferably in an amount of 0.06 - 0.28 mM.

4. The perfusion fluid according to any of claims 1 - 3, wherein the aqueous phase comprises taurine in an amount of 5.00 - 15.00 mM, most preferably 10.00 mM; L-arginine in an amount of 0.05 - 0.20 mM, most preferably 0.10 mM; L-tryptophan in an amount of 1.00 - 3.00 mM, most preferably 2.00 mM; and L-glutamine in an amount of 0.10 - 1.00 mM, most preferably 0.50 mM.

5. The perfusion fluid according to any of claims 1 - 4, wherein the oxygen carrier is one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin.

6. The perfusion fluid according to any of claims 1 - 5, wherein the main surfactant is Pluronic F-68 (Kolliphor P188), or Pluronic F- 108, or Pluronic F- 127, or bovine serum albumin (BSA), or human serum albumin (HSA), or recombinant human serum albumin, or derivatives thereof.

7. The perfusion fluid according to any of claims 1 - 6, wherein the co-surfactant is an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt or 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt.

8. The perfusion fluid according to any of claims 1 - 7, wherein the oncotic agent is bovine serum albumin (BSA), or human serum albumin (HSA), or recombinant human serum albumin, or succinylated gelatine, and more preferably bovine serum albumin (BSA), human serumalbumin (HSA), or recombinant human serum albumin, in an amount of: 20 - 40 g / L, most preferably 40 g / L.

9. The perfusion fluid according to any of claims 1 - 8, wherein the main surfactant is Pluronic F-68 or homologs thereof or derivatives thereof, or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin and derivatives thereof, and the co-surfactant is (lH,lH,2H,2H-perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains.

10. The perfusion fluid according to any of claims 1 - 9,27.wherein the main buffer component is one or more compounds selected from28.- pharmacologically active substances, preferably allopurinol; and / or29.- antioxidants, preferably glutathione, N-acetylcysteine, vitamin C and vitamin E; whereas the buffer components constituting salts are salts selected from Na+, K+, Ca2+, Mg2+.

11. The perfusion fluid according to any of claims 1 - 10, wherein the buffer is mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine, and Na2HPO4, Na^PCU, NaCl, KC1, CaCh, MgCh • 6H2O and more preferably mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L- tryptophan, L-glutamine, and Na2HPO4, NaftPCU, NaCl, KC1, CaCh, MgCh • 6H2O.

12. The perfusion fluid according to any of claims 1 - 11, wherein contains perfluorooctyl bromide (PFOB) in an amount of 187.21 g / L; perfluorodecyl bromide (PFDB) in an amount of 5.79 g / L; Pluronic F-68 (Kolliphor P188) in an amount of 27.20 g / L; 1H, 2H, 2H-Perfluorooctyl)phosphocholine in an amount of 4.00 g / L; bovine albumin in an amount of 40.00 g / L; L-arginine in an amount of 0.10 mM; allopurinol in an amount of 1.00 mM; mannitol in an amount of 50.00 mM; taurine in an amount of 10.00 mM; L-tryptophan in an amount of 2.00 mM; D-glucose • H2O in an amount of 10.00 mM; NaCl in an amount of 60.00 mM; KC1 in an amount of 4.60 mM; Na2HPO4 in an amount of 8.00 mM; Na^PCU in an amount of 1.50 mM; CaCh in an amount of 0.60 mM; MgCh • 6H2O in an amount of 0.40 mM; L-glutamine in an amount of 0.50 mM; glutathione in an amount of 3.00 mM; DL-P-hydroxybutyric acid sodium salt in an amount of 0.20 mM; NaOH to adjust a pH in the range of 6.8 - 7.4.

13. The perfusion fluid according to any of claims 1 - 11, wherein contains perfluorooctyl bromide (PFOB) in an amount of 374.42 g / L; perfluorodecyl bromide (PFDB) in an amount of 11.58 g / L; Pluronic F-68 (Kolliphor P188) in an amount of 34.00 g / L; 1H, 2H, 2H-Perfluorooctyl)phosphocholine in an amount of 5.00 g / L; bovine albumin in an amount of 40.00 g / L; L-arginine in an amount of 0.10 mM; allopurinol in an amount of 1.00 mM; mannitol in an amount of 21.40 mM; taurine in an amount of 10.00 mM; L-tryptophan in an amount of 2.00 mM; D-glucose • H2O in an amount of 10.00 mM; NaCl in an amount of 60.00 mM; KC1 in an amount of 4.60 mM; Na2HPO4 in an amount of 8.00 mM; NaFLPCU in an amount of 1.50 mM; CaCh in an amount of 0.60 mM; MgCL • 6H2O in an amount of 0.40 mM; L-glutamine in an amount of 0.50 mM; glutathione in an amount of 3.00 mM; DL-P -hydroxybutyric acid sodium salt in an amount of 0.20 mM; NaOH to adjust a pH in the range of 6.8 - 7.4.

14. A method for producing a perfusion fluid in the form of a PFC oil-in-water nanoemulsion, comprising the steps of1. preparation of the components of the primary perfusion fluid including34.la. preparation of the solution of surfactant or surfactants,35.lb. preparation of perfluorinated phase,36.lc. preparation of the buffer2. mechanical homogenization of the components prepared in step 1. to obtain a primary perfusion fluid;3. microfluidization of the primary perfusion fluid obtained in step 2. to obtain the perfusion fluid,39.wherein:40.in step la. the solution of surfactant or surfactants is:41.ultrapure water and42.the main surfactant or at least one main surfactant and at least one co-surfactant wherein:43.the main surfactant is Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F-127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof, diblock / triblock / multiblock Pluronics, such as e.g. Pluronics (condensation polymers of polypropylene glycols with polyoxyethylene glycols) and derivatives thereof, perfluorinated copolymers of the hydrocarb on-fluorocarb on type, gelatine and derivatives thereof, preferably Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or Pluronic F-127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof;44.co-surfactant is: an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or 1,2-dimyristoyl-sn- glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, perfluorinated phospholipids (e.g. phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids, preferably (1H, 1H, 2H, 2H- Perfluorooctyl)phosphocholine or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D- maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3- phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl- methoxy polyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt45.in step lb. the perfluorinated phase is one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane, preferably perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin in step 1c. the solid component of the buffer is:46.a. an oncotic agent which is one oncotic agent selected from the group comprising of bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch or recombinant human serum albumin or derivatives thereof, Dextran 30 kDa, Dextran 50 kDa, or derivatives thereof, gelatine derivatives, cyclodextrins, preferably bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch;47.b. the main osmotic agent, which is mannitol or raffinose or sucrose or glucose or citrate chelates or salts of lactobionic acid or gluconic acid, preferably mannitol;48.c. the main component of a buffer which is one or more compounds such as49.- sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O, - DL-P-hydroxybutyric acid sodium salt,50.- antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N- acetylcysteine or catalase or peroxidase, preferably glutathione, and51.- pharmacologically active substances - allopurinol or adenine or adenosine or phosphates or hormones, preferably insulin or steroids preferably dexamethasone, most preferably allopurinol;52.d. buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more, most preferably all, of: NaCl, KC1, CaCl2, MgCh • 6H2O;53.e. buffering components maintaining the pH in the range 7.1 - 7.6, preferably one or more compounds selected from the group: Na2HPO4 and NaH2PO4, buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine HCl and L-histidine, preferably Na2HPO4 and / or Na2HPO4; f. possibly other components, that are amino acids including preferably one or more amino acids selected from the group: taurine, L-arginine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine.

15. The method according to claim 14,wherein in step 3. the primary perfusion fluid obtained in step 2. is pumped at a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled using a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C;55.and / or56.in step 3. the preliminary perfusion fluid obtained in step 2. is passed 1 - 15 times through the microfluidizer chamber, most preferably 3 - 9 passes through the microfluidizer chamber.

16. A method for producing a perfusion fluid in the form of a PFC oil-in-water nanoemulsion, comprising the steps of1. preparation of the primary perfusion fluid components comprising1. preparation of the pre-emulsion components comprising60.la. preparation of the solution of surfactant or surfactants61.lb. preparation of the perfluorinated phase62.II. mechanical homogenization of the components prepared in step I to obtain a primary preemulsion,63.III. microfluidization of the primary pre-emulsion obtained in step II to obtain the preemulsion,64.IV. the preparation of the buffer comprising65.IVa. preparation of buffer components66.IVR. pH adjustment67.IVQ. quality control of the buffer obtained2. mixing the pre-emulsion obtained in step III with the buffer obtained in step IV to obtain the primary perfusion fluid, wherein the components being preferably mixed in a ratio of pre-emulsion to buffer from 1:1 to 1:2;3. microfluidization of the primary perfusion fluid obtained in step 2. to obtain the perfusion fluid;70.wherein:71.in step la. the solution of surfactant or surfactants is: ultrapure water and72.the main surfactant or at least one main surfactant and at least one co-surfactant wherein:73.the main surfactant is Pluronic F-68 (Kolliphor P188) or Pluronic F-108 or PluronicF- 127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof, diblock / triblock / multiblock Pluronics, such as e.g. Pluronics (condensation polymers of polypropylene glycols with polyoxyethylene glycols) and derivatives thereof, perfluorinated copolymers of the hydrocarbon-fluorocarbon type, gelatine and derivatives thereof, preferably Pluronic F-68 (Kolliphor Pl 88) or Pluronic F-108 or Pluronic F- 127 or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or derivatives thereof74.co-surfactant is: an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or 1,2-dimyristoyl-sn- glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, perfluorinated phospholipids (e.g. phosphatidylethanolamines, phosphatidylserine, phosphatidylinositol, PEGylated phospholipids, sphingomyelins, glycerophosphocholine), perfluorinated glycolipids, preferably (1H, 1H, 2H, 2H- Perfluorooctyl)phosphocholine or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D- maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3- phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl- methoxy polyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt75.in step lb. the perfluorinated phase is of one or more compounds selected from perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin, perfluorooctane, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorobutylcyclohexane, perfluoro- 15 -crown ether (PFCE), perfluorocyclohexane, preferably perfluorooctyl bromide, perfluorodecyl bromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributylamine, perfluorodecalin in step 1c. the solid component of the buffer is:76.a. an oncotic agent, which is one oncotic agent selected from the group comprising of bovine serum albumin (BSA) or human serum albumin (HSA) or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch or recombinant human serum albumin or derivatives thereof, Dextran 30 kDa, Dextran 50 kDa or derivatives thereof, gelatine derivatives, cyclodextrins, preferably bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin or PEG 35 kDa or PEG 20 kDa or Dextran 40 kDa or succinylated gelatine or hydroxy ethylated starch;77.b. the main osmotic agent, which is mannitol or raffinose or sucrose or glucose or citrate chelates or salts of lactobionic acid or gluconic acid, preferably mannitol;78.c. the main component of the buffer, which is one or more compounds selected from:79.- sugars - D-glucose • H2O or fructose or sucrose or dextran, preferably D-glucose • H2O, - DL-P-hydroxybutyric acid sodium salt,80.- antioxidants - glutathione or vitamin C or vitamin E or deferoxamine or N- acetylcysteine or catalase or peroxidase, preferably glutathione, and81.- pharmacologically active substances - allopurinol or adenine or adenosine or phosphates or hormones, preferably insulin or steroids preferably dexamethasone, most preferably allopurinol;82.d. buffer components constituting salts of Na+, K+, Mg2+, Ca2+, Cl", preferably selected from the group comprising: chlorides, sulphates(VI), phosphates, lactobionates, citrates, gluconates, preferably containing ions Na+, K+, Mg2+, Ca2+, Cl", preferably one or more, most preferably all, of: NaCl, KC1, CaCl2, MgCh • 6H2O;83.e. buffering components maintaining the pH in the range 7.1 - 7.6, preferably one or more compounds selected from the group: Na2HPO4 and NaH2PO4, buffers such as: bicarbonate consisting of a mixture of NaHCOs and H2CO3, phosphate consisting of a mixture of KH2PO4 and K2HPO4, citrate consisting of a mixture of NasCeHsO? 2H2O and CeHsO? H2O, histidine consisting of a mixture of histidine HCl and L-histidine, preferably Na2HPO4 and / or Na2HPO4; f. possibly other components, that are amino acids including preferably one or more amino acids selected from the group: taurine, L-arginine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine.

17. The method according to claim 16, wherein85.in step III. preferably the primary pre-emulsion obtained in step (II) is pumped at a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled by means of a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C;86.and in step 3. preferably the primary perfusion fluid obtained in step 2. is pumped at a pressure of 2065 bar through a polycrystalline diamond Y-type microfluidizer chamber with an inner diameter of 75 pm microchannels and cooled by means of a water bath so that the temperature of the fluid leaving the microfluidizer chamber does not exceed 30 °C;87.and / or88.in step III. microfluidization of the primary pre-emulsion obtained in step II to obtain the preemulsion is carried out in 1 - 10 passes of the pre-emulsion PFC through the microfluidizer chamber, most preferably 8 passes89.whereby90.in step 3. microfluidization of the primary perfusion fluid obtained in step 2. is carried out in 1-10 passes of the pre-emulsion obtained in step 2. through the microfluidizer chamber, most preferably 1-5 passes.

18. The method for producing according to any of claims 14 - 17, wherein the oncotic agent is bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin, or succinylated gelatine, and more preferably bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin, in an amount of 20 - 40 g / L, most preferably 40 g / L.

19. The method for producing according to any of claims 14 - 18, wherein other buffer components which are amino acids include taurine, L-arginine, L-tryptophan, and L-glutamine.

20. The method for producing according to any of claims 14 - 19, wherein the perfluorinated phase is one or more compounds selected from perfluorooctyl bromide, perfluorodecylbromide; perfluoropentane, perfluorohexane, perfluoroheptane, perfluorotributyl amine, perfluorodecalin.

21. The method for producing according to any of claims 14 - 20, wherein the main surfactant is Pluronic F-68 (Kolliphor P188), or Pluronic F-108, or Pluronic F-127, or bovine serum albumin (BSA), or human serum albumin (HSA), or recombinant human serum albumin, or derivatives thereof.

22. The method for producing according to any of claims 14 - 21, wherein the co-surfactant is an organic compound containing perfluorinated chains: (1H, 1H, 2H, 2H- Perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains, or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-maltopyranoside or Sphingomyelin or modified lipids: l,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or 1,2-dipalmitoyl-sn-glycero-3 -phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl-methoxy polyethyleneglycol -2000)- l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt.

23. The method for producing according to any of claims 14 - 22, wherein the main surfactant is Pluronic F-68 or homologs thereof or derivatives thereof, or bovine serum albumin (BSA) or human serum albumin (HSA) or recombinant human serum albumin and derivatives thereof, and the co-surfactant is (lH,lH,2H,2H-perfluorooctyl)phosphocholine or homologs thereof or derivatives thereof, including phosphocholine derivatives containing, in various structural configurations, perfluorocarbon chains.

24. The method for producing according to any of claims 14 - 20,98.wherein the main buffer component is one or more compounds selected from99.- pharmacologically active substances, preferably allopurinol; and / or100.- antioxidants, preferably glutathione, N-acetylcysteine, vitamin C and vitamin E; whereas the buffer components constituting salts are salts selected from Na+, K+, Ca2+, Mg2+.

25. The method for producing according to any of claims 14 -24, wherein the buffer is mannitol, D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L-tryptophan, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-valine, L-histidine, L-alanine, L-glycine, L-aspartic acid, L-proline, L-serine, L-tyrosine, and Na2HPO4, Na^PCU, NaCl, KC1, CaCh, MgCh • 6H2O, more preferably mannitol,D-glucose • H2O, glutathione, allopurinol, DL-P-hydroxybutyric acid sodium salt, L-arginine, taurine, L-tryptophan, L-glutamine, andNa2HPO4, NaFLPC NaCl, KC1, CaCh, MgCh • 6H2O.

26. The method for producing according to any of claims 14 -24, wherein the primary surfactant is: PluronicF-68 (Kolliphor P188) orPluronicF-108 orPluronicF127 or bovine serum albumin or human serum albumin or recombinant human serum albumin, in an amount of: 23.80 - 40.08 g / L, most preferably 23.80 - 34.00 g / L, and the co-surfactant is: - (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine or (1H, 1H, 2H, 2H-Perfluorooctyl)-P-D-Maltopyranoside or Sphingomyelin or modified lipid: l,2-Dioleoyl-sn-glycero-3-phospho-L-serine sodium salt or l,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt or l,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol sodium salt or N-(carbonyl -methoxy polyethylene glycol-2000)-l,2-distearoyl-sn-glycero-3 -phosphoethanolamine sodium salt, in amounts: 0.35 - 0.60 g / L, preferably 0.35 -0.50 g / L.

27. The method for producing according to any of claims 14 - 26, wherein104.steps 1 - 3 of the method are carried out under sterile / sterile conditions and / or105.step 3 shall be followed by step 4 involving sterilization of the perfusion fluid obtained in step 3106.to obtain sterile perfusion fluid,107.wherein sterilization of the perfusion fluid obtained in step 3 is carried out by sterilization filtration, gamma radiation, sterilization by ethylene oxide, most preferably by sterilizing filtration,108.alternatively, after steps 1 - 3 or after step 4, packaging of the resulting sterile perfusion fluid is carried out.

28. A perfusion fluid obtained by the method according to any of claims 14 - 27, preferably having a qualitative and quantitative composition and physicochemical properties as disclosed in claims 1 - 13.

29. The perfusion fluid according to any of claims 1 - 13 or 28, for use as a medicament.

30. The perfusion fluid according to any of claims 1 - 13 or 28, for use a blood substitute preparation.

31. The perfusion fluid according to any of claims 1 - 13 or 28, for use in the storage of organs intended for transplantation.

32. The perfusion fluid according to any of claims 1 - 13 or 28, for use in the treatment of acute renal failure, and chronic renal failure, in particular end-stage renal disease.

33. The perfusion fluid according to any of claims 1 - 13 or 28, for use in the treatment of acute and subacute liver failure, liver fibrosis and cirrhosis, alcoholic liver disease, toxic liver injury classified, and liver failure associated with liver cell cancer (hepatocellular carcinoma).

34. The perfusion fluid according to any of claims 1 - 13 or 28, for use in the treatment of heart failure, including acute systolic (congestive) heart failure and unspecified heart failure.

35. The perfusion fluid according to any of claims 1 - 13 or 28, for use in the treatment or prevention of a pathological condition that can be prevented, alleviated, or eliminated by administration of blood substitute preparations.

36. The perfusion fluid according to claim 33, wherein the pathological condition includes shock, including hypovolemic shock, traumatic, and other shock, including haemorrhagic shock, and unspecified shock.

37. The perfusion fluid according to any of claims 1 - 13 or 28, for use in ex vivo machine perfusion of organs intended for transplantation.

38. The perfusion fluid according to claim 37, wherein the organs intended for transplantation include organs retrieved after brain death - DBD, organs retrieved from donors after irreversible circulatory arrest - DCD, organs retrieved from donors aged 60 years or older, and donors in the age range 50 - 59 years with at least two of the following risk factors: arterial hypertension, death caused by stroke, blood creatinine level at time of death above 1.5 mg / dL - ECD.

39. The perfusion fluid according to any of claims 1 - 13 or 28, for use for use in a method of treatment by transplantation.

40. The perfusion fluid according to any of claims 1 - 13 or 28, for use in organ storage before and / or during transplantation.

41. The perfusion fluid according to any of claims 1 - 13 or 28, for use in organ reconditioning before and / or during transplantation.

42. The perfusion fluid according to any of claims 1 - 13 or 28, for use in ex vivo machine perfusion of organs, including most preferably kidney, liver, and heart.

43. The perfusion fluid according to any of claims 1 - 13 or 28, for use in ex vivo machine perfusion of organs under subnormothermic and normothermic conditions, preferably underhybrid conditions comprising periods of hypothermic perfusion, periods of controlled linear temperature increase or decrease, and periods of subnormothermic or normothermic perfusion, within the entire application range of 4 - 37°C and mean arterial pressure - MAP of 30 - 120 mmHg.

44. A method of treatment of acute renal failure and chronic renal failure, in particular endstage renal disease, using the perfusion fluid according to any of claims 1 - 13 or 28.

45. A method of treatment of acute and subacute liver failure, liver fibrosis and cirrhosis, alcoholic liver disease, toxic liver injury, and liver failure associated with liver cell cancer (hepatocellular carcinoma) using the perfusion fluid according to any of claims 1 - 13 or 28.

46. A method of treatment of heart failure, including acute systolic (congestive) heart failure and unspecified heart failure, using the perfusion fluid according to any of claims 1 - 13 or 28.

47. A method of treatment, or prevention of a pathological condition that can be prevented, alleviated, or eliminated by administration of blood substitute preparations, using the perfusion fluid according to any of claims 1 - 13 or 28.

48. The method of treatment according to claim 47, wherein the pathological condition includes shock, including hypovolemic shock, traumatic shock, other shock including haemorrhagic shock, and unspecified shock.

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