A normothermic extracorporeal perfusate for maintaining liver viability

Abstract

The application discloses a normal-temperature extracorporeal perfusion solution for maintaining liver activity, and mainly relates to the technical field of organ perfusion. The normal-temperature extracorporeal perfusion solution for maintaining liver activity comprises the following components: high-efficiency oxygen-carrying carriers HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids and vitamins. Compared with the prior art, the perfusion solution can make an isolated liver maintain a high bile secretion amount and stable portal vein pressure during normal-temperature perfusion, reduce the rising range of ALT and AST, reduce hepatocyte apoptosis and protect the structural integrity of mitochondria, thereby effectively reducing hepatocyte damage, maintaining liver activity, and being suitable for isolated liver preservation, pre-transplantation evaluation and related extracorporeal experiments.

Description

Technical Field

[0001] This invention relates to the field of organ perfusion technology, and in particular to a normothermic extracorporeal perfusion solution for maintaining liver activity. Background Technology

[0002] With the continuous advancement of in vitro organ perfusion technology, ex vivo liver preservation has become a core element in improving the success rate of liver transplantation and ensuring the smooth conduct of related in vitro experiments. Currently, the commonly used ex vivo liver perfusion media in clinical practice and experiments are mostly basal culture media and traditional classic perfusion media, including Williams E solution, Krebs-Henseleit solution, UW solution, Celsior solution, etc. These perfusion media can only maintain the basic physiological activity of the ex vivo liver for a short period of time, and usually have problems such as insufficient oxygen supply, limited energy metabolism support, insufficient microcirculation protection, and incomplete cell protection.

[0003] In existing technologies, if the perfusion fluid lacks a highly efficient oxygen carrier, it can easily lead to insufficient oxygen supply during liver perfusion, accelerating energy depletion and damage to hepatocytes; its energy metabolism support is weak, lacking diverse energy substrates and a complete antioxidant system, making it difficult to maintain normal hepatocyte metabolism and mitochondrial function; its support for liver microcirculation and bile secretion is limited, and long-term perfusion can easily lead to increased portal vein pressure, aggravating liver tissue damage; prolonged perfusion can promote increased hepatocyte apoptosis, destroy cell structure, and reduce liver tissue integrity.

[0004] Therefore, it is still necessary to develop an optimized perfusion solution that can provide efficient oxygen supply, maintain energy metabolism, enhance antioxidant capacity, and protect liver microcirculation, in order to prolong the in vitro viability of isolated livers and provide more stable liver conditions for transplantation or in vitro experiments. Summary of the Invention

[0005] This invention proposes a room-temperature extracorporeal perfusion solution for maintaining liver activity, which solves the technical problems of aggravated hepatocyte damage, decreased bile secretion capacity, and short duration of liver activity maintenance during long-term extracorporeal perfusion.

[0006] To achieve the above objectives, this invention proposes a normothermic in vitro perfusion solution for maintaining liver activity, comprising the following components: highly efficient oxygen carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, and vitamins.

[0007] In one embodiment, the high-efficiency oxygen-carrying carrier HBOC has a concentration of 70 g / L, the energy metabolism component concentration is 3.72 g / L, the hormone and growth factor concentration is 65 μg / L, the antioxidant component concentration is 3.95 g / L, the microcirculation protection factor concentration is 12 g / L, the HEPES buffer concentration is 7.15 g / L, the inorganic salt concentration is 1.09 g / L, the amino acid concentration is 6.45 g / L, and the vitamin concentration is 10.12 mg / L.

[0008] In one embodiment, the energy metabolism component includes at least one of adenosine, glucose, sodium pyruvate, L-carnitine, and nicotinamide.

[0009] In one embodiment, the hormone and growth factor include at least one of insulin and dexamethasone.

[0010] In one embodiment, the antioxidant component includes at least one of ascorbic acid 2-phosphate, reduced glutathione (GSH), allopurinol, deferoxamine, N-acetylcysteine, sodium selenite, and zinc sulfate.

[0011] In one embodiment, the microcirculation protective factor includes at least one of PGE2, ulinastatin, nitroglycerin, PEG20000, and heparin.

[0012] In one embodiment, the inorganic salt includes at least one of NaCl, KCl, CaCl2, MgSO4, magnesium chloride hexahydrate, Na2HPO4, and KH2PO4.

[0013] In one embodiment, the amino acid includes at least one selected from L-arginine, L-histidine, L-lysine, L-glutamine, L-proline, L-serine, glycine, L-alanine, L-asparagine monohydrate, L-cysteine, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0014] In one embodiment, the vitamin includes at least one of folic acid, thiamine HCl, riboflavin, calcium pantothenate, choline, inositol, vitamin A, α-tocopherol phosphate, nicotinamide, pyridoxine hydrochloride, and sodium riboflavin 5'-phosphate.

[0015] In one embodiment, the room-temperature in vitro perfusion solution for maintaining liver viability is used in the preservation of ex vivo livers.

[0016] In one embodiment, a method for preparing a room-temperature in vitro perfusion solution for maintaining liver activity is as follows: A high-efficiency oxygen carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, vitamins, and water are mixed, the osmotic pressure is adjusted to 290-310 mOsm / kg, and the volume is brought to a final volume to obtain the room-temperature in vitro perfusion solution for maintaining liver activity.

[0017] This invention introduces a highly efficient oxygen-carrying carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, and vitamins into a normothermic in vitro perfusion solution. This allows the perfusion solution to simultaneously provide oxygen supply support, energy metabolism support, antioxidant protection, microcirculation protection, and basic nutritional support during normothermic ex vivo liver perfusion. Compared to conventional perfusion solutions, this invention's perfusion solution can reduce ALT and AST release, maintain continuous bile secretion, stabilize portal vein pressure, reduce hepatocyte apoptosis, and protect the structural integrity of hepatocytes and mitochondria, thereby effectively mitigating ex vivo liver perfusion injury and prolonging the duration of liver viability. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram showing the ALT and AST detection results of the experimental and control groups of a normothermic in vitro perfusion solution for maintaining liver activity according to an embodiment of the present invention. Figure 2 This is a schematic diagram of HE staining results of liver tissues from the experimental group and the control group in an embodiment of a normothermic in vitro perfusion solution for maintaining liver viability according to the present invention. Figure 3 This is a schematic diagram of the TUNEL staining results of liver tissues from the experimental group and the control group in an embodiment of a normothermic in vitro perfusion solution for maintaining liver viability according to the present invention. Figure 4 This is a schematic diagram of transmission electron microscopy observation of hepatocytes in the experimental group and control group of an embodiment of a normothermic in vitro perfusion solution for maintaining liver viability according to the present invention. Figure 5 This is a schematic diagram of the cumulative bile production in the experimental and control groups of an embodiment of a normothermic extracorporeal perfusion solution for maintaining liver activity according to the present invention. Figure 6This is a schematic diagram of portal vein pressure in the experimental and control groups of an embodiment of a normothermic extracorporeal perfusion solution for maintaining liver viability according to the present invention. Figure 7 This is a schematic diagram of the liver perfusion appearance of the experimental group and the control group in an embodiment of an in vitro perfusion solution for maintaining liver activity according to the present invention.

[0020] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Ex vivo livers require high metabolic activity during in vitro perfusion at room temperature, placing high demands on oxygen supply, energy substrates, ion balance, antioxidant capacity, and microcirculatory status. Current conventional perfusion media are mostly basal culture media or classic organ preservation media. While these can maintain the liver's basic state for a certain period, their oxygen-carrying capacity is limited and cannot adequately meet the continuous metabolic needs of hepatocytes. Furthermore, existing perfusion media do not adequately support mitochondrial function, the antioxidant system, bile secretion, and portal vein pressure stability. Prolonged perfusion can easily lead to hepatocyte damage, tissue structure disruption, increased apoptosis, and microcirculatory disturbances.

[0023] In view of this, the present invention proposes a normothermic in vitro perfusion solution for maintaining liver activity, comprising the following components: highly efficient oxygen carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, and vitamins.

[0024] By simultaneously introducing highly efficient oxygen-carrying carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids and vitamins into the in vitro perfusion solution at room temperature, the perfusion solution of this invention can form a comprehensive protective system integrating oxygen supply support, energy metabolism support, antioxidant protection, microcirculation protection, buffering stability and nutrition maintenance during in vitro perfusion at room temperature, thereby reducing hepatocyte damage and maintaining the integrity of liver tissue structure and liver secretory function.

[0025] In the following embodiments, the concentration of the high-efficiency oxygen-carrying carrier HBOC is 70 g / L, the concentration of energy metabolism components is 3.72 g / L, the concentration of hormones and growth factors is 65 μg / L, the concentration of antioxidant components is 3.95 g / L, the concentration of microcirculation protection factor is 12 g / L, the concentration of HEPES buffer is 7.15 g / L, the concentration of inorganic salts is 1.09 g / L, the concentration of amino acids is 6.45 g / L, and the concentration of vitamins is 10.12 mg / L.

[0026] HBOC, as a highly efficient oxygen carrier, improves the problem of insufficient oxygen supply during isolated liver perfusion at room temperature, reducing the risk of hepatocyte energy depletion, mitochondrial damage, and cell necrosis caused by hypoxia. Selecting a high-efficiency oxygen carrier HBOC concentration of 70 g / L can effectively enhance the oxygen-carrying and oxygen-releasing capacity of the perfusion fluid under room temperature perfusion conditions, fully meeting the oxygen demand of highly metabolic hepatocytes and avoiding energy depletion, mitochondrial damage, and cell apoptosis caused by hypoxia. Simultaneously, this concentration avoids the risks of excessively high colloid osmotic pressure, abnormal viscosity, increased microcirculatory resistance, and cytotoxicity caused by high-concentration HBOC, maintaining the stability of the perfusion fluid's physicochemical properties, matching the overall osmotic pressure (290-310 mOsm / kg), ensuring stable portal vein pressure and unobstructed microcirculation, and providing a safe, efficient, and stable oxygen supply guarantee for long-term room temperature perfusion.

[0027] Regulating the concentration of energy metabolism components can provide a sufficient and balanced energy supply, avoiding energy deficiency or overload and maintaining a stable metabolic state. If the concentration is too low, there will be insufficient energy substrates, leading to rapid depletion of the energy reserves of hepatocytes, and a decline in mitochondrial function and activity. If the concentration is too high, it will easily cause the accumulation of metabolic substrates and an increase in osmotic pressure, which will increase the metabolic burden on hepatocytes and inhibit cell function.

[0028] Regulating the concentration of hormones and growth factors can effectively maintain the physiological homeostasis of hepatocytes. If the concentration is too low, the regulatory effect is weak, glucose metabolism will be disordered, hepatocyte polarity will be difficult to maintain, and functional decline will easily occur. If the concentration is too high, it will easily cause metabolic disorders, inhibit normal physiological activities of cells, and even cause hormone toxicity damage.

[0029] Regulating the concentration of antioxidant components can build an effective antioxidant barrier, balancing protection and safety. If the concentration is too low, the antioxidant capacity is insufficient, and it cannot effectively remove oxidative stress products, which can easily aggravate mitochondrial damage and hepatocyte apoptosis; if the concentration is too high, it can easily lead to an excessively strong reducing environment, interfere with the normal redox balance of cells, and affect the activity of metabolic enzymes.

[0030] Adjusting the concentration of microcirculation protective factors can effectively improve microcirculation, stabilize portal pressure, and ensure uniform perfusion. If the concentration is too low, the vasodilatory, anti-inflammatory, and anticoagulant effects will be insufficient, leading to increased portal vein pressure, uneven perfusion of the hepatic sinusoids, and microthrombus formation. If the concentration is too high, it can easily cause excessive vasodilation, increase the risk of bleeding, or make the perfusion fluid too viscous, affecting circulatory stability.

[0031] Adjusting the concentration of HEPES buffer can maintain a stable acid-base environment. If the concentration is too low, the buffering capacity is insufficient, and metabolic acidic substances are easily accumulated during perfusion, leading to a decrease in pH, reduced enzyme activity, and impaired cell function. If the concentration is too high, it will significantly increase osmotic pressure, produce cytotoxicity, and affect hepatocyte survival.

[0032] Regulating inorganic salt concentrations helps maintain normal cell morphology and function. Too low a concentration leads to insufficient ions and low osmotic pressure, easily causing hepatocyte edema and decreased cell membrane stability. Too high a concentration results in high osmotic pressure, causing cell shrinkage, ion imbalance, and interference with signal transduction and enzyme activity. If the amino acid concentration is too low, nutrient supply is insufficient, protein synthesis is hindered, repair capacity is reduced, and hepatocyte activity is difficult to maintain. If the concentration is too high, osmotic pressure increases significantly, metabolic burden is increased, and normal cell metabolism is inhibited. If the vitamin concentration is too low, coenzyme deficiency and weak antioxidant capacity occur, energy metabolism is hindered, and hepatocytes are easily damaged. If the concentration is too high, excess of some vitamins can produce cytotoxicity, interfering with normal physiological functions.

[0033] In the following embodiments, the energy metabolism components include at least one of adenosine, glucose, sodium pyruvate, L-carnitine, and nicotinamide.

[0034] Adenosine, glucose, sodium pyruvate, L-carnitine, and nicotinamide can provide multi-pathway energy metabolism support for hepatocytes. Among them, glucose and sodium pyruvate can serve as basal energy substrates, L-carnitine is beneficial for fatty acid metabolism, adenosine helps regulate cellular energy metabolism, and nicotinamide helps maintain NAD+. + Related metabolic processes, thereby maintaining hepatocyte mitochondrial function and basal metabolic activity.

[0035] In the following embodiments, the hormones and growth factors include at least one of insulin and dexamethasone.

[0036] Insulin and dexamethasone can work together to maintain the functional status of hepatocytes. Insulin helps promote glucose metabolism and glycogen synthesis, while dexamethasone helps maintain hepatocyte polarity and cell function stability, thereby reducing the decline of hepatocyte function during perfusion.

[0037] In the following embodiments, the antioxidant component includes at least one of ascorbic acid 2-phosphate, reduced glutathione (GSH), allopurinol, deferoxamine, N-acetylcysteine, sodium selenite, and zinc sulfate.

[0038] Ascorbic acid 2-phosphate, reduced glutathione (GSH), allopurinol, deferoxamine, N-acetylcysteine, sodium selenite, and zinc sulfate together constitute an antioxidant protection system that can scavenge or inhibit reactive oxygen species generated during perfusion, supplement glutathione-related antioxidant capacity, and reduce iron-mediated oxidative damage, thereby alleviating hepatocyte membrane damage, mitochondrial damage, and apoptosis.

[0039] In the following embodiments, the microcirculation protective factor includes at least one of PGE2, ulinastatin, nitroglycerin, PEG20000, and heparin.

[0040] PGE2, ulinastatin, nitroglycerin, PEG20000, and heparin can work together to improve the microcirculation during isolated liver perfusion. PGE2 and nitroglycerin help maintain vasodilation and reduce perfusion resistance, ulinastatin helps reduce inflammatory damage, PEG20000 helps maintain colloid osmotic pressure and reduce tissue edema, and heparin helps reduce coagulation and microcirculatory obstruction, thereby stabilizing portal vein pressure and improving uniform liver perfusion.

[0041] In the following embodiments, the inorganic salt includes at least one of NaCl, KCl, CaCl2, MgSO4, magnesium chloride hexahydrate, Na2HPO4, and KH2PO4.

[0042] NaCl, KCl, CaCl2, MgSO4, magnesium chloride hexahydrate, Na2HPO4, and KH2PO4 can maintain the osmotic pressure, ion balance, and buffering capacity of the perfusion fluid. Among them, sodium, potassium, calcium, and magnesium ions help maintain hepatocyte membrane potential, cell signal transduction, and enzymatic reactions, while phosphate components help stabilize the perfusion fluid buffer system, thereby providing a perfusion environment close to physiological conditions for isolated livers.

[0043] In the following embodiments, the amino acid includes at least one selected from L-arginine, L-histidine, L-lysine, L-glutamine, L-proline, L-serine, glycine, L-alanine, L-asparagine monohydrate, L-cysteine, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0044] A variety of amino acids can provide basic nutritional support and metabolic substrates for isolated livers. Among them, L-glutamine can support the urea cycle and nitrogen metabolism, while L-serine, glycine, and L-cysteine ​​can serve as precursors for glutathione synthesis. L-arginine, L-histidine, and other amino acids help maintain basic nutrition and buffer stability, thereby supporting hepatocyte metabolic function and reducing perfusion injury.

[0045] In the following examples, the vitamins include at least one of folic acid, thiamine HCl, riboflavin, calcium pantothenate, choline, inositol, vitamin A, α-tocopherol phosphate, nicotinamide, pyridoxine hydrochloride, and sodium riboflavin 5'-phosphate.

[0046] Folic acid, thiamine HCl, riboflavin, calcium pantothenate, choline, inositol, vitamin A, α-tocopherol phosphate, nicotinamide, pyridoxine hydrochloride, and riboflavin 5'-phosphate sodium salt can provide coenzyme, lipid metabolism, redox reaction, and antioxidant support for hepatocytes. Among them, vitamin A helps maintain the storage function of hepatocytes, and α-tocopherol phosphate has a lipid-soluble antioxidant effect, thereby further enhancing the activity maintenance effect of isolated liver.

[0047] In the following embodiments, the application of the ambient temperature in vitro perfusion solution for maintaining liver viability in the preservation of ex vivo liver is described.

[0048] When the perfusion solution of this invention is used for the preservation of ex vivo livers, it can maintain the liver's oxygen supply, energy metabolism, antioxidant capacity and microcirculation stability under normal temperature in vitro perfusion conditions, thereby delaying the damage process of ex vivo livers, prolonging the maintenance time of liver activity, and providing a stable perfusion environment for liver preservation before transplantation, functional assessment and in vitro experiments.

[0049] In the following embodiments, a method for preparing a room-temperature in vitro perfusion solution for maintaining liver activity is as follows: A high-efficiency oxygen carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, vitamins, and water are mixed, the osmotic pressure is adjusted to 290-310 mOsm / kg, and the volume is brought to a final volume to obtain the room-temperature in vitro perfusion solution for maintaining liver activity.

[0050] A method for preparing a normothermic in vitro perfusion solution for maintaining liver viability involves mixing a highly efficient oxygen-carrying carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, and vitamin components in a specific ratio. The osmotic pressure is adjusted to 290–310 mOsm / kg, resulting in a perfusion solution with an osmotic pressure and ion environment close to physiological conditions. This avoids hepatocyte edema or shrinkage caused by abnormal osmotic pressure and allows various active components to coexist stably, synergistically exerting their functions of oxygen delivery, energy supply, anti-oxidative damage, and microcirculation protection. The preparation process is simple, the components are clearly defined, and the reproducibility is high, making it convenient for application in normothermic in vitro liver perfusion experiments or in vitro liver preservation.

[0051] Experimental materials Rat erythrocytes were purchased from Hongquan Biotechnology Co., Ltd., model number HQ80086-020, with a concentration of 20%.

[0052] Processed rat red blood cells: Centrifuge rat red blood cells at 3000 rpm for 10 minutes, discard the upper layer of plasma and the components used to preserve the red blood cells, and retain the lower layer to obtain the processed rat red blood cells.

[0053] Example 1 A method for preparing a normothermic extracorporeal perfusion solution for maintaining liver viability is as follows: The following ingredients were added: 70g high-efficiency oxygen carrier HBOC, 18.7mg adenosine, 1.0g glucose, 1.0g sodium pyruvate, 483.6mg L-carnitine, 1221.3mg nicotinamide, 0.035mg insulin, 0.03mg dexamethasone, 0.1mg ascorbic acid 2-phosphate, 3.07g reduced glutathione (GSH), 13.6mg allopurinol, 65.7mg deferoxamine, 0.816g N-acetylcysteine, 0.003mg sodium selenite, 0.03mg zinc sulfate, 0.05mg PGE2, 7g ulinastatin, 100μg nitroglycerin, 5g PEG20000, 0.001mg heparin, 7.15g HEPES buffer, 400mg KCl, 140mg CaCl2, 97.67mg MgSO4, 200mg magnesium chloride hexahydrate, and 190mg... Na₂HPO₄, 60mg KH₂PO₄, 1.0g L-arginine, 1.0g L-histidine, 125mg L-lysine, 584mg L-glutamine, 30mg L-proline, 200mg L-serine, 200mg glycine, 225mg L-alanine, 284mg L-asparagine monohydrate, 120mg L-cysteine, 250mg L-isoleucine, 125mg L-leucine, 94mg L-lysine hydrochloride, 75mg L-methionine, 125mg L-phenylalanine, 300mg L-threonine, 20mg L-tryptophan, 300mg L-tyrosine and 100mg L-valine, 1mg folic acid, 1mg thiamine HCl, 1mg riboflavin, 1mg calcium pantothenate, 1.7mg choline, 2.02mg inositol, 0.1mg vitamin A, 0.1mg α-Tocopherol phosphate, 1 mg nicotinamide, 1 mg pyridoxine hydrochloride, 0.1 mg riboflavin 5'-phosphate sodium salt were mixed with 800 mL of water, the osmotic pressure was adjusted to 300 mOsm / kg with NaCl, and water was added to a final volume of 1 L to obtain the ambient temperature in vitro perfusion solution used to maintain liver activity.

[0054] Comparative Example 1 A method for preparing a conventional perfusion fluid is as follows: Mix 20g bovine serum albumin (BSA), 2.1g NaHCO3, and 800mL Williams E medium (1×). Adjust the CaCl2 solution to the desired concentration. 2+ The concentration was adjusted to 1.1 mM, and Williams E medium (1×) was added to bring the volume to 1 L to obtain the conventional perfusion solution.

[0055] The difference between Comparative Example 1 and Example 1 is that the conventional perfusion solution of Comparative Example 1 mainly provides Williams E medium (1×) basal medium, bovine serum albumin (BSA) protein carrier and NaHCO3 bicarbonate buffer system, and does not contain HBOC, adenosine, glucose, sodium pyruvate, L-carnitine, nicotinamide, insulin, dexamethasone, antioxidant components, microcirculation protective factors, amino acids and vitamins added in Example 1.

[0056] Test Example 1 The ambient temperature in vitro perfusion solution prepared by the method in Example 1 for maintaining liver viability was used as the perfusion solution for the experimental group, and the conventional perfusion solution prepared by the method in Comparative Example 1 was used as the perfusion solution for the control group. Three parallel samples were set up, each with 46.5 mL, designated as experimental group 1-3 and control group 1-3. Each parallel sample corresponded to one isolated SD rat liver. 20 mL of treated rat red blood cells were added to experimental group 1-3 and control group 1-3 to ensure oxygenation and circulatory function during perfusion.

[0057] The isolated SD rat livers obtained in 1.1 and samples collected during in vitro perfusion at room temperature were compared with the experimental groups 1-3 and the control group 1-3 in maintaining the viability of isolated rat livers using the detection methods described in 1.2-1.8.

[0058] 1.1 Obtaining and perfusing isolated SD rat livers at room temperature 1.1.1 Pretreatment of SD rats: SPF-grade healthy male SD rats weighing 250–300g were selected. They were fasted for 12 hours before the operation but allowed free access to water. They were anesthetized with isoflurane gas. After the anesthesia took effect, the rats were fixed on the operating table, the hair on their abdomen was removed, and the abdomen was disinfected with 75% alcohol.

[0059] 1.1.2 Ex vivo liver: A transverse abdominal incision was made along the lower edge of the costal arch. After opening the abdominal cavity, the xiphoid process was flipped upwards and fixed with hemostatic forceps. A cotton swab moistened with normal saline was used to gently push the gastrointestinal tract to the left side, and the area was covered with normal saline gauze for moisture retention. The perihepatic ligaments and the inferior vena cava were separated, and 1 mL of heparinized saline with a concentration of 100 IU / mL was injected through the inferior vena cava. The bile duct was separated and a bile duct cannula was connected. The left and right branches of the portal vein were freed and ligated, and the portal vein cannula was connected and fixed. The diaphragm was cut to induce cardiac arrest. The inferior vena cava was ruptured, and 10 mL of the experimental group perfusion fluid / control group perfusion fluid at 4℃ was injected through the portal vein cannula for pre-rinsing. The liver was then fully freed and weighed.

[0060] 1.1.3 Normative extracorporeal perfusion: After ex vivo, the livers were rinsed and rewarmed via the portal vein with room temperature saline. The ex vivo livers were then connected to room temperature mechanical perfusion systems containing the experimental group perfusion solution and the control group perfusion solution, respectively, and connected to an oxygen generation system. The oxygen flow rate was set at 1 L / min, and the portal vein perfusion solution flow rate was 1–1.5 mL / min / g liver weight. The ex vivo livers of SD rats in experimental groups 1–3 and control groups 1–3 were subjected to room temperature in vitro mechanical perfusion using the experimental group perfusion solution and the control group perfusion solution, respectively. Bile secretion and portal vein pressure were recorded during perfusion, and circulating perfusion fluid was collected for subsequent analysis.

[0061] 1.2 The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the perfusion fluid were measured to observe the degree of hepatocellular damage. During the normothermic in vitro perfusion process (1.1.3), perfusion fluid was collected from the perfusion fluid circulation system of the experimental group at 0h, 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h; perfusion fluid was collected from the perfusion fluid circulation system of the control group at 0h, 0.5h, 1h, 2h, and 3h. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the perfusion fluid of both groups were detected using a fully automated biochemical analyzer. Figure 1 ).

[0062] The results of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities showed that during normothermic in vitro perfusion, the activities of ALT and AST in the liver perfusion fluid of the experimental groups increased slightly over time. At 6 hours, ALT in experimental groups 1, 2, and 3 was approximately 120, 15, and 5 U / L, respectively, and AST was approximately 180, 140, and 70 U / L, respectively. The overall increase was slow, indicating milder hepatocellular damage. In the control group, ALT and AST activities increased significantly at 3 hours of perfusion, with ALT at approximately 530, 470, and 310 U / L, and AST at approximately 870, 780, and 500 U / L, respectively, far exceeding those in the experimental groups, suggesting significant and early-onset hepatocellular damage in the control group.

[0063] The results showed that the room-temperature in vitro perfusion solution prepared by the method in Example 1, used to maintain liver activity, could effectively reduce hepatocyte damage and maintain stable liver function, while the conventional perfusion solution prepared by the method in Comparative Example 1 significantly aggravated hepatocyte damage and rapidly increased enzyme activity.

[0064] 1.3 Liver tissue was stained with hematoxylin and eosin (HE) to observe liver tissue structure. In the normothermic in vitro perfusion process (1.1.3), the perfusion fluid circulation system for the experimental group was shut down 6 hours after perfusion, and the perfusion fluid circulation system for the control group was shut down 3 hours after perfusion. Liver tissues from both groups were collected after the perfusion was stopped. The tissues were prepared by routine formaldehyde fixation and paraffin embedding, and then stained with hematoxylin and eosin (HE). The integrity of liver lobule structure, hepatocyte arrangement, and necrosis / vacuolation were observed under a low-power microscope (5×), and the morphology of hepatocyte nuclei, chromatin distribution, and cell boundaries were observed under a high-power microscope (40×). By comparing the sections from the experimental and control groups, the protective effect of the optimized perfusion fluid on liver tissue integrity and hepatocyte morphology was evaluated. Figure 2 ).

[0065] Low-magnification (5×) observation showed that the experimental group had intact liver tissue structure, clear lobular outlines, regular hepatocyte arrangement, and uniform distribution of blood vessels and bile ducts, with no obvious necrosis or large areas of vacuolation. The control group's liver lobular structure remained partially intact, but local liver tissue disorder was observed, along with mild vacuolation and necrosis areas.

[0066] Further observation at high magnification (40×) revealed that the experimental group showed intact hepatocyte nuclei with uniform chromatin distribution and clear intercellular boundaries, with only occasional scattered vacuolated cells, and overall good cellular integrity. In the control group, some hepatocytes showed mild nuclei enlargement, uneven chromatin distribution, and blurred cell boundaries and vacuolation in some areas, suggesting that the control group liver tissue had significant perfusion injury or early necrosis.

[0067] The results showed that the structural integrity of the isolated liver in the ambient temperature mechanical perfusion system of the ambient temperature in vitro perfusion fluid for maintaining liver viability prepared by the method of Example 1 was significantly better than that of the conventional perfusion fluid prepared by the method of Comparative Example 1. This indicates that the ambient temperature mechanical perfusion system composed of the ambient temperature in vitro perfusion fluid for maintaining liver viability prepared by the method of Example 1 can effectively reduce hepatocyte damage and maintain liver tissue viability.

[0068] 1.4 The TUNEL assay was used to determine liver cell apoptosis. In the 1.1.3 in vitro perfusion process at room temperature, the perfusion fluid circulation system of the experimental group was shut down 6 hours after perfusion, and the perfusion fluid circulation system of the control group was shut down 3 hours after perfusion. Liver tissues from both groups were collected after the perfusion was stopped. Liver tissues were fixed in formaldehyde, routinely embedded in paraffin in the pathology department, and then the paraffin blocks were prepared into tissue sections. The sections were gently collected on poly-L-lysine slides and dried in a 37°C oven. Before detection, the sections were baked in a 60°C oven for 2 hours. Hepatocyte apoptosis was determined using the TdT-mediated dUTP nick end labeling (TUNEL) method and observed under a light microscope. Five fields of view were randomly selected from each tissue section. In each field of view, the number of red fluorescent cells was observed and counted at a wavelength of 520±20 nm, and the average value represented the number of apoptotic cells. The number of blue fluorescent cells was observed and counted at a wavelength of 620 nm, and the average value was taken as the total number of cells in that field of view. Figure 3 The formula for the apoptosis index (AI) is as follows: Apoptosis index (AI) = Number of apoptotic cells / Total number of cells in the field of view × 100%.

[0069] TUNEL staining results showed that in the experimental group, only a small number of scattered TUNEL-positive cells were observed 6 hours after perfusion, and the overall morphology of the hepatocyte nuclei was relatively intact, indicating a low level of hepatocyte apoptosis. In the control group, TUNEL-positive signals increased significantly 3 hours after perfusion, and more positive fluorescent signals were observed in local necrotic areas, indicating significant DNA breaks and cell death.

[0070] The results showed that the room-temperature in vitro perfusion method prepared in Example 1, used to maintain liver viability, could effectively reduce hepatocyte apoptosis levels and alleviate cell death during room-temperature perfusion of isolated liver, thereby maintaining liver viability.

[0071] 1.5 Transmission electron microscopy observation of hepatocyte mitochondrial status In the 1.1.3 room temperature in vitro perfusion process, the perfusion fluid circulation system of the experimental group was shut down 6 hours after perfusion, and the perfusion fluid circulation system of the control group was shut down 3 hours after perfusion. Liver tissues from both groups were collected after the perfusion was stopped. Blood in the liver tissue was rinsed with PBS, and liver tissue slices approximately 1cm × 1cm × 1mm in size were cut and fixed in electron microscopy fixative at room temperature for 1 hour, followed by fixation at 4℃. The samples were then sent to Wuhan Saiwei Company for transmission electron microscopy sample processing and image acquisition. Figure 4 ).

[0072] Transmission electron microscopy revealed that the experimental group hepatocytes exhibited intact overall structure, homogeneous cytoplasm, and clear cell boundaries. The nuclei were oval-shaped with intact nuclear membranes and slightly increased heterochromatin. Mitochondrial damage was mild; most mitochondria maintained normal morphology, with only a few showing slight dissolution of the intracellular matrix, cristae breakage, or reduction. Rough endoplasmic reticulum was abundant, with slight local swelling, and no obvious degranulation of surface ribosomes. A small number of autolysosomes were observed. Overall, the experimental group hepatocytes showed mild damage and largely intact mitochondrial structure. In contrast, the control group showed blurred cell boundaries, uneven cytoplasmic dissolution, and significant organelle damage. The nuclear membranes were blurred, and heterochromatin was increased. Mitochondria showed severe damage, with numerous swollen and enlarged mitochondria exhibiting membrane structure disruption, severe matrix dissolution, and cristae breakage or disappearance. A small number of lipid droplets and autolysosomes were also observed.

[0073] The results showed that the room-temperature in vitro perfusion solution prepared by the method in Example 1, used to maintain liver viability, could effectively protect the ultrastructure of hepatocytes, especially reducing mitochondrial damage; while the conventional perfusion solution prepared by the method in Comparative Example 1 caused more severe hepatocyte damage and significant destruction of mitochondrial structure.

[0074] 1.6 Detection of bile secretion During the normothermic extracorporeal perfusion process in 1.1.3, the bile status in the bile drainage tube of the experimental group was observed and the cumulative bile production was recorded at 0h, 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h. The bile status in the bile drainage tube of the control group was observed and the cumulative bile production was recorded at 0h, 0.5h, 1h, 2h, and 3h. Higher bile secretion indicated better maintenance of liver function. Figure 5 ).

[0075] The results of bile secretion analysis showed that during in vitro perfusion, the total amount of bile accumulated in the liver of the experimental groups continuously increased over time. The bile volume in experimental groups 1, 2, and 3 increased from 0 mL at 0 h to approximately 2.9-3.5 mL at 6 h, indicating that liver function remained good and the perfusion system effectively maintained bile secretion. The control group was perfused only up to 3 h, and its total bile accumulation was significantly lower than that of the experimental groups. In control groups 1, 2, and 3, the total bile volume at 3 h was approximately 0.6-0.8 mL, suggesting a rapid decline in hepatocyte function within a short period.

[0076] The results showed that the ambient temperature mechanical perfusion system for maintaining liver activity using the ambient temperature in vitro perfusion solution prepared by the method in Example 1 could better maintain liver secretory function, reduce hepatocyte damage, and prolong liver activity.

[0077] 1.7 Portal vein pressure measurement During the normothermic extracorporeal perfusion process in 1.1.3, pressure values ​​were recorded in the experimental group at 0h, 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h, while pressure values ​​in the control group were recorded at 0h, 0.5h, 1h, 2h, and 3h. Portal vein pressure was used to evaluate hepatic microcirculatory resistance during perfusion; elevated portal vein pressure usually indicates impaired hepatic microcirculation or increased blood flow resistance. Figure 6 ).

[0078] Portal vein pressure (PVP) monitoring results showed that the PVP in the experimental group remained relatively stable during in vitro liver perfusion. In experimental groups 1, 2, and 3, the PVP fluctuated slightly from approximately 9-10 mmHg at 0 h to approximately 8-12 mmHg at 6 h, with relatively small overall changes, suggesting that optimizing the perfusion system can maintain stable hepatic microcirculation. In the control group, the PVP significantly increased at 3 h of perfusion, reaching approximately 15-20 mmHg in control groups 1, 2, and 3, significantly higher than in the experimental group, indicating impaired hepatic microcirculation and increased blood flow resistance in the control group.

[0079] The results showed that the ambient temperature mechanical perfusion system of the ambient temperature in vitro perfusion fluid prepared by the method of Example 1 for maintaining liver activity could maintain the stability of liver microcirculation during in vitro perfusion, while the ambient temperature mechanical perfusion system of the conventional perfusion fluid prepared by the method of Comparative Example 1 was prone to causing portal vein pressure increase, reflecting liver function impairment.

[0080] 1.8 Observation of liver perfusion appearance Observe through real-life photos of the infusion process ( Figure 7 In the experimental group, the liver remained uniformly red and smooth during the 6-hour perfusion period, with no obvious blood stasis or dark spots, indicating that the perfusion system maintained good liver perfusion. After perfusion, the liver volume and texture remained largely intact, with no obvious swelling or discoloration. In the control group, slight dark spots and blood stasis were visible on the liver surface during the 3-hour perfusion period, and the liver volume was slightly enlarged. After perfusion, localized dark red areas appeared, suggesting that the liver perfusion effect in the control group was not as good as that in the experimental group, and microcirculation may have been impaired.

[0081] The results showed that the ambient temperature in vitro perfusion solution prepared by the method in Example 1 could maintain uniform perfusion and good appearance of the liver throughout the perfusion process, while the conventional perfusion solution prepared by the method in Comparative Example 1 had poor perfusion uniformity and may be accompanied by early perfusion injury.

[0082] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A normothermic extracorporeal perfusate for maintaining liver viability, characterized in that, It includes the following components: high-efficiency oxygen carrier HBOC, energy metabolism components, hormones and growth factors, antioxidant components, microcirculation protection factors, HEPES buffer, inorganic salts, amino acids, and vitamins.

2. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The high-efficiency oxygen-carrying carrier HBOC has a concentration of 70 g / L, an energy metabolism component concentration of 3.72 g / L, a hormone and growth factor concentration of 65 μg / L, an antioxidant component concentration of 3.95 g / L, a microcirculation protection factor concentration of 12 g / L, a HEPES buffer concentration of 7.15 g / L, an inorganic salt concentration of 1.09 g / L, an amino acid concentration of 6.45 g / L, and a vitamin concentration of 10.12 mg / L.

3. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The energy metabolism components include at least one of adenosine, glucose, sodium pyruvate, L-carnitine, and nicotinamide.

4. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The hormones and growth factors include at least one of insulin and dexamethasone.

5. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The antioxidant components include at least one of the following: ascorbic acid 2-phosphate, reduced glutathione (GSH), allopurinol, deferoxamine, N-acetylcysteine, sodium selenite, and zinc sulfate.

6. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The microcirculation protective factor includes at least one of PGE2, ulinastatin, nitroglycerin, PEG20000, and heparin.

7. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The inorganic salt includes at least one of NaCl, KCl, CaCl2, MgSO4, magnesium chloride hexahydrate, Na2HPO4, and KH2PO4.

8. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The amino acid includes at least one of L-arginine, L-histidine, L-lysine, L-glutamine, L-proline, L-serine, glycine, L-alanine, L-asparagine monohydrate, L-cysteine, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

9. The normothermic extracorporeal perfusion solution for maintaining liver viability as described in claim 1, characterized in that, The vitamins include at least one of folic acid, thiamine HCl, riboflavin, calcium pantothenate, choline, inositol, vitamin A, α-tocopherol phosphate, nicotinamide, pyridoxine hydrochloride, and sodium riboflavin 5'-phosphate.

10. The use of the ambient temperature in vitro perfusion solution for maintaining liver viability as described in any one of claims 1-9 in the preservation of ex vivo livers.

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