Organ perfusate and preparation method thereof

Abstract

The invention provides an organ perfusate and a preparation method thereof. The Na ion concentration of the organ perfusate is 110-130 mmol / L, and the K ion concentration of the organ perfusate is 20-30 mmol / L; the organ perfusate is prepared from 0.024 mmol / L to 0.036 mmol / L of a colloid osmotic pressure substance, 100 mmol / L to 103 mmol / L of an impermeable substance, 0.001 mmol / L to 0.025 mmol / L of a cell protecting agent, 23 mmol / L to 38 mmol / L of a buffering substance, 3.015 mmol / L to 8.06 mmol / L of calcium and magnesium inorganic salt, 2.5 mmol / L to 6.5 mmol / L of an antioxidant and the balance of an energy substrate. Wherein the molar osmotic pressure of the organ perfusate is 290-320 mOsm / kg, and the viscosity of the organ perfusate at a preset temperature is lower than 2.0 cP; the colloid osmotic pressure substance is Pluronic F-68; and the cell protective agent is thyroxine.

Description

Technical Field

[0001] This invention belongs to the field of organ perfusion fluid technology, and particularly relates to an organ perfusion fluid and its preparation method. Background Technology

[0002] Organ transplantation is the only effective treatment for various end-stage organ diseases. With the rapid development of organ transplant medicine, the survival rate of organ transplant patients has significantly improved, and organ transplantation is now widely practiced worldwide. The number of patients waiting for transplants is constantly increasing, far exceeding the number of organ donors, resulting in a severe shortage of organ supply. High-quality donors are a prerequisite and fundamental guarantee for successful organ transplantation. Static cryopreservation is currently the most widely used method of organ preservation in clinical practice.

[0003] Currently, ischemia-reperfusion injury caused by cryopreservation of donor organs will affect the prognosis of liver transplant recipients. Avoiding or reducing this injury can improve the function of transplanted organs and the survival of recipients. There are two existing methods to reduce the damage caused by ischemia-reperfusion of transplanted organs: (1) cryopreservation of organs using organ preservation solution; (2) preservation of organs using mechanical perfusion. Although there is evidence that mechanical perfusion is superior to cryopreservation, there is no consensus among centers on many key parameters, including temperature, flow rate, pressure, perfusion pathway and oxygenation. Therefore, cryopreservation is still the gold standard for ex vivo liver preservation. At present, the improvement of donor liver preservation methods is mainly focused on adjusting the different components of organ preservation solution. The main functions that low-temperature organ perfusion solution needs to have include: maintaining osmotic pressure balance to avoid tissue and cell edema, maintaining pH balance to avoid acidosis, providing energy metabolism substrates, and anti-oxidation. Low-temperature organ perfusion fluid is used for surface flushing and internal irrigation of kidneys, livers, and pancreas during organ removal from donors, as well as for cryopreservation or low-temperature mechanical perfusion during transport and storage, in order to prepare the organs for preservation, transport, and eventual transplantation into recipients.

[0004] While static cryopreservation is effective in maintaining lung graft survival for approximately 6-8 hours, prolonged exposure to cold ischemia ultimately leads to irreversible organ damage. The graft is further damaged when oxygenated blood flow is restored at body temperature. To address organ ischemia-reperfusion (IR) injury caused by cellular stress during cryopreservation and ischemia-reperfusion, we introduced a novel cytoprotective agent, thyroxine. As a naturally occurring hormone secreted by the thyroid gland, thyroxine's physiological functions are closely related to cellular metabolic regulation, structural stability, and anti-oxidative stress, providing a potential pathway to solve the core problems of cryopreservation: thyroxine binds to intracellular thyroxine receptors (TR), regulating the expression of mitochondrial respiratory chain-related genes (such as cytochrome c oxidase and ATP synthase), thereby enhancing mitochondrial function and promoting aerobic metabolic efficiency. Under cryogenic conditions, it does not "activate" metabolism (avoiding excessive energy consumption), but rather maintains basal cellular metabolic activity through "fine-tuning," delaying ATP depletion, reducing lactic acid accumulation caused by anaerobic glycolysis, and alleviating intracellular acidosis. Studies have shown that thyroxine has a specific protective effect on metabolically active organs (such as the heart, liver, and kidneys), maintaining the activity of core function-related enzymes (such as bile synthases in hepatocytes and contraction-related proteins in cardiomyocytes). In the cryopreservation of ex vivo organs, this property helps reduce the denaturation or degradation of function-related proteins, laying the foundation for rapid organ function recovery after reperfusion.

[0005] Common organ preservation solutions include UW solution, HTK solution, Celsior solution, and IGL-1 solution. UW solution and HTK solution are the most commonly used. UW solution (University of Wisconsin solution) was initially developed by Belzer et al. at the University of Wisconsin for the preservation of the liver and pancreas. UW solution is an intracellular fluid-type preservation solution, high in potassium (125 mmol / L) and low in sodium (36 mmol / L), using non-osmotic lactobionate and raffinose to prevent cell edema. Hydroxyethyl starch (HES) acts as a colloid to prevent tissue edema, phosphate acts as a buffer system to prevent cellular acidosis, glutathione and allopurinol scavenge oxygen free radicals, and adenosine provides a precursor for ATP synthesis as an energy substrate. UW solution has shown good preservation effects on various organs, including the heart, lungs, liver, kidneys, pancreas, and small intestine. Upon its emergence, it quickly replaced Euor-Collins solution, becoming the most widely used organ preservation solution and gradually becoming the "gold standard" for organ preservation. However, UW fluid also has some limitations, such as the risk of cardiac arrest due to high potassium; the high viscosity of the fluid and the presence of insoluble particles can lead to microcirculatory ischemia, which in turn can cause ischemic complications in the liver tissue and biliary system. The high viscosity problem is mainly due to the colloidal substance HES. Attached Figure Description

[0006] Figure 1The diagram shows LDH data from Examples 1-4 and Comparative Example 1, which are embodiments of the present invention.

[0007] Figure 2 The figures provided are schematic diagrams of MTT data from Examples 1-4 and Comparative Example 1, which are embodiments of the present invention.

[0008] Figure 3 Schematic diagram of mitochondrial superoxide data from Examples 1-4 and Comparative Example 1 provided for embodiments of the present invention. Summary of the Invention

[0009] This invention provides an organ perfusion fluid and its preparation method, which is used for low-temperature static refrigeration or low-temperature mechanical perfusion of organs. It can solve the problems of high viscosity, high potassium risk and high cost of the above-mentioned commercially available products, and can effectively reduce organ damage caused by cold ischemia and ischemia-reperfusion.

[0010] The technical solutions provided by the embodiments of the present invention are as follows:

[0011] On the one hand, an organ perfusion solution is provided, wherein the Na ion concentration of the organ perfusion solution is 110-130 mmol / L and the K ion concentration is 20-30 mmol / L;

[0012] The organ perfusion fluid comprises: 0.024–0.036 mmol / L of a colloidal osmotic pressure substance with a viscosity less than 2.0 cP; 100–103 mmol / L of a non-permeable substance; 0.005–0.025 mmol / L of a cell protectant; 23–38 mmol / L of a buffer; 3.015–8.06 mmol / L of calcium and magnesium inorganic salts; 2.5–6.5 mmol / L of an antioxidant; and the balance being an energy substrate. The colloidal osmotic pressure substance is Pluronic F-68; and the cell protectant is thyroxine.

[0013] The organ perfusion fluid has a molar osmotic pressure of 290–320 mOsm / kg and a viscosity of less than 2.0 cP at a preset temperature.

[0014] In one alternative embodiment, the colloidal osmotic pressure material is Pluronic F-68.

[0015] The non-permeable substance includes at least one of raffinose pentahydrate, lactobionic acid, and mannitol.

[0016] In one optional embodiment, when the non-permeable substance comprises a mixture of raffinose pentahydrate, lactobionic acid, and mannitol, the mixing ratio of raffinose pentahydrate, lactobionic acid, and mannitol is 23–25:45–55:30.

[0017] In one optional embodiment, the cell protectant is triiodothyronine or tetraiodothyronine;

[0018] The concentration of triiodothyronine or tetraiodothyronine is 0.005–0.025 mmol / L.

[0019] In one alternative embodiment, the buffering substance comprises: potassium dihydrogen phosphate and / or hydroxyethylpiperazine ethanesulfonic acid or sodium bicarbonate.

[0020] In one optional embodiment, the molar concentration of potassium dihydrogen phosphate is 20-30 mmol / L, and the molar concentration of hydroxyethylpiperazine ethanesulfonic acid is 3-8 mmol / L.

[0021] In one optional embodiment, the calcium-magnesium inorganic salt comprises calcium chloride and / or magnesium chloride, wherein the concentration of calcium chloride is 0.015–0.06 mmol / L and the concentration of magnesium chloride is 3–8 mmol / L.

[0022] In one optional embodiment, the antioxidant comprises: allopurinol and / or glutathione;

[0023] The energy substrates include glucose and adenine nucleoside;

[0024] The molar concentrations of allopurinol and glutathione are 2.5–6.5 mmol / L.

[0025] The molar concentration of glucose is 5–15 mmol / L;

[0026] The molar concentration of the adenine nucleoside is 2–10 mmol / L.

[0027] On the other hand, a method for preparing organ perfusion fluid is provided, in which a buffer substance of 23 mmol / L to 38 mmol / L is added to water for injection to dissolve the fluid.

[0028] Dissolve in inorganic salts at concentrations of 3.015 mmol / L to 8.06 mmol / L;

[0029] Dissolve in 2.5–6.5 mmol / L of antioxidant;

[0030] Dissolve in 0.001–0.025 mmol / L of cell protectant;

[0031] Add energy substrate to dissolve;

[0032] After all other components have completely dissolved, add the colloidal osmotic pressure substance to dissolve it.

[0033] Sodium hydroxide was added to adjust the pH value to a preset value to obtain the organ perfusion solution;

[0034] The organ perfusion fluid has a molar osmotic pressure of 290–320 mOsm / kg and a viscosity of less than 2.0 cP at a preset temperature.

[0035] The organ perfusion solution provided by the embodiments of the present invention has at least the following beneficial effects:

[0036] The organ perfusion solution provided in this embodiment of the invention provides the required colloidal osmotic pressure through the preferred colloidal substance Pluronic F-68, and also has a relatively low viscosity, which is less than 2.0 cP. It can stabilize and protect the cell membrane, reduce membrane permeability, and thus prevent permeable cell swelling and vascular endothelial damage. The addition of thyroxine as a cell protectant promotes mitochondrial function and reduces cell apoptosis to protect organ cells during the cryopreservation process. Detailed Implementation

[0037] Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art.

[0038] The term "comprising" and its variations as used herein signify open inclusion, i.e., "including but not limited to". Unless otherwise stated, the term "or" means "and / or". The term "based on" means "at least partially based on". The terms "one example embodiment" and "one embodiment" mean "at least one example embodiment". The term "another embodiment" means "at least one additional embodiment". The terms "first", "second", etc., may refer to different or the same objects. Other explicit and implicit definitions may also be included below.

[0039] The organ perfusion fluid provided by the present invention will be further explained and described below through optional embodiments.

[0040] The organ perfusion fluid provided in this embodiment of the invention has a high Na ion concentration and a low K ion concentration. For example, the Na ion concentration of the organ perfusion fluid provided in this embodiment of the invention can be 110 mmol / L, 113 mmol / L, 115 mmol / L, 117 mmol / L, 119 mmol / L, 120 mmol / L, 122 mmol / L, 124 mmol / L, 125 mmol / L, 126 mmol / L, 127 mmol / L, 128 mmol / L, 129 mmol / L, or 130 mmol / L, etc., and the K ion concentration can be 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, or 30 mmol / L.

[0041] In organ perfusion fluid, the molar concentration of colloidal osmotic substances can be 0.024 mmol / L, 0.025 mmol / L, 0.026 mmol / L, 0.027 mmol / L, 0.028 mmol / L, 0.029 mmol / L, 0.030 mmol / L, 0.032 mmol / L, 0.033 mmol / L, 0.034 mmol / L, or 0.036 mmol / L.

[0042] The molar concentration of the cytoprotective agent in the organ perfusion fluid can be 0.001 mmol / L, 0.002 mmol / L, 0.003 mmol / L, 0.004 mmol / L, 0.005 mmol / L, 0.006 mmol / L, 0.007 mmol / L, 0.008 mmol / L, 0.009 mmol / L, 0.01 mmol / L, 0.011 mmol / L, 0.012 mmol / L, 0.013 mmol / L, 0.014 mmol / L, 0.015 mmol / L, 0.016 mmol / L, 0.017 mmol / L, 0.018 mmol / L, 0.019 mmol / L, 0.020 mmol / L, 0.021 mmol / L, 0.022 mmol / L, 0.023 mmol / L, 0.024 mmol / L, or 0.025 mmol / L.

[0043] The colloidal osmotic pressure-regulating substance is Pluronic F-68; the cell protectant is thyroxine.

[0044] Pluronic F-68 is a triblock copolymer composed of polyethylene oxide (PEO), polypropylene oxide (PPO), and polyethylene oxide (PEO). It possesses good water solubility and low viscosity, and is commonly used as an excipient in various formulations and drug delivery systems in the pharmaceutical industry. It can also effectively repair damaged cell membranes. Related studies have shown that Pluronic F-68 can reduce platelet aggregation and may aid microcirculation during organ reperfusion; it can also be used as a plasma substitute for pre-filling cardiopulmonary bypass devices; and it exhibits significant cytoprotective effects on plant cells recovered from cryopreservation. Therefore, Pluronic F-68 has the potential to replace HES as a colloidal substance in organ perfusion fluids.

[0045] In one alternative implementation, Pluronic F-68, as a colloidal osmotic pressure agent, provides the required colloidal osmotic pressure to the perfused tissue while possessing relatively low viscosity. Pluronic F-68 stabilizes and protects cell membranes, reducing membrane permeability and thus preventing osmotic cell swelling and vascular endothelial damage. In this embodiment of the invention, the molecular weight of Pluronic F-68 is 8400. Pluronic F-68 can be replaced to some extent by polyethylene glycol (molecular weight 35000) and dextran 40 (molecular weight 40kDa). In this embodiment of the invention, Pluronic F-68 is preferred. For example, the amount of Pluronic F-68 added can be 0.024 mmol / L, 0.025 mmol / L, 0.026 mmol / L, 0.027 mmol / L, 0.028 mmol / L, 0.029 mmol / L, 0.030 mmol / L, 0.032 mmol / L, 0.033 mmol / L, 0.034 mmol / L or 0.036 mmol / L.

[0046] In one alternative embodiment, the non-permeable substance includes at least one of raffinose pentahydrate, lactobionic acid, and mannitol.

[0047] Non-permeable substances can reduce edema in perfused tissues. Lactobionic acid was chosen as a non-permeable substance because it not only reduces tissue edema but also has certain antioxidant capacity. Raffinose pentahydrate was chosen as a non-permeable substance because it not only reduces tissue edema but is also not directly metabolized by cells.

[0048] For example, the molar concentration of the non-permeable substance can be 100 mmol / L, 102 mmol / L, or 103 mmol / L.

[0049] In one optional embodiment, when the non-permeable substance comprises a mixture of raffinose pentahydrate, lactobionic acid, and mannitol, the mixing ratio of raffinose pentahydrate, lactobionic acid, and mannitol is 23–25:45–55:30. Exemplary examples include mixing ratios of raffinose pentahydrate, lactobionic acid, and mannitol such as 23:50:30, 25:43:30, 23:55:30, 25:51:30, 24:53:30, and 24:54:30.

[0050] In one alternative embodiment, the buffering substance includes: potassium dihydrogen phosphate and / or hydroxyethylpiperazine ethanesulfonic acid or sodium bicarbonate.

[0051] The molar concentration of the buffer substance can be 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, 31 mmol / L, 32 mmol / L, 33 mmol / L, 34 mmol / L, 35 mmol / L, 36 mmol / L, 37 mmol / L, or 38 mmol / L, etc.

[0052] This invention employs potassium dihydrogen phosphate as a buffer substance, which can stabilize the pH of the organ perfusion fluid and provide an appropriate amount of potassium ions, making the organ perfusion fluid an extracellular fluid type. Hydroxyethylpiperazine ethanesulfonic acid (HEPES) can further enhance buffering capacity and stabilize the pH of the organ perfusion fluid. Furthermore, the HEPES buffer zone is closer to physiological values ​​and has better biocompatibility.

[0053] In one optional embodiment, the molar concentration of potassium dihydrogen phosphate is 20–30 mmol / L, and the molar concentration of hydroxyethylpiperazine ethanesulfonic acid is 3–8 mmol / L.

[0054] In this invention, both potassium dihydrogen phosphate and HEPES are used as buffering substances. The molar concentration of potassium dihydrogen phosphate is strictly controlled at 20–30 mmol / L. Exemplarily, the molar concentration of potassium dihydrogen phosphate can be 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, or 30 mmol / L. HEPES can be replaced by sodium bicarbonate, preferably HEPES, and the molar concentration can be 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, or 8 mmol / L.

[0055] In one optional embodiment, the calcium-magnesium inorganic salt comprises calcium chloride and / or magnesium chloride, wherein the concentration of calcium chloride is 0.015–0.06 mmol / L and the concentration of magnesium chloride is 3–8 mmol / L.

[0056] The molar concentrations of calcium and magnesium inorganic salts can be 3.015 mmol / L, 4.015 mmol / L, 5.015 mmol / L, 6.015 mmol / L, 7.015 mmol / L, 8.015 mmol / L, or 8.06 mmol / L.

[0057] Furthermore, in the calcium and magnesium inorganic salts, the concentration of calcium chloride can be 0.015 mmol / L, 0.018 mmol / L, 0.019 mmol / L, 0.025 mmol / L, 0.027 mmol / L, 0.029 mmol / L, 0.035 mmol / L, 0.045 mmol / L, 0.055 mmol / L, or 0.06 mmol / L, and the concentration of magnesium chloride can be 3 mmol / L, 3.5 mmol / L, 3.6 mmol / L, 3.8 mmol / L, 3.9 mmol / L, 4 mmol / L, 5.12 mmol / L, 6.3 mmol / L, 7.4 mmol / L, or 8 mmol / L.

[0058] Calcium chloride can add a small amount of calcium ions to organ perfusion fluid to inhibit calcium overload at low temperatures, while magnesium chloride can provide magnesium ions to organ perfusion fluid. Magnesium sulfate can be used as a substitute, but magnesium chloride is preferred.

[0059] In one optional embodiment, the antioxidant includes allopurinol and / or glutathione; allopurinol and glutathione, as antioxidants, can combat oxygen free radicals. The molar concentration of allopurinol and glutathione is 2.5–6.5 mmol / L; exemplaryly, the molar concentration of the mixture of allopurinol and glutathione can be 2.5 mmol / L, 2.6 mmol / L, 2.8 mmol / L, 3.9 mmol / L, 4 mmol / L, 5.2 mmol / L, 5.3 mmol / L, 5.4 mmol / L, or 6.5 mmol / L.

[0060] The energy substrates include glucose and adenine nucleoside; glucose serves as the energy substrate for cellular metabolism, with a molar concentration of 5–15 mmol / L; exemplarily, the molar concentration of glucose can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, or 15 mmol / L. Adenine nucleoside can be used to address glucose metabolism restriction, rapidly synthesizing ATP under hypoxic conditions, with a molar concentration of 2–10 mmol / L; exemplarily, it can be 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, or 10 mmol / L. Adenine nucleoside can be replaced by a combination of adenine and D-ribose.

[0061] On the other hand, a method for preparing organ perfusion fluid is provided, which involves adding a buffer substance of 20 mmol / L to 30 mmol / L to water for injection to dissolve the fluid.

[0062] Dissolve in inorganic salts at concentrations of 3.015 mmol / L to 8.06 mmol / L;

[0063] Dissolve in 2.5–6.5 mmol / L of antioxidant;

[0064] Add energy substrate to dissolve;

[0065] After all other components have completely dissolved, add the colloidal osmotic pressure substance to dissolve it.

[0066] Sodium hydroxide was added to adjust the pH value to the preset value to obtain the organ perfusion solution;

[0067] The organ perfusion fluid has a molar osmotic pressure of 290–320 mOsm / kg and a viscosity of less than 2.0 cP at a preset temperature.

[0068] The preparation method of organ perfusion fluid provided by the present invention will be described in detail below through specific embodiments.

[0069] Example 1 Preparation method: Taking the preparation of 1L perfusion solution as an example, take 900mL of water for injection, (1) first add the buffer substance potassium dihydrogen phosphate and HEPES and stir until completely dissolved; (2) add the inorganic salts magnesium chloride and calcium chloride and stir until completely dissolved; (3) add the antioxidants allopurinol and glutathione and stir until completely dissolved; (4) add the nutrient glucose and stir until completely dissolved; (5) add raffinose pentahydrate, lactobionic acid, mannitol, adenine nucleoside and tetraiodothyronine in sequence and stir until completely dissolved; (6) after the other components are completely dissolved, add Pluronic F-68 and stir until completely dissolved. (7) Dissolve sodium hydroxide in 100mL of water for injection and add it to the prepared solution. After mixing, wait for 1h and detect the pH value. If necessary, use hydrochloric acid to fine-tune the pH to 7.4. (8) The prepared perfusion solution is filtered through a 0.22μm filter membrane and then sealed for storage.

[0070] The content of the substances added in Example 1 is shown in Table 1.

[0071] Table 1

[0072] Example 1 g / L mmol / L Pluronic F-68 2.5 0.3 Raftilose® pentahydrate 13.7 23.1 Lactobionic acid 17.9 50 Mannitol 5.4 30 L-Thyroxine 0.0077 0.01 Magnesium chloride 0.476 5 Calcium chloride 0.0055 0.05 Potassium dihydrogen phosphate 3.402 25 Allopurinol 0.136 1 Glutathione 0.922 3 Sodium hydroxide 3.2 80 Hydrochloric acid to pH 7.4 / / Glucose 1.8 10 Adenine 1.34 5 HEPES 1.19 5 NaCl 2.34 40

[0073] The operational steps in Examples 2-4 are the same as those in Example 1, except for the specific amounts of substances. The content of each substance in the preparation method of Example 2 is shown in Table 2, and the content of each substance in the preparation method of Example 3 is shown in Table 3.

[0074] Table 2 (Example 2)

[0075] Example 2 g / L mmol / L Pluronic F-68 2.5 0.3 Raftilose® pentahydrate 13.7 23.1 Lactobionic acid 17.9 50 Mannitol 5.4 30 L-Thyroxine 0.0004 0.005 Magnesium chloride 0.476 5 Calcium chloride 0.0055 0.05 Potassium dihydrogen phosphate 3.402 25 Allopurinol 0.136 1 Glutathione 0.922 3 Sodium hydroxide 3.2 80 Hydrochloric acid to pH 7.4 / / Glucose 1.8 10 Adenine 1.34 5 HEPES 1.19 5 NaCl 2.34 40

[0076] Table 3 (Example 3)

[0077]

[0078]

[0079] Table 4 (Example 4)

[0080] Example 4 g / L mmol / L Pluronic F-68 2.5 0.3 Raftilose® pentahydrate 13.7 23.1 Lactobionic acid 17.9 50 Mannitol 5.4 30 Magnesium chloride 0.476 5 Calcium chloride 0.0055 0.05 Potassium dihydrogen phosphate 3.402 25 Allopurinol 0.136 1 Glutathione 0.922 3 Sodium hydroxide 3.2 80 Hydrochloric acid to pH 7.4 / / Glucose 1.8 10 Adenine 1.34 5 HEPES 1.19 5 NaCl 2.34 40

[0081] In addition, the embodiments of the present invention also provide corresponding comparative examples, the specific parameters of each substance in the comparative examples are shown in Table 5.

[0082] Table 5

[0083]

[0084] The method used in Comparative Example 1 is as follows:

[0085] Taking 1L of Comparative Example 1 as an example, take 900mL of water for injection, (1) first add the buffer substance potassium dihydrogen phosphate and stir until completely dissolved; (2) add the inorganic salt magnesium sulfate heptahydrate and stir until completely dissolved; (3) add the antioxidants allopurinol and glutathione and stir until completely dissolved; (4) add raffinose pentahydrate, lactobionic acid and adenine nucleoside in sequence and stir until completely dissolved; (6) after the other components are completely dissolved, add hydroxyethyl starch and stir until completely dissolved. (7) Dissolve potassium hydroxide and sodium hydroxide in 100mL of water for injection, add to the prepared solution, mix well and wait for 1h, detect the pH value, and if necessary, finely adjust the pH to 7.4 with hydrochloric acid. (8) The prepared perfusion solution is filtered through a 0.22μm filter membrane and then sealed for storage.

[0086] Experimental demonstrations showed that the organ perfusion fluid prepared in Examples 1 to 4 of this invention, compared to Comparative Example 1, had the advantage of significantly reducing the viscosity of the perfusion fluid (Table 6) and decreasing cell apoptosis. Figure 1 Promote mitochondrial function Figure 2 ), reduced mitochondrial superoxide production ( Figure 3 The cell protection effect is positively correlated with the amount of thyroxine added as a cell protectant. After reaching a certain amount, the protective effect tends to stabilize. Example 1 is preferred.

[0087] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0088] Viscosity determination: Viscosity was measured at 4°C using a rotational viscometer.

[0089] Table 6

[0090] Sample Viscosity (cP) Example 1 1.82±0.03 Example 2 1.84±0.05 Example 3 1.83±0.02 Example 4 1.82±0.02 Control Example 1 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 Example 18 Example 19 Example 20 Example 21 Example 22 Example 23 Example 24 Example 25 Example 26 Example 27 Example 28 Example 29 Example 30 Example 31 Example 32 Example 33 Example 34 Example 35 Example 36 Example 37 Example 38 Example 39 Example 40 Example 41 Example 42 Example 43 Example 44 Example 45 Example 46 Example 47 Example 48 Example 49 Example 50 Example 51 Example 52 Example 53 Example 54 Example 55 Example 56 Example 57 Example 58 Example 59 Example 60 Example 61 Example 62 Example 63 Example 64 Example 65 Example 66 Example 67 Example 68 Example 69 Example 70 Example 71 Example 72 Example 73 Example 74 Example 75 Example 76 Example 77 Example 78 Example 79 Example 80 Example 81 Example 82 Example 83 Example 84 Example 85 Example 86 Example 87 Example 88 Example 89 Example 90 Example 91 Example 92 Example 93 Example 94 Example 95 Example 96 Example 97 Example 98 Example 99 Example 100 Example 101 Example 102 Example 103 Example 104 Example 105 Example 106 Example 107 Example 108 Example 109 Example 110 Example 111 Example 112 Example 113 Example 114 Example 115 Example 116 Example 117 Example 5.31±0.12

[0091] Liver cell preservation experiment

[0092] Experimental methods:

[0093] Cultured human hepatocytes were washed three times at room temperature with Hank's balanced salt solution free of calcium and magnesium, and then placed in the culture media of Examples 1-4 and Control Example 1, respectively. The culture dishes were then placed in sealed plastic bags and stored at 4°C for 12 h to simulate clinical conditions of cryopreservation of organs. After ischemia, the cells were washed three times at room temperature with Hank's balanced salt solution free of calcium and magnesium. Subsequently, complete cell culture medium (37°C) was added, and reperfusion was simulated by continuing incubation at 37°C under 5% CO2 gas for 4 h. Preservation damage was assessed for each solution using indicators of lactate dehydrogenase (LDH) release, mitochondrial function (MTT), and mitochondrial superoxide levels. Hepatocytes cultured under normal conditions were used as a heat control.

Claims

1. An organ perfusion fluid, characterized in that, The concentration of Na ions in the organ perfusion fluid is 110–130 mmol / L, and the concentration of K ions is 20–30 mmol / L. The organ perfusion solution comprises: 0.024–0.036 mmol / L of colloidal osmotic substances, 100 mmol / L–103 mmol / L of non-osmotic substances, 0.001–0.025 mmol / L of cell protectants, 23–38 mmol / L of buffer substances, 3.015–8.06 mmol / L of calcium and magnesium inorganic salts, 2.5–6.5 mmol / L of antioxidants, and the balance being energy substrates; The colloidal osmotic pressure-regulating substance is Pluronic F-68; the cell protectant is thyroxine. The organ perfusion fluid has a molar osmotic pressure of 290–320 mOsm / kg and a viscosity of less than 2.0 cP at a preset temperature.

2. The organ perfusion fluid according to claim 1, characterized in that, The Pluronic F-68 is a triblock copolymer in the form of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) with a molecular weight of 8400.

3. The organ perfusion fluid according to claim 1, characterized in that, The cell protectant is thyroxine; The thyroxine includes triiodothyronine or tetraiodothyronine, wherein the molar concentration of triiodothyronine or tetraiodothyronine is 0.001 to 0.025 mmol / L.

4. The organ perfusion solution according to claim 1, characterized in that, The non-permeable substance includes at least one of raffinose pentahydrate, lactobionic acid, and mannitol.

5. The organ perfusion solution according to claim 1, characterized in that, When the non-permeable substance comprises a mixture of raffinose pentahydrate, lactobionic acid, and mannitol, the mixing ratio of raffinose pentahydrate, lactobionic acid, and mannitol is 23-25:45-55:

30. The buffering substances include: potassium dihydrogen phosphate and / or hydroxyethylpiperazine ethanesulfonic acid or sodium bicarbonate.

6. The organ perfusion fluid according to claim 5, characterized in that, The molar concentration of potassium dihydrogen phosphate is 20–30 mmol / L, and the molar concentration of hydroxyethylpiperazine ethanesulfonic acid is 3–8 mmol / L.

7. The organ perfusion fluid according to claim 1, characterized in that, The calcium and magnesium inorganic salts include calcium chloride and / or magnesium chloride, wherein the concentration of calcium chloride is 0.015–0.06 mmol / L and the concentration of magnesium chloride is 3–8 mmol / L.

8. The organ perfusion fluid according to claim 1, characterized in that, The antioxidants include: allopurinol and glutathione; The total molar concentration of the antioxidant is 2.5–6.5 mmol / L, the molar concentration of allopurinol is 0.5–1.5 mmol / L, and the molar concentration of glutathione is 2–5 mmol / L. The energy substrates include glucose and adenine nucleoside; The molar concentration of glucose is 5–15 mmol / L; The molar concentration of the adenine nucleoside is 2–10 mmol / L.

9. A method for preparing an organ perfusion solution, characterized in that, Add a buffer solution of 23 mmol / L to 38 mmol / L to the water for injection to dissolve it; Dissolve in inorganic salts at concentrations of 3.015 mmol / L to 8.06 mmol / L; Dissolve in 2.5–6.5 mmol / L of antioxidant; Add energy substrates, non-permeable substances, and cell protectants to dissolve; After all other components have completely dissolved, add the colloidal osmotic pressure substance to dissolve it. Sodium hydroxide was added to adjust the pH value to a preset value to obtain the organ perfusion solution; The organ perfusion fluid has a molar osmotic pressure of 290–320 mOsm / kg and a viscosity of less than 2.0 cP at a preset temperature.

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