Mesenchymal stem cell extracellular vesicle-based organ perfusion preservation solution, preparation and application

Abstract

The application provides an organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles, a preparation method and application of the extracellular vesicles in preparation of the organ perfusion preservation solution. The organ perfusion preservation solution is used for perfusion in ex vivo preservation and transplantation surgery. The organ perfusion preservation solution comprises extracted, purified and freeze-dried umbilical cord mesenchymal stem cell extracellular vesicles. The treated extracellular vesicles contain bioactive components including proteins, lipids, mRNA and miRNA. The organ perfusion preservation solution further comprises a basic HTK solution, standard HTK solution components and excipients. The organ perfusion preservation solution of the application provides more extensive protection at the cellular level by various bioactive components such as proteins, lipids, mRNA and miRNA in the extracellular vesicles, and reduces oxidative stress and metabolic damage during ex vivo preservation.

Description

Technical Field

[0001] This invention relates to organ perfusion preservation solutions, and more particularly to ex vivo organ perfusion preservation solutions, preparation methods, and application technologies. Background Technology

[0002] Liver transplantation is currently the only effective treatment for end-stage liver disease and acute liver failure. However, during transplantation, the liver experiences severe ischemia-reperfusion injury (IRI) due to oxidative stress and inflammatory responses during organ preservation and reperfusion, significantly impacting the functional recovery of the transplanted liver and the patient's postoperative prognosis. Traditional HTK (Histidine-Tryptophan-Ketoglutarate) solutions primarily protect the liver by reducing cellular metabolism and oxidative stress, but they still have significant limitations in anti-inflammatory effects and promoting hepatocyte regeneration and repair. Current research indicates that while existing HTK preservation solutions can provide basic protection in liver transplantation, they cannot effectively prevent inflammatory responses, apoptosis, and impaired functional recovery caused by IRI. In particular, breakthroughs are needed in promoting hepatocyte regeneration and repair and in anti-inflammatory immune regulation.

[0003] Existing modified HTK solution technology is evolving along three main directions: (1) Antioxidant enhancement: such as CN108849861A adding free radical scavengers such as glutathione, but it is limited by the metabolic inactivation and penetration efficiency of small molecule antioxidants; (2) Enhanced metabolic support: by supplementing adenosine, basic amino acids, etc. to improve ATP regeneration efficiency, but it faces the risk of metabolic acidosis caused by increased liquid ammonia; (3) Optimized immune regulation: combined with anti-inflammatory drugs such as glucocorticoids or TNF-α antagonists, but there are side effects of interfering with receptor immune homeostasis. However, the above strategies are mostly single-target interventions, and lack the ability to systematically regulate key immune response events in the IRI process, such as Th1 / Th17 polarization, Kupffer cell activation, and CD4+ T lymphocyte CD154 overexpression.

[0004] Although traditional HTK solution improvement schemes have made progress in metabolic support and local anti-apoptosis, they still have significant limitations in solving the following problems: (1) Single dimension of immune regulation: Existing anti-inflammatory additives only target specific inflammatory mediators and are difficult to block core pathological links such as abnormal T cell activation; (2) Lack of regenerative repair function: There is a lack of regulatory elements for the activation of hepatic progenitor cells and vascular endothelial repair; (3) Formulation compatibility conflict: The stability of small molecule drugs and bioactive components in the preservation solution is mutually restrictive.

[0005] Therefore, a more effective organ preservation solution is needed to address the problems existing in current technologies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an organ perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells, a preparation method thereof, and the application of extracellular vesicles in the preparation of organ perfusion preservation solutions, in view of the problems existing in the background art.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides an organ perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells, wherein the organ perfusion preservation solution is used for perfusion in ex vivo preservation and transplantation surgery, and the organ perfusion preservation solution comprises extracellular vesicles of mesenchymal stem cells that have been extracted, purified and freeze-dried, and the processed extracellular vesicles contain bioactive components including proteins, lipids, mRNA and miRNA. The organ perfusion preservation solution also includes basic HTK solution, standard HTK solution components, and excipients.

[0008] Furthermore, the base HTK solution comprises histidine, tryptophan, and α-ketoglutarate.

[0009] Furthermore, the excipient is selected from at least one of antioxidants, energy metabolism substrates, anti-apoptotic agents, cell protectants, and anti-inflammatory and immunomodulatory agents.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned organ perfusion preservation solution, comprising the following steps: (1) Extraction and purification of extracellular vesicles from umbilical cord-derived mesenchymal stem cells; (2) The purified extracellular vesicle suspension was freeze-dried and made into freeze-dried powder; (3) Preparation of organ perfusion preservation solution: (3.1) Prepare HTK solution according to the standard formula, ensuring that all components are fully dissolved and mixed evenly; (3.2) Add the freeze-dried powder of extracellular vesicles of stem cells prepared in step (2) to the HTK solution at a ratio of 100 mg / L and shake it thoroughly to dissolve it completely.

[0011] Furthermore, the extraction and purification of extracellular vesicles specifically includes the following steps: (1.1) Culture P3-P7 generation umbilical cord-derived mesenchymal stem cells and collect the supernatant; (1.2) Preliminary centrifugation: Centrifugation removes cell debris; (1.3) Secondary centrifugation: Centrifugation removes large particles; (1.4) High-speed centrifugation: Centrifuge the treated supernatant to remove fine particles and microparticles; (1.5) Ultracentrifugation: Centrifugation is used to collect extracellular vesicle particles, which will be deposited at the bottom of the tube; (1.6) After resuspending in PBS, the concentration of extracellular vesicle proteins was determined and stored at -80°C for later use.

[0012] Furthermore, the preparation of the lyophilized stem cell extracellular vesicle powder specifically includes the following steps: (2.1) Solution treatment: The extracted and purified extracellular vesicle particles were suspended in an appropriate buffer (such as PBS buffer) to ensure their stability before lyophilization; (2.2) Rapid freezing: The extracellular vesicle solution is rapidly frozen at -80°C or below for several hours, or rapidly frozen in liquid nitrogen to ensure that the extracellular vesicles are not damaged during the freeze-drying process; (2.3) Preparation of freeze-dried powder: The frozen sample is placed in a freeze dryer and freeze-dried in a low temperature and high vacuum environment. The water sublimates, leaving dry extracellular vesicle powder, which is the freeze-dried powder of stem cell extracellular vesicles.

[0013] Furthermore, the preparation method also includes adding excipients to the organ perfusion preservation solution obtained in step (3) and distributing them evenly through operations such as stirring, centrifugation, and shaking.

[0014] Thirdly, the present invention provides a method for using the above-mentioned organ perfusion preservation solution, comprising: When not in use, the extracellular vesicle lyophilized powder should be stored in ampoules at room temperature. The organ perfusion preservation solution of the present invention is mainly used for liver transplantation surgery. Before or during liver transplantation surgery, step (3) of the preparation method is performed, in which lyophilized extracellular vesicles are mixed into HTK solution and used immediately. Then, the liver is perfused using the mixed organ perfusion preservation solution. During perfusion, it is ensured that the organ perfusion preservation solution flows evenly through the liver tissue; and / or, During liver transplantation, organ perfusion preservation solution is used to maintain the liver in optimal condition until the surgery is completed.

[0015] Fourthly, the present invention also provides the application of extracellular vesicles of umbilical cord-derived mesenchymal stem cells in the preparation of organ perfusion preservation solutions.

[0016] The beneficial effects of this invention are: 1. The present invention introduces the lyophilized preparation of umbilical cord mesenchymal stem cell extracellular vesicles (MSC-EVs) into the HTK preservation system, which can not only retain the functional proteins and miRNAs carried by the extracellular vesicles, but also solve the problem of vesicle activity preservation through the lyophilization process, providing full-cycle protection for the liver during the ischemic phase covering antioxidation, anti-inflammatory immunomodulation, and pro-regenerative repair. By applying the MSC-EVs lyophilized powder to improve the preservation effect and functional recovery of the transplanted liver, the common ischemia-reperfusion injury (IRI) and other complications after liver transplantation can be effectively prevented and treated, representing a significant technical breakthrough compared with existing single-mechanism improvement schemes.

[0017] 2. Verified by methods such as sequencing analysis and animal experiments, the present invention has the following distinct technical effects: (1) Significantly improving liver preservation effect: A variety of bioactive components such as proteins, lipids, mRNAs and miRNAs in extracellular vesicles provide more extensive protection at the cellular level, and reduce oxidative stress and metabolic injury during ex vivo preservation.

[0018] (2) Enhancing anti-apoptotic and anti-inflammatory capabilities: The natural antioxidant factors and anti-inflammatory components in extracellular vesicles can effectively scavenge free radicals and inhibit inflammatory responses, thereby alleviating cell damage and dysfunction caused by ischemia-reperfusion injury (IRI). Adding EVs to the HTK solution can significantly reduce the hepatocyte apoptosis index shown by TUNEL staining through delivering specific factors.

[0019] (3) Promoting liver function repair: The regenerative factors in extracellular vesicles can accelerate hepatocyte repair, and improve the recovery speed and quality of the overall function of the transplanted liver.

[0020] (4) Reducing postoperative complications: By reducing postoperative inflammatory responses and cell apoptosis, the incidence of common postoperative complications is lowered, and the survival rate of the transplanted liver and the prognosis of patients are improved.

[0021] (5) Prolonging preservation time: The optimized formula of the modified HTK solution maintains the biological activity of the liver during preservation, prolongs the preservation time, and provides more operation time and flexibility for transplantation surgery. Brief Description of the Drawings

[0022] Figure 1 is the flow chart of 4D-label-free sequencing for extracellular vesicle proteomics in Example 3 of the present invention.

[0023] Figure 2 is the VENN diagram of 4D-label-free sequencing for extracellular vesicle proteomics in Example 3 of the present invention.

[0024] Figure 3This is a graph showing the enrichment results of 4D-label-free sequencing of extracellular vesicle proteomics in Example 3 of this invention.

[0025] Figure 4 This is a transmission electron microscope image of the lyophilized MSC-EVs powder of stem cell extracellular vesicles in Example 4 of the present invention.

[0026] Figure 5 This is a transmission electron microscope image of the control group in Example 4 of the present invention.

[0027] Figure 6 This is a nanoparticle tracking analysis diagram of the lyophilized stem cell extracellular vesicle powder MSC-EVs in Example 4 of the present invention.

[0028] Figure 7 This is a nanoparticle tracking analysis diagram of the control group in Example 4 of the present invention.

[0029] Figure 8 This is a comparison diagram of Western blot experiments between the lyophilized MSC-EVs (stem cell extracellular vesicles) powder and the control group in Example 4 of this invention.

[0030] Figure 9 This is an immunofluorescence image of ordinary HTK solution (control group) labeled with Dir dye perfused for 1 hour in Example 5 of the present invention.

[0031] Figure 10 This is an immunofluorescence image of ordinary HTK solution (control group) labeled with Dir dye perfused for 3 hours in Example 5 of this invention.

[0032] Figure 11 This is an immunofluorescence image of ordinary HTK solution (control group) labeled with Dir dye perfused for 5 hours in Example 5 of this invention.

[0033] Figure 12 This is an immunofluorescence image of HTK solution (experimental group) labeled with Dir dye and containing MSC-EVs perfused for 1 hour in Example 5 of this invention.

[0034] Figure 13 This is an immunofluorescence image of HTK solution (experimental group) labeled with Dir dye and containing MSC-EVs perfused for 3 hours in Example 5 of this invention.

[0035] Figure 14 This is an immunofluorescence image of HTK solution (experimental group) labeled with Dir dye and containing MSC-EVs perfused for 5 hours in Example 5 of this invention.

[0036] Figure 15 This is a graph showing the ALT results of liver function testing in Example 6 of the present invention.

[0037] Figure 16This is a graph showing the AST results of liver function testing in Example 6 of the present invention.

[0038] Figure 17 This is a graph showing the LDH results of liver function testing in Example 6 of the present invention.

[0039] Figure 18 This is a HE staining result of tissues from the non-damaged group (control group) in Example 7 of the present invention.

[0040] Figure 19 This is a HE staining result of the tissue in the ordinary HTK solution perfusion group (comparison group) in Example 7 of the present invention.

[0041] Figure 20 This is a HE staining result of the tissue in the organ perfusion preservation solution perfusion group (experimental group) in Example 7 of the present invention.

[0042] Figure 21 This is a graph showing the Suzuki score results for each group in Embodiment 7 of the present invention.

[0043] Figure 22 This is a Tunel staining result image of the non-damaged group (control group) in Example 7 of the present invention.

[0044] Figure 23 This is a Tunel staining result of the ordinary HTK solution perfusion group (comparison group) in Example 7 of the present invention.

[0045] Figure 24 This is a Tunel staining result of the organ perfusion preservation fluid perfusion group (experimental group) in Example 7 of the present invention.

[0046] Figure 25 This is a schematic diagram of the in vitro perfusion mode in Embodiment 8 of the present invention.

[0047] Figure 26 This is a schematic diagram of the surgical procedure in Embodiment 8 of the present invention. Detailed Implementation

[0048] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be described in detail, clearly, and completely below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Furthermore, based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of the present invention.

[0049] Example 1: An organ perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells.

[0050] This embodiment provides an organ perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells. The components of the organ perfusion preservation solution include: (1) Basic HTK solution, including: histidine, tryptophan and α-ketoglutarate. (2) Other standard HTK solution components, such as buffer solutions, ionic components, etc.; (3) Freeze-dried extracellular vesicle powder of stem cells: extracellular vesicles secreted by umbilical cord mesenchymal stem cells are extracted, purified and freeze-dried. The extracellular vesicles contain bioactive components such as proteins, lipids, mRNA and miRNA. (4) Excipients: Antioxidants (such as glutathione, vitamin C, vitamin E); energy metabolism substrates (such as adenosine, glucose, amino acids); anti-apoptotic agents (such as cyclosporine A); cell protectants (such as HSP, ATP); anti-inflammatory and immunomodulatory agents (such as glucocorticoids, anti-TNF-α antibodies). Excipients can be selected and added according to actual needs, or they can be omitted.

[0051] Example 2: A method for preparing an organ perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells.

[0052] This embodiment provides a method for preparing the organ perfusion preservation solution of Example 1, including the following steps: 1. Extraction and freeze-drying of extracellular vesicles from stem cells.

[0053] The specific steps are as follows: (1.1) Culture P3-P7 generation umbilical cord-derived mesenchymal stem cells and collect serum-free supernatant.

[0054] (1.2) Preliminary centrifugation: remove cell debris by centrifuging at 300-500G for about 10 minutes.

[0055] (1.3) Secondary centrifugation: Centrifuge at 3000G for about 30 minutes to remove large particles.

[0056] (1.4) High-speed centrifugation: Centrifuge the treated supernatant at 10000G for about 30 minutes to remove fine particles and microparticles, and collect the supernatant.

[0057] (1.5) Final ultracentrifugation: Centrifuge at 100,000 G for about 70 min to collect extracellular vesicle particles. The extracellular vesicle particles will be deposited at the bottom of the tube and the precipitate will be collected.

[0058] (1.6) After resuspending with PBS, step (1.5) can be repeated 1-2 times. Then, the extracellular vesicle protein concentration is determined by 4D-label-free and stored below -80℃ for later use.

[0059] 2. Prepare lyophilized extracellular vesicle powder using purified extracellular vesicle suspension.

[0060] The specific steps are as follows: (2.1) Solution treatment: The extracted and purified extracellular vesicle particles are suspended in an appropriate buffer (such as PBS buffer) to ensure that they remain stable before lyophilization.

[0061] (2.2) Rapid freezing: The extracellular vesicle solution is rapidly frozen at -80°C for several hours, or rapidly frozen in liquid nitrogen to ensure that the extracellular vesicles are not damaged during the freeze-drying process.

[0062] (3) Freeze-drying: The frozen sample is placed in a freeze dryer and freeze-dried in a low temperature and high vacuum environment. The water sublimates, leaving dry extracellular vesicle powder.

[0063] The entire process of preparing the lyophilized powder typically takes 24-48 hours. The resulting product is a lyophilized powder of extracellular vesicles from stem cells.

[0064] (3) Preparation of organ perfusion preservation solution.

[0065] (3.1) Prepare HTK solution according to standard formula, ensuring that all components are fully dissolved and mixed evenly.

[0066] (3.2) Add a predetermined amount of lyophilized extracellular vesicle powder of umbilical cord stem cells to the HTK solution, and shake thoroughly to completely dissolve the lyophilized extracellular vesicle powder at a ratio of 100 mg / L to HTK.

[0067] Dissolution rate requirement: The time required for the prepared stem cell extracellular vesicle lyophilized powder to completely dissolve in HTK solution should not exceed 5 minutes. Rapid dissolution can ensure that the extracellular vesicles retain their activity as much as possible before use, and avoid structural damage or reduced activity of the extracellular vesicles due to excessively long dissolution process.

[0068] Quality control indicators after dissolution: The particle size of the dissolved extracellular vesicle lyophilized powder should be between 100-1000 nanometers. Extracellular vesicles of this size are suitable for interacting with cells and are conducive to their function.

[0069] (3.3) Depending on the actual use scenario, other excipients (antioxidants, energy metabolism substrates, etc.) may be added, and their uniform distribution shall be ensured.

[0070] 3. Storage method In this embodiment, under normal conditions, the extracellular vesicle lyophilized powder and the standard HTK solution are stored separately and used immediately. Generally, the extracellular vesicle lyophilized powder is stored at room temperature in ampoules at 100 mg / vial (the dosage can be adjusted according to actual needs). The HTK solution should be stored in a light-protected, sealed container at 4°C to ensure that its active ingredients do not degrade during storage.

[0071] 4. Instructions for use As described in the preservation method, the organ perfusion preservation solution of the present invention is used immediately upon application, and the specific application scenarios are as follows: (1) Ex vivo liver perfusion: Before liver transplantation or during preservation, the liver is used immediately by mixing freeze-dried extracellular vesicles into HTK solution and perfusing the liver with modified HTK solution. During perfusion, ensure that the modified HTK solution flows evenly through the liver tissue to fully exert its protective and repairing effects.

[0072] (2) Transplantation process: During the liver transplantation surgery, the modified HTK solution was continued to be used for perfusion in order to maintain the optimal condition of the liver.

[0073] Example 3: Sequencing of extracellular vesicle protein contents.

[0074] To verify the role of extracellular vesicles in HTK solution, in this embodiment, the prepared lyophilized extracellular vesicle powder was dissolved in HTK solution at a ratio of 100 mg / L, stored on ice, and sent to Lianchuan Biotechnology Co., Ltd. for 4D-label-free protein sequencing.

[0075] The specific steps of sequencing include: Label-free quantification methods utilize mass spectrometry to analyze peptide fragments from enzymatic protein digestion. The basic principle is based on the peak area extracted from the peptide precursor ion (XIC). First, peptides and proteins in the sample are identified. Then, the identified peptides (proteins) are quantitatively analyzed. This allows for the analysis of differences in the same protein across multiple samples using label-free quantification. A 4D label-free sequencing flowchart is shown below. Figure 1 As shown.

[0076] Based on the raw files obtained from mass spectrometry detection, corresponding databases were searched. Protein identification was then performed based on the search results, along with peptide and protein distribution analysis to evaluate the quality of the mass spectrometry data and the database search. Identified proteins were annotated using common functional databases, including the GO database, KEGG database, Reactome database, and subcellular localization. Quantitative analysis, sample correlation analysis, and protein differential analysis were conducted, and GO functional enrichment analysis was performed on differentially expressed proteins. Figure 2As shown, the protein profiles of extracellular vesicles (MSC-EVs) from P5, P6, and P7 passage umbilical cord-derived mesenchymal stem cells were analyzed using VENN plots to comprehensively describe the expression of common and differentially expressed proteins. 997 common protein molecules were identified. GO enrichment results for these common proteins are shown below. Figure 3 As shown, the gene sets for protein folding (GO:0006457) and protein stability (GO:0050821) were significantly enriched, and these functions may play a central role in the mechanism of downstream phenotypes of extracellular vesicles in stem cells.

[0077] It is evident that extracellular vesicles exert the following effects on downstream molecules: (1) enhancing the functional stability of downstream proteins; (2) reducing the toxicity of misfolding or aggregation to cells; and (3) regulating key signaling pathways of oxidative stress, affecting cell survival, metabolism, and differentiation. All of these results suggest that vesicles play a crucial role in post-transplant injury repair.

[0078] Example 4: Transmission electron microscopy and nanoparticle tracking analysis (NTA).

[0079] To determine the effectiveness of the lyophilized powder, this embodiment uses transmission electron microscopy and nanoparticle tracking analysis to analyze the lyophilized stem cell extracellular vesicle powder (experimental group) and the control group (unlyophilized mesenchymal stem cell extracellular vesicles) prepared according to Example 2.

[0080] Using standard HTK solution as the solute, the lyophilized extracellular vesicle powder of stem cells prepared according to Example 2 (stored at room temperature for 1 month) was dissolved and compared with the un-lyophilized group (fresh extracellular vesicles). Figure 4 and Figure 5 As shown, under electron microscopy, the morphology of lyophilized and non-lyophilized extracellular vesicles did not show significant changes, both being biconcave saucer-shaped (scale bar: 100nm).

[0081] Further transmission electron microscopy and nanoparticle tracking analysis (NTA) were performed, such as... Figure 6 and Figure 7 As shown, the peak diameter of the vesicles is all around 100 nm, and the diameter range is 20-1000 nm; Figure 8 As shown, Western blot experiments revealed that, compared with mesenchymal stem cells (MSCs), both groups of MSC-EVs (the lyophilized experimental group and the non-lyophilized control group) expressed CD9 (23 Kda), CD81 (25 Kda), and TSG101 (44 Kda) proteins, meeting the criteria for extracellular vesicle identification.

[0082] Example 5, Immunofluorescence experiment.

[0083] To further verify the effect of MSC-EVs on stem cells during in vitro perfusion, this embodiment used Dir-labeled HTK solution containing MSC-EVs to perfuse rat livers in vitro, and used Dir-labeled ordinary HTK solution as a control group for in vitro rat liver perfusion. Figure 9 , Figure 10 , Figure 11 It was found that after perfusion for 1 hour, 3 hours, and 5 hours, rat hepatocytes phagocytosed Dir-MSC-EVs (…). Figure 12 , Figure 13 , Figure 14 This indicates that MSC-EVs can enter the liver with HTK solution and be absorbed to perform their functions under in vitro perfusion conditions (scale bar: 50 μm, blue in this example represents DAPI fluorescent dye, and red represents Dir dye).

[0084] Example 6: Liver function test.

[0085] To verify the protective effect of the organ perfusion preservation solution of the present invention on liver function, liver function tests were performed on the serum of rats in the no-damage group (control group), the ordinary HTK solution perfusion group (comparative group), and the organ perfusion preservation solution perfusion group of the present invention (experimental group). The test results are as follows: Figure 15 , Figure 16 , Figure 17 As shown in the figure, the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly elevated in the transplant group 24 hours post-surgery, indicating severe post-operative liver damage. In contrast, the transplanted livers perfused with the organ perfusion preservation solution of this invention showed a significant decrease in AST and ALT levels. Figure 17 This demonstrated that there was no statistically significant difference in lactate dehydrogenase (LDH) among the groups (n=5).

[0086] Example 7, staining experiment.

[0087] To verify the histological effects of this invention, this embodiment performed HE staining experiments on the control group (no-damage group), the ordinary HTK solution perfusion group (comparative group), and the organ perfusion preservation solution perfusion group of this invention (experimental group). The results are as follows: Figures 18-20 As shown.

[0088] contrast Figure 18 The control group shown is Figure 19 The standard HTK group (comparison group) showed significant hepatocellular necrosis, portal inflammation, destruction of liver lobule structure, and congestion, with higher Suzuki scores and more severe damage; in contrast, from... Figure 20It can also be clearly seen that after using the organ perfusion preservation solution of the present invention, the above-mentioned phenomena in the liver transplant group (experimental group) were significantly improved, and the Suzuki score was lower. Figure 21 (Scale bar: 200μm) The Suzuki score (mSS) here is a grading system based on clinical imaging examinations.

[0089] from Figure 22 , Figure 23 and Figure 24 The results of TUNEL staining for each group in the previous paragraph are shown. DAPI is a standard fluorescent dye, and Merge is a merged image. The results of TUNEL staining show that, compared with the control group, the apoptosis of the ordinary HTK solution perfusion group (comparison group) was significantly increased; while the apoptotic cells of the organ perfusion preservation solution of the present invention (experimental group) were significantly reduced (scale bar: 200μm).

[0090] Example 8, Animal Experiment (Rat Liver Transplantation Experiment).

[0091] This embodiment provides a complete animal experimental process to enable those skilled in the art to better implement the technical solution of the present invention, and also to more comprehensively demonstrate the technical effects of the present invention. However, those skilled in the art should understand that the specific values ​​in the embodiment are only for better illustrating the technical solution of the present invention and should not be regarded as a limitation of the present invention.

[0092] First, the organ perfusion preservation solution required by the present invention is prepared according to the method of Example 2. The organ perfusion preservation solution formula used in this example is as follows: NaCl 15 mmol / L KCl 8 mmol / l <![CDATA[MnCl2]]> 8 mmol / L Histidine 180 mmol / L Histidine hydrochloride (Histidine-HCl) 15 mmol / L Tryptophan 2 mmol / L Mannitol 20 mmol / L MSC-EVs 100 mg / l water margin In this embodiment, the perfusion method involves pumping the isolated liver through the portal vein and pumping it out through the vena cava for circulation, maintaining the fluid temperature at 4 degrees Celsius during the process. The perfusion pattern is as follows: Figure 25 As shown.

[0093] The entire experimental process is as follows: 1. Liver procurement and preoperative preparation Healthy SD rats, weighing 250-300g, of any sex, were selected. All experimental procedures complied with the "Regulations on the Management of Laboratory Animals" and the "Animal Welfare Requirements".

[0094] Anesthetize donor rats, routinely perform laparotomy to expose the liver ( Figure 26 In step A), a catheter is inserted into the portal vein, and in-situ flushing is performed with low-temperature (4°C) heparinized saline to remove blood and rapidly cool the area to reduce thermal ischemia injury. Simultaneously, the hepatic artery is ligated, and the inferior vena cava and bile duct are severed. Figure 26 (Step B) After completely freeing the liver, it is removed.

[0095] 2. Ex vivo perfusion preservation Mechanical perfusion system connection: The ex vivo donor liver is connected to the cryogenic mechanical continuous perfusion device via portal vein cannulation. Figure 25 This ensures that the temperature of the organ perfusion preservation solution is maintained at 4°C throughout the process. Monitoring parameters: The device includes a branch tube, a pressure sensor, and a flow control valve to monitor portal vein pressure in real time, with the flow rate set to 1 ml / min. Continuous perfusion: The perfusion pressure is maintained stable by adjusting the flow rate (e.g., by controlling the valve) for 1-5 hours (depending on actual needs). This stage replaces traditional static cold preservation and may improve the quality of the donor liver. Figure 26 Middle step E, Figure 26 Middle step F, Figure 26 (Step G, the values ​​in the figure are for illustrative purposes only).

[0096] 3. Receptor surgery and vascular anastomosis Recipient liver resection: The recipient rat was anesthetized, and its own liver was removed, while the inferior vena cava segment of the diaphragm was preserved for anastomosis.

[0097] Portal vein and inferior vena cava: rapid anastomosis using end-to-end suture or "two-cuff method" (cannula connection) is employed to reduce operation time and the risk of thrombosis.

[0098] Inferior and superior vena cava: manual microsurgical suturing to ensure vascular patency. Bile duct cannulation reconstruction.

[0099] 4. Reperfusion and Functional Assessment After opening the blood vessels, observe the changes in liver color and bile secretion. Figure 26 Middle step C, Figure 26 (Step D)

[0100] Monitoring reperfusion injury indicators, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, histopathological examination (HE staining), and hepatocyte apoptosis. (The detection methods and comparisons of these indicators have been described in the preceding examples.) Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A liver perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells, characterized in that, The liver perfusion preservation solution is used for perfusion in ex vivo preservation and transplantation surgery. The liver perfusion preservation solution includes extracellular vesicles of umbilical cord mesenchymal stem cells that have been extracted, purified and freeze-dried. The processed extracellular vesicles of umbilical cord mesenchymal stem cells contain bioactive components including proteins, lipids, mRNA and miRNA. The liver perfusion preservation solution also includes basic HTK solution, standard HTK solution components and excipients; the basic HTK solution includes: histidine, tryptophan, α-ketoglutarate and mannitol; The method for preparing the liver perfusion preservation solution is as follows: (1) Extraction and purification of extracellular vesicles from umbilical cord-derived mesenchymal stem cells; (1.1) Culture P3-P7 generation umbilical cord-derived mesenchymal stem cells and collect the supernatant; (1.2) Preliminary centrifugation: Centrifugation removes cell debris; (1.3) Secondary centrifugation: Centrifugation removes large particles; (1.4) High-speed centrifugation: Centrifuge the treated supernatant to remove fine particles and microparticles; (1.5) Ultracentrifugation: Centrifugation is used to collect extracellular vesicle particles, which will be deposited at the bottom of the tube; (1.6) After resuspending in PBS, the concentration of extracellular vesicle proteins was determined and stored at -80℃ or below for later use; It also includes the step of sequencing the contents of extracellular vesicle proteins. The specific sequencing steps include: Label-free quantitative methods involve mass spectrometry analysis of protein enzymatically digested peptides. The basic principle is based on the peak area of ​​peptide precursor ions, first identifying peptides and proteins in the sample, and then performing quantitative analysis on the identified peptides. Label-free quantitative analysis is used to analyze the differences of the same protein between multiple samples. Based on the raw files obtained from mass spectrometry detection, corresponding databases are searched, and proteins are further identified based on the database search results. Peptide and protein distribution analyses are performed to evaluate the quality of the mass spectrometry detection data and the database search. The identified proteins are annotated with common functional databases, including the GO database, KEGG database, Reactome database, and subcellular localization. Quantitative analysis of proteins, sample correlation analysis, and protein differential analysis are performed, and GO functional enrichment analysis is conducted for differentially expressed proteins. (2) The purified extracellular vesicle suspension was freeze-dried and made into freeze-dried powder; (2.1) Solution treatment: The extracted and purified extracellular vesicle particles were suspended in buffer solution to ensure their stability before lyophilization; (2.2) Rapid freezing: The extracellular vesicle solution is rapidly frozen at -80°C or in liquid nitrogen to ensure that the extracellular vesicles are not damaged during the freeze-drying process; (2.3) Preparation of freeze-dried powder: The frozen sample is placed in a freeze dryer and freeze-dried in a low temperature and high vacuum environment to sublimate the water and leave the dry extracellular vesicle powder, which is the freeze-dried powder of stem cell extracellular vesicles. (3) Preparation of liver perfusion preservation solution: (3.1) Prepare HTK solution according to the standard formula, ensuring that all components are fully dissolved and mixed evenly; (3.2) Add the freeze-dried powder of extracellular vesicles of stem cells prepared in step (2) to the HTK solution at a ratio of 100 mg / L and shake it thoroughly to dissolve it completely.

2. The liver perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells according to claim 1, characterized in that, The excipients are selected from at least one of antioxidants, energy metabolism substrates, anti-apoptotic agents, cell protectants, and anti-inflammatory and immunomodulatory agents.

3. The liver perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells according to claim 1, characterized in that, The preparation method further includes adding excipients to the liver perfusion preservation solution obtained in step (3) and distributing them evenly through operations such as stirring, centrifugation, and shaking.

4. The liver perfusion preservation solution based on extracellular vesicles of mesenchymal stem cells according to claim 1, characterized in that, The method of using the liver perfusion preservation solution includes: The lyophilized extracellular vesicle powder was stored in ampoules at room temperature. Before or during liver transplantation, perform step (3) of the preparation method, mix the lyophilized extracellular vesicles into HTK solution, and use the mixed liver perfusion preservation solution to perfuse the liver; during the perfusion process, ensure that the liver perfusion preservation solution flows evenly through the liver tissue. And / or, During liver transplantation, liver perfusion preservation solution is used to maintain the liver in optimal condition until the end of the surgery.

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