Organ perfusion preserving fluid based on extracellular vesicles of mesenchymal stem cells as well as preparation and application of organ perfusion preserving fluid

Abstract

The invention provides an organ perfusion preserving fluid based on mesenchymal stem cell extracellular vesicles, a preparation method and application of the extracellular vesicles in preparation of the organ perfusion preserving fluid. The organ perfusion preserving fluid is used for perfusion in in-vitro preservation and transplantation operations, the organ perfusion preserving fluid comprises umbilical cord mesenchymal stem cell extracellular vesicles subjected to extraction, purification and freeze-drying treatment, and the treated extracellular vesicles contain bioactive components including protein, lipid, mRNA and miRNA. The organ perfusion preserving fluid further comprises basic HTK liquid, standard HTK liquid components and auxiliary materials. The organ perfusion preserving fluid provides wider protection at the cellular level through various bioactive components such as protein, lipid, mRNA and miRNA in extracellular vesicles, and reduces oxidative stress and metabolic damage during in-vitro preservation.

Description

Technical Field

[0001] The invention relates to an organ perfusion preservation solution, in particular to an isolated organ perfusion preservation solution, a preparation method and an application technology. Background Art

[0002] Liver transplantation is currently the only effective means of treating end-stage liver disease and acute liver failure. However, during the transplantation process, due to oxidative stress and inflammatory response during organ preservation and reperfusion, the liver will experience severe ischemia-reperfusion injury (IRI), which significantly affects the functional recovery of the transplanted liver and the patient's postoperative prognosis. Traditional HTK (Histidine-Tryptophan-Ketoglutarate) solution mainly protects the liver by reducing cell metabolism and reducing oxidative stress, but it still has obvious deficiencies in anti-inflammation and promoting liver cell regeneration and repair. Current studies have shown that although the existing HTK preservation solution can provide basic protection in liver transplantation, it still cannot effectively prevent the inflammatory response, cell apoptosis and functional recovery disorders caused by IRI. In particular, in promoting the regeneration and repair of liver cells and anti-inflammatory immune regulation, the existing technology still needs to be broken through.

[0003] The existing improved HTK solution technology has evolved in three major directions: (1) Antioxidant enhancement: For example, CN108849861A adds free radical scavengers such as glutathione, but is limited by the metabolic inactivation and penetration efficiency of small molecule antioxidants; (2) Metabolic support enhancement: By supplementing adenosine, basic amino acids, etc. to improve ATP regeneration efficiency, it faces the risk of metabolic acidosis caused by increased liquid ammonia; (3) Immunomodulatory optimization: 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 such as Th1 / Th17 polarization, Kupffer cell activation, and CD4+ T lymphocyte CD154 overexpression in the IRI process.

[0004] Although the traditional HTK solution improvement program has made progress in metabolic support, local anti-apoptosis and other aspects, it still has obvious 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: Lack of regulatory elements for liver progenitor cell activation and vascular endothelial repair; (3) Conflict of formulation compatibility: The stability of small molecule drugs and bioactive components in the preservation solution restricts each other.

[0005] Therefore, a better organ preservation solution is needed to solve the problems existing in the prior art. 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 mesenchymal stem cell extracellular vesicles, a preparation method thereof, and the application of extracellular vesicles in the preparation of organ perfusion preservation solutions, aiming at the problems existing in the background technology.

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

[0008] Further, the basic HTK solution includes: Histidine, Tryptophan, and α-Ketoglutarate.

[0009] Further, the excipients are selected from at least one of antioxidants, energy metabolism substrates, anti-apoptotic agents, cytoprotective agents, and anti-inflammatory and immunomodulatory agents.

[0010] In the second aspect, the present invention provides a preparation method for the above organ perfusion preservation solution, including the following steps: (1) Extract and purify extracellular vesicles from umbilical cord-derived mesenchymal stem cells; (2) Subject the purified extracellular vesicle suspension to freeze-drying treatment and make it into a freeze-dried powder; (3) Prepare the organ perfusion preservation solution: (3.1) Prepare the HTK solution according to the standard formula to ensure that all components are fully dissolved and mixed evenly; (3.2) Add the stem cell extracellular vesicle freeze-dried powder prepared in step (2) to the HTK solution at a ratio of 100 mg / L, and shake it well to completely dissolve it.

[0011] Further, the extraction and purification of extracellular vesicles specifically include the following steps: (1.1) Culture umbilical cord-derived mesenchymal stem cells at passages P3 - P7 and collect the supernatant; (1.2) Perform primary centrifugation: Centrifuge to remove cell debris; (1.3) Perform secondary centrifugation: Centrifuge to remove large particulate matter; (1.4) Perform high-speed centrifugation: Centrifuge the processed supernatant to remove fine particles and microparticles; (1.5)Ultracentrifugation: Centrifuge to collect extracellular vesicle particles, and the obtained extracellular vesicle particles will deposit at the bottom of the tube; (1.6)Resuspend with PBS and then measure the protein concentration of extracellular vesicles, and store at -80 °C or below for later use.

[0012] Furthermore, the preparation of the freeze-dried powder of stem cell extracellular vesicles specifically includes the following steps: (2.1)Solution treatment: Suspend the extracted and purified extracellular vesicle particles in an appropriate buffer (such as PBS buffer) to ensure their stability before lyophilization; (2.2)Quick freezing: Quick-freeze the extracellular vesicle solution at -80 °C or below for several hours, or quickly freeze it in liquid nitrogen to ensure that the extracellular vesicles are not damaged during the lyophilization process; (2.3)Preparation of freeze-dried powder: Put the frozen sample into a lyophilizer and perform lyophilization treatment in a low-temperature and high-vacuum environment. The water sublimes, leaving behind a 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 making them evenly distributed through operations such as stirring, centrifugation, and shaking.

[0014] In a third aspect, the present invention provides a method for using the above organ perfusion preservation solution, including: In the case of non-use, the freeze-dried powder of extracellular vesicles is stored at room temperature in an ampoule bottle; The organ perfusion preservation solution of the present invention is mainly used for liver transplantation surgery. Before or during the liver transplantation surgery, perform step (3) of the preparation method, mix the freeze-dried extracellular vesicles into the HTK solution, use it immediately, and then use the mixed organ perfusion preservation solution for liver perfusion; during the perfusion process, ensure that the organ perfusion preservation solution evenly flows through the liver tissue; and / or, During the liver transplantation surgery, use the organ perfusion preservation solution for perfusion to maintain the optimal state of the liver until the end of the surgery.

[0015] In a fourth aspect, the present invention also provides the application of umbilical cord-derived mesenchymal stem cell extracellular vesicles in the preparation of an organ perfusion preservation solution.

[0016] The beneficial effects of the present invention are: 1. The present invention introduces a freeze-dried 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 freeze-drying process, providing full-cycle protection against oxidation, anti-inflammatory immune regulation, and promoting regeneration and repair for the ischemic liver during the ischemic period. By applying the freeze-dried powder of MSC-EVs, the preservation effect and functional recovery of the transplanted liver are improved to effectively prevent and treat common ischemia-reperfusion injury (IRI) and other complications after liver transplantation, showing a significant technological breakthrough compared with the existing single-mechanism improvement schemes.

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

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

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

[0020] (4) Reduce postoperative complications: By reducing postoperative inflammatory responses and apoptosis, reduce the occurrence of common postoperative complications, and improve the survival rate of the transplanted liver and the prognosis of patients.

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

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

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

[0024] Figure 3It is the enrichment result diagram of extracellular vesicle proteomics 4D-label-free sequencing in Example 3 of the present invention.

[0025] Figure 4 It is the transmission electron microscope image of the freeze-dried powder of stem cell extracellular vesicle MSC-EVs in Example 4 of the present invention.

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

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

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

[0029] Figure 8 It is the comparison diagram of Western blot experiments between the freeze-dried powder of stem cell extracellular vesicle MSC-EVs and the control group in Example 4 of the present invention.

[0030] Figure 9 It is the immunofluorescence image of the perfusion of normal HTK solution (control group) labeled with Dir dye for 1H in Example 5 of the present invention.

[0031] Figure 10 It is the immunofluorescence image of the perfusion of normal HTK solution (control group) labeled with Dir dye for 3H in Example 5 of the present invention.

[0032] Figure 11 It is the immunofluorescence image of the perfusion of normal HTK solution (control group) labeled with Dir dye for 5H in Example 5 of the present invention.

[0033] Figure 12 It is the immunofluorescence image of the perfusion of HTK solution added with MSC-EVs (experimental group) labeled with Dir dye for 1H in Example 5 of the present invention.

[0034] Figure 13 It is the immunofluorescence image of the perfusion of HTK solution added with MSC-EVs (experimental group) labeled with Dir dye for 3H in Example 5 of the present invention.

[0035] Figure 14 It is the immunofluorescence image of the perfusion of HTK solution added with MSC-EVs (experimental group) labeled with Dir dye for 5H in Example 5 of the present invention.

[0036] Figure 15 It is the result diagram of ALT for liver function determination in Example 6 of the present invention.

[0037] Figure 16It is the AST result diagram of liver function measurement in Example 6 of the present invention.

[0038] Figure 17 It is the LDH result diagram of liver function measurement in Example 6 of the present invention.

[0039] Figure 18 It is the tissue HE staining result diagram of the non-injured group (control group) in Example 7 of the present invention.

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

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

[0042] Figure 21 It is the Suzuki score result diagram of each group in Example 7 of the present invention.

[0043] Figure 22 It is the Tunel staining result diagram of the non-injured group (control group) in Example 7 of the present invention.

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

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

[0046] Figure 25 It is the ex vivo perfusion mode diagram of Example 8 of the present invention.

[0047] Figure 26 It is the surgical procedure schematic diagram of Example 8 of the present invention. Detailed implementation manners

[0048] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be specifically, clearly and completely described below in the form of embodiments with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. And all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

[0049] Example 1, an organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles.

[0050] This embodiment provides an organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles. The composition of the organ perfusion preservation solution includes: (1) Basic HTK solution, including: Histidine, Tryptophan, and α-Ketoglutarate; (2) Other standard HTK solution components, such as buffers, ionic components, etc.; (3) Freeze-dried powder of stem cell extracellular vesicles: The extracellular vesicles secreted by umbilical cord mesenchymal stem cells are used, and after extraction, purification, and freeze-drying treatment, the extracellular vesicles contain bioactive components such as proteins, lipids, mRNA, miRNA, etc.; (4) Auxiliary materials: Antioxidants (such as glutathione, vitamin C, vitamin E); Energy metabolism substrates (such as adenosine, glucose, amino acids); Anti-apoptosis agents (such as cyclosporine A); Cell protectants (such as HSP, ATP); Anti-inflammatory and immunomodulators (such as glucocorticoids, anti-TNF-α antibodies). The auxiliary materials can be selectively added according to actual needs or not added.

[0051] Example 2, a preparation method of an organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles.

[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 stem cell extracellular vesicles.

[0053] The specific steps are as follows: (1.1) Culture umbilical cord-derived mesenchymal stem cells at passages P3 - P7, and collect the 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: Remove large particulate matter by centrifuging at 3000G for about 30 minutes.

[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 100000G for about 70 minutes to collect extracellular vesicle particles. The obtained extracellular vesicle particles will deposit at the bottom of the tube, and collect the precipitate.

[0058] (1.6) After resuspending with PBS, steps (1.5) can be repeated 1 - 2 times, and then detected on the machine. Determine the protein concentration of extracellular vesicles by 4D-label-free, and store for later use at -80°C or below.

[0059] 2. Preparation of extracellular vesicle lyophilized powder using the purified extracellular vesicle suspension.

[0060] The specific steps are as follows: (2.1) Solution treatment: Suspend the extracted and purified extracellular vesicle particles in an appropriate buffer (such as PBS buffer) to ensure their stability before lyophilization.

[0061] (2.2) Quick freezing: Quickly freeze the extracellular vesicle solution at -80 °C for several hours or in liquid nitrogen to ensure that the extracellular vesicles are not damaged during the lyophilization process.

[0062] (3) Lyophilization: Place the frozen sample in a lyophilizer and perform lyophilization treatment in a low-temperature and high-vacuum environment. The water sublimes, leaving behind dry extracellular vesicle powder.

[0063] The entire process of preparing the lyophilized powder usually takes 24 - 48 hours. Prepare the lyophilized powder of stem cell extracellular vesicles.

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

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

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

[0067] Dissolution speed requirement: The time required for the prepared lyophilized powder of stem cell extracellular vesicles to be completely dissolved in the HTK solution should not exceed 5 minutes. Quick dissolution can ensure that the extracellular vesicles maintain their activity as much as possible before use and avoid damage to the structure or reduction of the activity of the extracellular vesicles due to too long a dissolution process.

[0068] Quality control index after dissolution: The particle size of the lyophilized powder of stem cell extracellular vesicles after dissolution should be between 100 - 1000 nanometers. At this particle size, the extracellular vesicles are suitable for interacting with cells and are beneficial for function exertion.

[0069] (3.3) According to the actual usage scenario requirements, other excipients (such as antioxidants, energy metabolism substrates, etc.) can be further added and ensure their uniform distribution.

[0070] 3. Preservation method In this embodiment, under normal conditions, the extracellular vesicle freeze-dried powder and the standard HTK solution are stored separately and used immediately upon retrieval. Generally speaking, the requirements for storage are as follows: The extracellular vesicle freeze-dried powder can be stored at room temperature in ampoules at a dosage of 100 mg / vial (the dosage can also be adjusted according to actual needs). The HTK solution should be stored in the dark, sealed, and refrigerated at 4°C to ensure that its active ingredients do not degrade during storage.

[0071] 4. Usage method As described in the storage method, the organ perfusion preservation solution of the present invention is used immediately upon retrieval. The specific usage scenarios are as follows: (1) Ex vivo perfusion of the liver: Before or during the liver transplantation surgery, use it immediately upon retrieval. Mix the freeze-dried extracellular vesicles into the HTK solution and use the modified HTK solution for liver perfusion. During the perfusion process, ensure that the modified HTK solution flows evenly through the liver tissue to fully exert its protective and reparative effects.

[0072] (2) Transplantation process: During the liver transplantation surgery, continue to use the modified HTK solution for perfusion to maintain the optimal state of the liver.

[0073] Example 3, sequencing of the protein content of extracellular vesicles.

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

[0075] The specific steps of the sequencing include: The label-free quantification method analyzes the mass spectrometry of protein enzymatic peptides. Its basic principle is based on the peak area (XIC) of the peptide precursor ion. First, identify the peptides and proteins in the sample, and then perform quantitative analysis on the identified peptides (proteins), and use label-free quantification to analyze the differences in the same protein between multiple samples. The flowchart of 4D-label-free sequencing is as Figure 1 shown.

[0076] Based on the Raw file obtained from mass spectrometry detection, search the corresponding database. Further, based on the results of database search, perform protein identification, and at the same time perform peptide and protein distribution analysis to evaluate the quality of mass spectrometry detection data and database search. Annotate the identified proteins with common functional databases, including GO database, KEGG database, Reactome database, and subcellular localization; perform quantitative analysis of proteins, sample correlation analysis, and protein difference analysis, and perform GO functional enrichment analysis on differentially expressed proteins. As Figure 2As shown in the figure, the protein profiles of umbilical cord-derived mesenchymal stem cell extracellular vesicles (MSC-EVs) of P5, P6, and P7 generations were described as a whole using a VENN diagram to show the expression of common proteins and differentially expressed proteins: It was found that 997 protein molecules were common. The GO enrichment results for this part of the common proteins are as shown in Figure 3 and the gene sets of protein folding (GO:0006457) and protein stability (GO:0050821) were significantly enriched, and these functions may play a core role in the mechanism of the downstream phenotypes of stem cell extracellular vesicles.

[0077] Thus, extracellular vesicles have the following effects on downstream effector molecules: (1) enhancing the functional stability of downstream proteins. (2) reducing the toxicity of misfolded or aggregated proteins to cells. (3) regulating key signaling pathways of oxidative stress and affecting cell survival, metabolism, and differentiation. The above results are all conducive to playing a crucial role in injury repair after transplantation.

[0078] Example 4, Transmission Electron Microscopy and Nanoparticle Tracking Analysis (NTA).

[0079] To determine the effect of the freeze-dried powder, in this example, transmission electron microscopy and nanoparticle tracking analysis were performed on the stem cell extracellular vesicle freeze-dried powder prepared according to Example 2 (experimental group) and the control group (mesenchymal stem cell extracellular vesicles without freeze-drying treatment).

[0080] Using standard HTK solution as the solute, the stem cell extracellular vesicle freeze-dried powder prepared according to Example 2 (stored at room temperature for 1 month) was dissolved, and compared with the non-freeze-dried treatment group (fresh extracellular vesicles), as shown in Figure 4 and Figure 5 As shown, there was no obvious change in the morphology of freeze-dried and non-freeze-dried extracellular vesicles under the electron microscope, and both were biconcave saucer-shaped (scale bar: 100 nm).

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

[0082] Example 5, Immunofluorescence Experiment.

[0083] To further verify the effect of MSC-EVs on stem cells during ex vivo perfusion, in this example, the rat isolated liver was perfused with HTK solution containing MSC-EVs labeled with Dir dye, and the common HTK solution labeled with Dir dye was used as a control group for rat liver ex vivo perfusion. Compared with the control group ( Figure 9 , Figure 10 , Figure 11 ), it was found that after 1H (1 hour), 3H (3 hours), and 5H (5 hours) of perfusion, rat hepatocytes phagocytosed Dir-MSC-EVs ( Figure 12 , Figure 13 , Figure 14 ), indicating that under ex vivo perfusion conditions, MSC-EVs can enter the liver with the HTK solution and be absorbed to exert their functions (scale bar: 50μm. In the figures of this example, blue represents DAPI fluorescent dye and red represents Dir dye).

[0084] Example 6, Liver function measurement experiment.

[0085] To verify the protective effect of the organ perfusion preservation solution of the present invention on liver function, in this example, liver function was measured in the sera of rats in the non-injury group (control group), the group perfused with common HTK solution (comparison group), and the group perfused with the organ perfusion preservation solution of the present invention (experimental group). The test results are as shown in Figure 15 , Figure 16 , Figure 17 . It can be seen from the figure that the aspartate aminotransferase AST and alanine aminotransferase ALT in the transplantation group were significantly increased 24H after surgery, indicating severe liver injury after surgery. However, the aspartate aminotransferase AST and alanine aminotransferase ALT of the transplanted liver perfused with the organ perfusion preservation solution of the present invention were significantly down-regulated. Figure 17 It was proved that there was no significant difference in lactate dehydrogenase LDH among the groups (n = 5).

[0086] Example 7, Staining experiment.

[0087] To verify the histological effect of the present invention, in this example, histological HE staining experiments were performed on the control group (non-injury group), the group perfused with common HTK solution (comparison group), and the group perfused with the organ perfusion preservation solution of the present invention (experimental group). The results are as shown in Figures 18 - 20 .

[0088] Compared with the control group shown in Figure 18 , obvious hepatocyte necrosis, portal area inflammation, destruction of the hepatic lobule structure, congestion, etc. occurred in the common HTK in Figure 19 (comparison group), and the Suzuki score was higher and the damage was more severe. 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 transplantation group (experimental group) have been significantly improved, and the Suzuki score is lower ( Figure 21 ). (Scale bar: 200 μm) The Suzuki score (mSS) here is a grading system based on clinical imaging examinations.

[0089] From Figure 22 , Figure 23 and Figure 24 are the Tunel staining results of each group in the previous paragraph. Among them, DAPI is a standard fluorescent dye, Merge is the merged image. From the Tunel staining results, it can be seen that compared with the control group, apoptosis in the normal HTK solution perfusion group (comparison group) increases significantly; while apoptosis cells decrease significantly in the perfusion with the organ perfusion preservation solution of the present invention (experimental group) (Scale bar: 200 μm).

[0090] Example 8, Animal experiment (rat liver transplantation experiment).

[0091] This example provides a complete animal experiment process so that those skilled in the art can better implement the technical solution of the present invention and can also more comprehensively show the technical effects of the present invention. However, those skilled in the art should know that the specific values in the examples are only for better interpreting the technical solution of the present invention and should not be regarded as a limitation to the present invention.

[0092] First, prepare the organ perfusion preservation solution required for the present invention according to the method of Example 2. The formula of the organ perfusion preservation solution used in this example is as follows: Component Concentration NaCl 15 mmol / l KCl 8 mmol / l <![CDATA[MnCl 2 > 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 The balance The perfusion method used in this example is that the isolated liver is pumped into through the portal vein and pumped out through the vena cava for circulation. During this period, the liquid temperature is maintained at 4 degrees Celsius, and the perfusion mode is as Figure 25 shown.

[0093] The whole experimental process is as follows: 1. Donor liver acquisition and preoperative preparation Select healthy SD rats, with a body weight of 250 - 300 g, regardless of gender. All experimental operations comply with the "Regulations on the Administration of Laboratory Animals" and the "Animal Welfare Requirements".

[0094] Anesthetize the donor rats, routinely open the abdomen to expose the liver ( Figure 26 step A in Figure 26 ), insert a catheter into the portal vein, and perform in-situ flushing with low-temperature (4°C) heparinized saline to remove blood and rapidly cool down to reduce warm ischemia injury. Synchronously ligate the hepatic artery, cut off the inferior vena cava and bile duct ( Figure 26 step B in

[0095] 2. Ex vivo perfusion preservation Mechanical perfusion system connection: Connect the excised donor liver to a hypothermic mechanical continuous perfusion device through portal vein cannulation ( Figure 25 ), and ensure that the temperature of the organ perfusion preservation solution is maintained at 4°C throughout the process. Monitoring parameters: The device includes a branch pipe, a pressure sensor, and a flow control valve, and the portal vein pressure is monitored in real time. The flow rate is set to 1 ml / min. Continuous perfusion: Maintain the perfusion pressure stable by adjusting the flow rate (such as controlling the valve) for 1 - 5 hours (according to actual needs). This stage replaces traditional static cold preservation and may improve the quality of the donor liver ( Figure 26 step E in Figure 26 step F in Figure 26 step G in, and the values in the figure are only for illustration).

[0096] 3. Recipient surgery and vascular anastomosis Recipient liver resection: Anesthetize the recipient rats and remove their own livers, leaving the diaphragmatic segment of the inferior vena cava for anastomosis.

[0097] Portal vein and infrahepatic vena cava: Use end-to-end suture or the "two-cuff method" for rapid anastomosis (cannula connection) to reduce the operation time and thrombus risk.

[0098] Suprahepatic vena cava: Hand microsuture to ensure vascular patency. Reconstruct the bile duct cannula.

[0099] 4. Reperfusion and function evaluation After opening the blood vessels, observe the color change of the liver and bile secretion ( Figure 26 step C in Figure 26 step D in).

[0100] Monitor reperfusion injury indicators: such as the levels of aspartate aminotransferase AST and alanine aminotransferase ALT, histopathology (HE staining), and hepatocyte apoptosis. (The detection methods and comparisons of the indicators have been recorded in the foregoing embodiments) 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 recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles, characterized in that: The organ perfusion preservation solution is used for perfusion in ex vivo preservation and transplantation surgery, and the organ perfusion preservation solution includes extracellular vesicles of umbilical cord mesenchymal stem cells that have been extracted, purified and freeze-dried, and the treated extracellular vesicles of umbilical cord mesenchymal stem cells contain biologically active components including proteins, lipids, mRNA, and miRNA; The organ perfusion preservation solution also includes basic HTK solution, standard HTK solution components and auxiliary materials.

2. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 1, characterized in that: The basic HTK solution includes: Histidine, Tryptophan and α-ketoglutarate.

3. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 1, characterized in that: The auxiliary material is selected from at least one of antioxidants, energy metabolism substrates, anti-apoptotic agents, cell protectants, and anti-inflammatory and immunomodulatory agents.

4. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 1, characterized in that: The preparation method of the organ perfusion preservation solution is as follows: (1) Extraction and purification of extracellular vesicles from umbilical cord-derived mesenchymal stem cells; (2) The purified extracellular vesicle suspension is freeze-dried to prepare freeze-dried powder; (3) Preparation of organ perfusion preservation solution: (3.1) Prepare HTK solution according to the standard formula, ensuring that all ingredients are fully dissolved and mixed evenly; (3.2) Add the stem cell extracellular vesicle freeze-dried powder prepared in step (2) into the HTK solution at a ratio of 100 mg / L and shake thoroughly to completely dissolve it.

5. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 4, characterized in that: The extraction and purification of extracellular vesicles specifically includes the following steps: (1.1) Cultivate P3-P7 umbilical cord-derived mesenchymal stem cells and collect the supernatant; (1.2) Preliminary centrifugation: centrifugation to remove cell debris; (1.3) Secondary centrifugation: centrifugation to remove large particles; (1.4) High-speed centrifugation: The treated supernatant is centrifuged to remove fine particles and microparticles; (1.5) Ultracentrifugation: Centrifuge to collect extracellular vesicle particles, which will settle at the bottom of the tube; (1.6) Resuspend in PBS and measure the extracellular vesicle protein concentration. Store at -80℃ or below for future use.

6. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 4, characterized in that: The preparation of the stem cell extracellular vesicle freeze-dried powder specifically comprises the following steps: (2.1) Solution treatment: The extracted and purified extracellular vesicle particles are suspended in a buffer to ensure their stability before lyophilization; (2.2) Quick freezing: Quickly freeze the extracellular vesicle solution 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, high-vacuum environment to sublimate the water and leave dry extracellular vesicle powder, which is the stem cell extracellular vesicle freeze-dried powder.

7. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 4, characterized in that: The preparation method further comprises adding auxiliary materials to the organ perfusion preservation solution prepared in step (3), and uniformly distributing the auxiliary materials through stirring, centrifugation, shaking, etc.

8. The organ perfusion preservation solution based on mesenchymal stem cell extracellular vesicles according to claim 4, characterized in that: The method for using the organ perfusion preservation solution comprises: The extracellular vesicle freeze-dried powder is placed in an ampoule and stored at room temperature; Before a liver transplantation operation or during the preservation process, perform step (3) in the preparation method, mix the freeze-dried extracellular vesicles into the HTK solution, and use the mixed organ perfusion preservation solution to perfuse the liver; during the perfusion process, ensure that the organ perfusion preservation solution flows evenly through the liver tissue; and / or, During liver transplant surgery, organ perfusion preservation solution is used for perfusion to maintain the liver in optimal condition until the end of the surgery.

9. Application of extracellular vesicles of umbilical cord-derived mesenchymal stem cells in the preparation of organ perfusion preservation fluid.

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