Preserving fluid, preserving method and application of alkyl gallate in preserving fluid

Abstract

The invention provides a preserving fluid, a preserving method and application of alkyl gallate in the preserving fluid. The components of the preserving fluid comprise colloidal substances and alkyl gallate, and the concentration of the alkyl gallate in the preserving fluid is 0.1 mu mol / L to 299 mu mol / L. The preserving fluid can be used for preserving cells, tissues and organs, can enable the cells, the tissues and the organs to have relatively strong viability, can also maintain a relatively high viability level in a relatively long preservation time, and is beneficial to use in activity protection of the cells, the tissues and the organs.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to preservation solutions, preservation methods, and the application of alkyl gallates in preservation solutions. Background Art

[0002] The active protection technologies for cells, tissues, and organs are applied in various different fields. For example, organ transplants such as kidneys, livers, hearts, pancreases, and lungs in transplantation medicine have become effective means to save the lives of patients with end-stage diseases, primary and fulminant organ failure. The success of organ transplantation depends on preventing ischemic injury to donor organs before transplantation. In related technologies, many preservation solutions have been developed to reduce ischemic injury and ensure the viability of cells, tissues, and organs. However, the allowable preservation time and preservation effect of existing preservation solutions cannot meet clinical needs and still need to be improved. Therefore, there is an urgent need to develop a new generation of preservation solutions so that cells, tissues, and organs have stronger viability during preservation and improve the success rate of their transplantation. Summary of the Invention

[0003] In view of this, this application provides a preservation solution, a preservation method, and the application of alkyl gallates in the preservation solution. This preservation solution can be used to preserve cells, tissues, and organs, prevent or reduce ischemic injury, enable cells, tissues, and organs to have stronger viability, and at the same time maintain a relatively high viability level during a relatively long preservation time, which is beneficial for their use in active protection.

[0004] In a first aspect, this application provides a preservation solution, which contains a colloidal substance and an alkyl gallate, and the concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L.

[0005] Optionally, the concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 150 μmol / L.

[0006] Further, the concentration of the alkyl gallate in the preservation solution is 1 μmol / L to 100 μmol / L.

[0007] Optionally, the alkyl gallate includes at least one of propyl gallate, octyl gallate, and lauryl gallate.

[0008] Optionally, the colloidal substance includes at least one of polyglycerol, polyethylene glycol, and hydroxyethyl starch.

[0009] Further, the preservation solution includes at least one of HPG preservation solution, UW preservation solution, and IGL-1 preservation solution. The HPG preservation solution includes the polyglycerol, the UW preservation solution includes the hydroxyethyl starch, and the IGL-1 preservation solution includes the polyethylene glycol.

[0010] Further, the HPG preservation solution includes galacturonic acid, sodium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, adenosine, reduced glutathione, allopurinol, and polyglycerol.

[0011] Optionally, the pH value of the preservation solution is 2 to 9.

[0012] Optionally, the osmotic pressure of the preservation solution is 150 mOsm / kg to 1500 mOsm / kg.

[0013] The preservation solution provided by the present application can extend the preservation time of cells, tissues, and organs, prevent or reduce ischemic injury, enable cells, tissues, and organs to have a high vitality level, and is beneficial to the use of cells, tissues, and organs in various fields including medicine.

[0014] In a second aspect, the present application provides a preservation method, including preserving at least one of cells, tissues, and organs in the preservation solution described in the first aspect.

[0015] The preservation method provided by the present application is simple to operate and can allow the extension of the existing preservation time and the preservation of the vitality of cells, tissues, and organs.

[0016] In a third aspect, the present application provides the use of alkyl gallate in a preservation solution, the preservation solution includes a colloidal substance and the alkyl gallate, and the concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L.

[0017] By adding alkyl gallate to the preservation solution, the present application improves the preservation effect of the preservation solution and is beneficial to the use of the preservation solution. Description of the Drawings

[0018] Figure 1 The LDH release amount of cells after being preserved by the preservation solution, where Figure 1 in (a) is the LDH release amount of EA.hy926 cells preserved for 3 days by the preservation solution containing different concentrations of propyl gallate, (b) is the LDH release amount of EA.hy926 cells preserved for different days by the preservation solution containing different concentrations of propyl gallate, (c) is the LDH release amount of HepG2 cells preserved for 3 days by the preservation solution containing different concentrations of propyl gallate, (d) is the LDH release amount of HepG2 cells preserved for different days by the preservation solution containing different concentrations of propyl gallate, (e) is the LDH release amount of HKC-8 cells preserved for 5 days by the preservation solution containing different concentrations of propyl gallate, and (f) is the LDH release amount of HKC-8 cells preserved for different days by the preservation solution containing different concentrations of propyl gallate.

[0019] Figure 2MTT detection results of cells after being preserved in the preservation solution, where Figure 2 In (a) of Figure 2 , the absorbance values detected by MTT after EA.hy926 cells were preserved in the preservation solution containing different concentrations of propyl gallate for 3 days; in (b), the absorbance values detected by MTT after HepG2 cells were preserved in the preservation solution containing different concentrations of propyl gallate for 3 days; in (c), the absorbance values detected by MTT after HKC-8 cells were preserved in the preservation solution containing different concentrations of propyl gallate for 8 days.

[0020] Figures 3a to 3f Cell viability detection results of cells after being preserved in the preservation solution, where Figure 3a Flow cytometry detection results after EA.hy926 cells were preserved in the preservation solution for 3 days Figure 3b Cell survival rate after EA.hy926 cells were preserved in the preservation solution for 3 days Figure 3c Flow cytometry detection results after HepG2 cells were preserved in the preservation solution for 3 days Figure 3d Cell survival rate after HepG2 cells were preserved in the preservation solution for 3 days Figure 3e Flow cytometry detection results after HKC-8 cells were preserved in the preservation solution for 3 days Figure 3f Cell survival rate after HKC-8 cells were preserved in the preservation solution for 5 days.

[0021] Figure 4 MDA content of cells after being preserved in the preservation solution, where Figure 4 In (a) of Figure 4 , the MDA content of EA.hy926 cells after being preserved in the preservation solution; in (b), the MDA content of HKC-8 cells after being preserved in the preservation solution.

[0022] Figures 5a to 5d ATP content of EA.hy926 cells after being preserved in the preservation solution, where Figure 5a iATP content Figure 5b Ratio of iATP to eATP Figure 5c Flow cytometry JC-1 results of cells after being preserved in the preservation solution Figure 5d Red / green ratio.

[0023] Figures 6a to 6d ATP content of HKC-8 cells after being preserved in the preservation solution, where Figure 6a iATP content Figure 6b Ratio of iATP to eATP Figure 6c Flow cytometry JC-1 results of cells after being preserved in the preservation solution Figure 6d Red / green ratio.

[0024] Figure 7 Preservation effect of the kidney after being preserved in the preservation solution, where Figure 7Among them, (a) is the content of LDH in the preservation solution, (b) is the microscopic image of the kidney section, (c) is the cumulative score of glomerular injury, (d) is the percentage of the number of intact glomeruli, and (e) is the ATP content.

[0025] Figure 8 is the protection effect of different additives, among which Figure 8 in (a) is the preservation effect of different additives on EA.hy926 cells, and (b) is the preservation effect of different additives on HepG2 cells.

[0026] Figure 9 is to compare the effects of different preservation solutions on the LDH release amount. Specific embodiments

[0027] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.

[0028] The meaning of the preservation solution in the present application is the common understanding of those of ordinary skill in the art, and generally refers to any liquid that can be used to reduce the damage effects brought by freezing, ischemia, and warm blood reperfusion to organs, tissues, or cells under any conditions or circumstances. Terms with similar meanings include transplantation solution, organ preservation solution, preservation solution for transplantation.

[0029] The cells in the present application can but are not limited to include endothelial cells, pancreatic cells, stem cells, blood cells, immune cells, etc. The tissues in the present application can but are not limited to include bones, tendons (referred to as musculoskeletal transplantation), corneas, skin, heart valves, islets of Langerhans, part or all of the face, nerves, blood vessels, etc. The organs in the present application can but are not limited to include the heart, kidney, liver, lung, pancreas, intestine, spleen, limbs (including fingers or toes), sex organs, thymus, etc.

[0030] The present application provides a preservation solution, which contains a colloidal substance and an alkyl gallate. The concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L. During the preservation of cells, tissues, and organs, ischemia can lead to a significant decrease in mitochondrial adenosine triphosphate (ATP) biosynthesis in cells due to hypoxia. The decrease in ATP levels will cause a chain loss of cell functions and then lead to cell death. Therefore, ischemic injury is an important reason for reducing the vitality and functions of cells, tissues, and organs. In cells, tissues, and organs preserved at low temperatures (such as 0°C to 5°C, etc.), the combined effects of hypothermic ischemic injury and additional injury caused by rewarming or reperfusion are the main causes of cell apoptosis resulting in loss of function after transplantation. Hypothermic preservation can cause the aerobic metabolism of cells, tissues, and organs to stop, avoiding warm ischemic injury. However, under hypothermic conditions, the cell membrane changes from the liquid crystal state to the gel state and lipid peroxidation occurs, altering specific lipid-protein interactions, changing phospholipid asymmetry and lipid composition, etc., making it lose stability. The cell membrane is the site where hypothermia-induced injury occurs. The alkyl gallate in the preservation solution of the present application can also prevent lipid peroxidation of the cell membrane, playing a role in protecting the mitochondrial membrane and the outer cell membrane, ensuring the production and consumption of ATP, avoiding ischemic injury, and also ensuring the structure and function of the cell membrane, which is beneficial to enhancing the protective effect on cells, tissues, and organs to avoid necrosis. Especially under hypothermic preservation, it can prevent or reduce the transformation of the cell membrane from the liquid crystal state to the gel state and lipid peroxidation of the cell membrane, prevent cell swelling, ensure the stability of the cell membrane structure and the production and consumption of ATP, thereby being beneficial to extending the preservation time of cells, tissues, and organs, reducing cold ischemic injury, and cell death.

[0031] In the present application, the concentration of alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L, which not only ensures the protective effect of alkyl gallate but also avoids the adverse consequences caused by excessive alkyl gallate, thereby improving the preservation time and protective effect of the preservation solution. Specifically, the concentration of alkyl gallate in the preservation solution can be, but is not limited to, 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 5 μmol / L, 10 μmol / L, 25 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 75 μmol / L, 90 μmol / L, 100 μmol / L, 135 μmol / L, 140 μmol / L, 150 μmol / L, 165 μmol / L, 175 μmol / L, 190 μmol / L, 200 μmol / L, 220 μmol / L, 245 μmol / L, 268 μmol / L, 280 μmol / L, 290 μmol / L or 295 μmol / L, etc. In an embodiment of the present application, the concentration of alkyl gallate in the preservation solution is 0.1 μmol / L to 150 μmol / L, which is beneficial to further improving the preservation effect of the preservation solution and increasing the viability of cells, tissues, and organs. In another embodiment of the present application, the concentration of alkyl gallate in the preservation solution is 1 μmol / L to 100 μmol / L, which is beneficial to further improving the preservation effect of the preservation solution and increasing the viability of cells, tissues, and organs. In yet another embodiment of the present application, the concentration of alkyl gallate in the preservation solution is 10 μmol / L to 100 μmol / L, which is beneficial to further improving the preservation effect of the preservation solution and increasing the viability of cells, tissues, and organs. In yet another embodiment of the present application, the concentration of alkyl gallate in the preservation solution is 20 μmol / L to 70 μmol / L, which is beneficial to further improving the preservation effect of the preservation solution and increasing the viability of cells, tissues, and organs. In yet another embodiment of the present application, the concentration of alkyl gallate in the preservation solution is 25 μmol / L to 50 μmol / L, which is beneficial to further improving the preservation effect of the preservation solution and increasing the viability of cells, tissues, and organs.

[0032] In one embodiment of the present application, the alkyl gallate includes at least one of propyl gallate, octyl gallate, and lauryl gallate. In one embodiment of the present application, the alkyl gallate includes at least one of propyl gallate, octyl gallate, lauryl gallate, and their derivatives. That is to say, the alkyl gallate includes at least one of propyl gallate, propyl gallate derivatives, octyl gallate, octyl gallate derivatives, lauryl gallate, and lauryl gallate derivatives. The present application does not limit the derivatives, which may be substances substituted by halogen, oxygen, sulfur, nitrogen, glycosyl, amino acid groups, etc. Among them, propyl gallate (PG) is n-propyl 3,4,5-trihydroxybenzoate, and its structural formula is shown in Formula (I); the structural formula of octyl gallate (OG) is shown in Formula (II); the structural formula of lauryl gallate (LG) is shown in Formula (III).

[0033]

[0034] In the present application, the colloidal substance refers to a substance that can generate a colloid in the preservation solution. A colloid, also known as a colloidal dispersion, is a relatively homogeneous mixture. In a colloid, there are two different states of matter, one is the dispersed phase and the other is the continuous phase. In one embodiment of the present application, the colloidal substance includes at least one of polyglycerol, polyethylene glycol, and hydroxyethyl starch, which can further enhance the protection of cell membranes, further improve the survival rate of cells, and thus improve the vitality and preservation effect of cells, tissues, and organs.

[0035] In one embodiment of the present application, the preservation solution includes at least one of HPG preservation solution, UW preservation solution, and IGL-1 preservation solution. The HPG preservation solution includes polyglycerol, the UW preservation solution includes hydroxyethyl starch, and the IGL-1 preservation solution includes polyethylene glycol, which is beneficial to further improve the effect of the preservation solution. Among them, the UW preservation solution is the University of Wisconsin solution, and the IGL-1 preservation solution is the Institute Georges Lopez-1 preservation solution; the HPG preservation solution is the polyglycerol preservation solution, that is, it can be understood as a preservation solution containing polyglycerol. The polyglycerol preservation solution can be named "HPG preservation solution" or other names, which are all within the protection scope of the present application.

[0036] In an embodiment of the present application, the HPG preservation solution includes galacturonic acid, sodium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, adenosine, reduced glutathione, allopurinol, and polyglycerol, which is beneficial to the preservation of cells, tissues, and organs and ensures the viability of cells, tissues, and organs. In an example of the present application, the molecular weight of polyglycerol is 0.5 kDa to 3.5 kDa, which is beneficial to preservation and will not cause adverse losses to cells, tissues, and organs. Specifically, the molecular weight of polyglycerol can be, but is not limited to, 0.5 kDa, 1 kDa, 1.2 kDa, 1.5 kDa, 1.8 kDa, 2 kDa, 2.1 kDa, 2.5 kDa, 2.7 kDa, 3 kDa, 3.3 kDa, or 3.5 kDa, etc. Among them, the HPG preservation solution can include polyglycerol with one molecular weight or multiple molecular weights of polyglycerol. In the present application, the HPG preservation solution can include hyperbranched polyglycerol. In an example of the present application, the branching degree of polyglycerol is 0.4 to 0.7. Specifically, the branching degree of polyglycerol can be, but is not limited to, 0.4, 0.5, 0.55, 0.6, 0.65, or 0.7, etc. In an example of the present application, the pH value of the HPG preservation solution is 4 to 8. Specifically, the pH value of the HPG preservation solution can be, but is not limited to, 4, 4.5, 5, 5.2, 5.5, 6, 6.2, 6.5, 7, 7.5, 7.6, or 8, etc. In an example of the present application, the osmotic pressure of the HPG preservation solution is 150 mOsm / kg to 500 mOsm / kg. Specifically, the osmotic pressure of the HPG preservation solution can be, but is not limited to, 150 mOsm / kg, 200 mOsm / kg, 240 mOsm / kg, 290 mOsm / kg, 300 mOsm / kg, 350 mOsm / kg, 370 mOsm / kg, 400 mOsm / kg, 430 mOsm / kg, 450 mOsm / kg, 480 mOsm / kg, 490 mOsm / kg, or 500 mOsm / kg, etc. In an example of the present application, the HPG preservation solution includes 100 mmol / L of galacturonic acid, 100 mmol / L of sodium hydroxide, 25 mmol / L of potassium dihydrogen phosphate, 5 mmol / L of magnesium sulfate, 5 mmol / L of adenosine, 3 mmol / L of reduced glutathione, 1 mmol / L of allopurinol, and 30 g / L of polyglycerol, which is beneficial to further improving the preservation effect of the preservation solution.

[0037] In one embodiment of the present application, the UW preservation solution comprises 100 mmol / L of galacturonic acid, 100 mmol / L of potassium hydroxide, 25 mmol / L of potassium dihydrogen phosphate, 5 mmol / L of magnesium sulfate, 5 mmol / L of adenosine, 3 mmol / L of reduced glutathione, 1 mmol / L of allopurinol, 30 mmol / L of raffinose and 50 g / L of hydroxyethyl starch. In one embodiment of the present application, the pH value of the UW preservation solution is 7.4. In one embodiment of the present application, the osmotic pressure of the UW preservation solution is 320 mOsm / kg.

[0038] In one embodiment of the present application, the preservation solution comprises a colloidal substance and propyl gallate, and further comprises at least one of a physiologically acceptable salt, a buffer, a diluent and an excipient. Specifically, the physiologically acceptable salt, buffer, diluent and excipient can be selected according to the use.

[0039] In one embodiment of the present application, the solvent of the preservation solution is water, that is, the preservation solution is an aqueous solution, which is safer and more reliable.

[0040] In one embodiment of the present application, the pH value of the preservation solution is 2 to 9. Specifically, the pH value of the preservation solution can be, but is not limited to, 2, 3, 4, 4.5, 5, 5.1, 5.5, 6, 6.2, 6.3, 6.4, 6.5, 7, 7.5, 7.6, 7.9, 8, 8.5 or 9, etc. Exemplarily, the pH value of the preservation solution can be 4 to 8, 5 to 8, 5 to 7.9, 5.1 to 7.9, 6 to 7.9, 6 to 7.6, 6.1 to 7.6, 6.2 to 7.6, 6.3 to 7.6, 6.4 to 7.6 or 6.5 to 7.5, etc. In one embodiment of the present application, the pH value of the preservation solution can be 6 to 7.6, which belongs to the neutral range and is more conducive to improving the preservation effect.

[0041] In an embodiment of the present application, the osmotic pressure of the preservation solution is 150 mOsm / kg to 1500 mOsm / kg. Specifically, the osmotic pressure of the preservation solution can be, but is not limited to, 150 mOsm / kg, 200 mOsm / kg, 240 mOsm / kg, 290 mOsm / kg, 300 mOsm / kg, 350 mOsm / kg, 370 mOsm / kg, 400 mOsm / kg, 430 mOsm / kg, 450 mOsm / kg, 480 mOsm / kg, 490 mOsm / kg, 500 mOsm / kg, 550 mOsm / kg, 580 mOsm / kg, 600 mOsm / kg, 800 mOsm / kg, 1000 mOsm / kg, 1250 mOsm / kg or 1400 mOsm / kg, etc. Exemplarily, the osmotic pressure of the preservation solution can be 180 mOsm / kg to 1250 mOsm / kg, 200 mOsm / kg to 800 mOsm / kg, 200 mOsm / kg to 500 mOsm / kg, 240 mOsm / kg to 600 mOsm / kg, 290 mOsm / kg to 580 mOsm / kg, 290 mOsm / kg to 480 mOsm / kg or 290 mOsm / kg to 450 mOsm / kg, etc.

[0042] The preservation solution provided by the present application can be used for transporting cells, tissues, organs, such as ex vivo transportation, etc., which is beneficial to the progress of transplantation, can improve the transplantation success rate, and has a good preservation effect especially on organs donated by elderly donors with diseases, low-quality organs, etc., and can maintain their high vitality. The transplantation in the present application can be allogeneic transplantation, syngeneic transplantation or autologous transplantation, etc., and can be used in the treatment of organ failure, tissue diseases, tissue damage, tissue failure, cell diseases, cell damage, cell failure, etc., and can be cell, tissue, organ transplantation of mammals, etc.

[0043] The preservation solution provided by the present application can be used for static preservation in a bag or machine perfusion preservation. In an embodiment of the present application, the preservation solution can be prepared by sterilizing and mixing each component in the preservation solution or sterilizing after mixing. Specifically, equipment for preparing the preservation solution, such as a cell or tissue storage instrument, an organ perfusion machine, etc., can be used for the preparation of the preservation solution.

[0044] The preservation solution provided by the present application can prevent or reduce ex vivo damage to cells, tissues, organs, such as preventing or reducing the damage effects caused by cold ischemia and warm blood reperfusion on cell function, tissue function, organ function, and maintaining cell function, tissue function, organ function.

[0045] The present application provides a preservation method, which includes placing at least one of cells, tissues, and organs in the preservation solution in any of the above embodiments for preservation. The preservation method provided by the present application is simple to operate, can extend the existing preservation time, and effectively improve the viability of the cells, tissues, and organs after preservation.

[0046] In one embodiment of the present application, the preservation temperature is -80°C to 80°C. The preservation solution provided by the present application can be used at various preservation temperatures and can ensure the preservation effect, with a wide range of applications and diverse application scenarios. For example, the preservation temperature can be ultra-low temperature, sub-zero temperature, refrigeration temperature, or mammalian body temperature, etc. Specifically, the preservation temperature can be, but is not limited to, -80°C, -70°C, -60°C, -50°C, -40°C, -30°C, -20°C, -10°C, 0°C, 1°C, 2°C, 4°C, 5°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 38°C, 40°C, 50°C, 60°C, 70°C, or 80°C, etc. Exemplarily, the preservation temperature can be, but is not limited to, -18°C to 40°C, -18°C to 37°C, -18°C to 10°C, 0°C to 37°C, 0°C to 25°C, 0°C to 10°C, 0°C to 8°C, 0°C to 4°C, 1°C to 6°C, 2°C to 5°C, 25°C to 40°C, 29°C to 38°C, 35°C to 38°C, etc.

[0047] In the present application, the preservation time can be selected according to actual needs. The preservation solution provided by the present application can preserve cells, tissues, and organs in vitro for a long time. In one embodiment of the present application, the maximum preservation time can be 8 days. In this case of the preservation duration, the cells, tissues, and organs can still maintain strong viability, ensuring the use of the cells, tissues, and organs. Specifically, the maximum preservation time can be, but is not limited to, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, etc.

[0048] The present application provides the application of alkyl gallate in the preservation solution. The preservation solution contains a colloidal substance and alkyl gallate. The concentration of alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L, which can improve the preservation effect of the preservation solution and is beneficial to the use of the preservation solution. The relevant introduction of the preservation solution is as described above and will not be elaborated here.

[0049] The effects of the technical solutions of the present application are further illustrated by specific examples below.

[0050] C57BL / 6j mice used in the experiments were purchased from Jackson Laboratory (Bar Harbor, ME, USA); EA.hy926 (human endothelial cells) and HepG2 (human hepatocytes) were purchased from ATCC (Manassas, VA, USA); HKC-8 (human proximal tubular epithelial cells) was provided by Dr. Lorraine Racusen of the Johns Hopkins University School of Medicine in the United States.

[0051] The preservation solutions used in the experiments included HPG preservation solution, UW preservation solution, HPG preservation solution containing propyl gallate (abbreviated as "PG"), HPG preservation solution containing octyl gallate, HPG preservation solution containing lauryl gallate, and UW preservation solution containing propyl gallate. Among them, the components of the HPG preservation solution (abbreviated as "HPG") included 100 mmol / L lacturonic acid, 100 mmol / L sodium hydroxide, 25 mmol / L potassium dihydrogen phosphate, 5 mmol / L magnesium sulfate, 5 mmol / L adenosine, 3 mmol / L reduced glutathione, 1 mmol / L allopurinol, and 30 g / L hyperbranched polyglycerol (molecular weight 1 kDa), with a pH value of 7.4 and an osmotic pressure of 320 mOsm / kg; the components of the UW preservation solution (abbreviated as "UW") included 100 mmol / L lacturonic acid, 100 mmol / L potassium hydroxide, 25 mmol / L potassium dihydrogen phosphate, 5 mmol / L magnesium sulfate, 5 mmol / L adenosine, 3 mmol / L reduced glutathione, 1 mmol / L allopurinol, 30 mmol / L raffinose, and 50 g / L hydroxyethyl starch, with a pH value of 7.4 and an osmotic pressure of 320 mOsm / kg.

[0052] Determination of lactate dehydrogenase (LDH): It was determined by the LDH method using a cytotoxicity detection device (Roche Applied Science, Laval, QC). In cultured cells, the LDH release amount in the preservation solution was presented as a percentage of the positive control (using 2% Triton X-100 to lyse the cells), or in cryopreservation of mouse kidneys, the LDH release content in the preservation solution was measured by absorbance (OD 490 ), and the data was expressed as mean ± standard deviation.

[0053] Determination of adenosine triphosphate (ATP): The amount of ATP in solutions or cell and tissue extracts was determined using an ATP determination device (Invitrogen-Life Technologies Inc., Burlington, ON, Canada). Briefly, in vitro human cells (1×10 6Cells (at a density of

[0054] Histological analysis of tissue damage after kidney cryopreservation: Kidney tissues were first fixed in 10% buffered formaldehyde. Then the samples were embedded in paraffin and used for hematoxylin-eosin staining after sectioning. Histological analysis was used to determine tissue damage in H&E-stained sections, and a single-blind form was adopted to pathologically score tissue damage after cryopreservation based on the severity of glomerular damage. According to microscopic observation: 0: normal glomeruli without damage; 1: glomeruli with less than 25% area damage; 2: glomeruli with 25%-50% area damage; 3: glomeruli with 50%-75% area damage; or 4: glomeruli with more than 75% area damage.

[0055] Example 1: Propyl gallate-containing preservation solution improves the protective effect on human cells during freezing

[0056] HPG preservation solution and UW preservation solution containing different concentrations of propyl gallate (PG concentrations were 0, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, or 100 μM) were used as preservation solutions, and EA.hy926 cells, HepG2 cells, and HKC-8 cells were respectively preserved at 4°C. The degree of cell death was detected by measuring the release amount of LDH in the preservation solution. The results are as Figure 1 shown, where Figure 1 is the release amount of LDH of cells after preservation with the preservation solution, Figure 1(a) shows the LDH release of EA.hy926 cells stored in preservation solutions containing different concentrations of propyl gallate for 3 days. Compared with the preservation solution without propyl gallate, the LDH content in the preservation solution with propyl gallate decreases, and the degree of cell death is low, indicating that the preservation solution containing propyl gallate plays a protective role in cells during refrigeration. By analyzing, the p - value of the HPG preservation solution containing propyl gallate is 0.0206 (one - way ANOVA, n = 4), the p - value of the UW preservation solution containing propyl gallate is 0.0032 (one - way ANOVA, n = 3), and the p - value for the comparison between the HPG preservation solution and the UW preservation solution with a propyl gallate concentration of 25 μM - 50 μM is 0.3127 (two - way ANOVA); Figure 1 (b) shows the LDH release of EA.hy926 cells stored in preservation solutions containing different concentrations of propyl gallate for different days. Compared with the preservation solution without propyl gallate, the LDH content in the preservation solution with propyl gallate decreases, and the degree of cell death is low, indicating that the preservation solution containing propyl gallate plays a protective role in cells during refrigeration, and as the storage time increases, the preservation solution containing propyl gallate can still play a good protective role. By two - way ANOVA, the p - value for the comparison between the preservation solution without propyl gallate and the preservation solution containing 10 μM propyl gallate is 0.0432, the p - value for the comparison between the preservation solution containing 10 μM propyl gallate and the preservation solution containing 25 μM propyl gallate is 0.0093, the p - value for the comparison between the preservation solution containing 10 μM propyl gallate and the preservation solution containing 50 μM propyl gallate is 0.0049, and the p - value for the comparison between the preservation solution containing 25 μM propyl gallate and the preservation solution containing 50 μM propyl gallate is 0.7167; Figure 1 (c) shows the LDH release of HepG2 cells stored in preservation solutions containing different concentrations of propyl gallate for 3 days. Compared with the preservation solution without propyl gallate, the LDH content in the preservation solution with propyl gallate decreases, and the degree of cell death is low, indicating that the preservation solution containing propyl gallate plays a protective role in cells during refrigeration. By analyzing, the p - value of the HPG preservation solution containing propyl gallate is less than 0.0001 (one - way ANOVA, n = 4), the p - value of the UW preservation solution containing propyl gallate is less than 0.0001 (one - way ANOVA, n = 3), and the p - value for the comparison between the HPG preservation solution and the UW preservation solution with a propyl gallate concentration of 25 μM - 50 μM is 0.1167 (two - way ANOVA); Figure 1(d) shows the LDH release of HepG2 cells preserved in preservation solutions containing different concentrations of propyl gallate for different days. Compared with the preservation solution without propyl gallate, the LDH content in the preservation solution with propyl gallate decreased, and the degree of cell death was low, indicating that the preservation solution containing propyl gallate played a protective role in cells during refrigeration. Moreover, as the preservation time increased, the preservation solution containing propyl gallate could still play a good protective role. Through two-way ANOVA, the p-value for comparing the preservation solution without propyl gallate with the preservation solution containing 10 μM propyl gallate was 0.0012, the p-value for comparing the preservation solution containing 10 μM propyl gallate with the preservation solution containing 25 μM propyl gallate was 0.0375, the p-value for comparing the preservation solution containing 10 μM propyl gallate with the preservation solution containing 50 μM propyl gallate was 0.4352, and the p-value for comparing the preservation solution containing 25 μM propyl gallate with the preservation solution containing 50 μM propyl gallate was 0.1052; Figure 1 (e) shows the LDH release of HKC-8 cells preserved in preservation solutions containing different concentrations of propyl gallate for 5 days. Compared with the preservation solution without propyl gallate, the LDH content in the preservation solution with propyl gallate decreased, and the degree of cell death was low, indicating that the preservation solution containing propyl gallate played a protective role in cells during refrigeration. By analyzing, the p-value of the HPG preservation solution containing propyl gallate was less than 0.0001 (one-way ANOVA, n = 4), the p-value of the UW preservation solution containing propyl gallate was 0.0010 (one-way ANOVA, n = 3), and the p-value for comparing the HPG preservation solution and the UW preservation solution with a propyl gallate concentration of 25 μM - 50 μM was 1.000 (two-way ANOVA); Figure 1 (f) shows the LDH release of HKC-8 cells preserved in preservation solutions containing different concentrations of propyl gallate for different days. Compared with the preservation solution without propyl gallate, the LDH content in the preservation solution with propyl gallate decreased, and the degree of cell death was low, indicating that the preservation solution containing propyl gallate played a protective role in cells during refrigeration. Moreover, as the preservation time increased, the preservation solution containing propyl gallate could still play a good protective role. Through two-way ANOVA, the p-value for comparing the preservation solution without propyl gallate with the preservation solution containing 10 μM propyl gallate was less than 0.0001, the p-value for comparing the preservation solution containing 10 μM propyl gallate with the preservation solution containing 25 μM propyl gallate was 0.2625, the p-value for comparing the preservation solution containing 10 μM propyl gallate with the preservation solution containing 50 μM propyl gallate was 0.4806, and the p-value for comparing the preservation solution containing 25 μM propyl gallate with the preservation solution containing 50 μM propyl gallate was 0.4949.

[0057] Although different cells have different sensitivities to hypothermic hypoxia injury and to propyl gallate, adding propyl gallate to UW or HPG preservation solutions enhanced their protective effects on these cells from death in a dose-dependent manner (p < 0.05, one-way ANOVA) ( Figure 1 (a), (c), (e) in Figure 1 ), and propyl gallate had the greatest protective effect on three different cells when its concentration in UW and HPG solutions was approximately 25 μM to 50 μM. At the same time, there was no statistical difference in the cytoprotective effect of propyl gallate (25 μM - 50 μM) between UW and HPG solutions ( Figure 1 (a), (c), (e) in

[0058] Example 2: Preservation solution containing propyl gallate improves the activity and survival rate of cells during re-warming and oxygen supply

[0059] Using HPG preservation solution and UW preservation solution containing different concentrations of propyl gallate (PG concentration is 0, 10 μM, 25 μM or 50 μM) as preservation solutions, after storing EA.hy926 cells for 3 days, HepG2 cells for 3 days, and HKC-8 cells for 8 days at 4°C respectively, changing to normal cell culture medium, where EA.hy926 cells and HepG2 cells use DMEM culture medium (Dulbecco's modified Eagle medium), and HKC-8 cells use K1 + / + medium, culturing at 37°C under the condition of containing 20% oxygen for 48 h, testing the cell activity during re-warming and oxygen supply by measuring the NAD(P)H-dependent cell redox enzyme activity using the MTT (thiazolyl blue) method, the data is expressed as mean ± standard deviation, and the results are as Figure 2 shown, where Figure 2 is the MTT detection result of cells after storing cells with the preservation solution, Figure 2(a) shows the absorbance values detected by MTT after EA.hy926 cells were stored in preservation solutions containing different concentrations of propyl gallate for 3 days. Compared with the preservation solution without propyl gallate, the OD values of EA.hy926 cells detected by MTT increased after being stored in the preservation solution with propyl gallate, indicating high EA.hy926 cell activity. The preservation solution containing propyl gallate played a protective role on the cells and could improve the cell activity after reoxygenation. By analysis, the p-value of the HPG preservation solution containing propyl gallate was less than 0.0001 (one-way ANOVA, n = 3), the p-value of the UW preservation solution containing propyl gallate was less than 0.001 (one-way ANOVA, n = 3), and the p-value for comparison between the HPG preservation solution and UW preservation solution containing propyl gallate was 0.9150 (two-way ANOVA); Figure 2 (b) shows the absorbance values detected by MTT after HepG2 cells were stored in preservation solutions containing different concentrations of propyl gallate for 3 days. Compared with the preservation solution without propyl gallate, the OD values of HepG2 cells detected by MTT increased after being stored in the preservation solution with propyl gallate, indicating high HepG2 cell activity. The preservation solution containing propyl gallate played a protective role on the cells and could improve the cell activity after reoxygenation. By analysis, the p-value of the HPG preservation solution containing propyl gallate was less than 0.0001 (one-way ANOVA, n = 3), the p-value of the UW preservation solution containing propyl gallate was less than 0.001 (one-way ANOVA, n = 3), and the p-value for comparison between the HPG preservation solution and UW preservation solution containing propyl gallate was 0.3208 (two-way ANOVA); Figure 2 (c) shows the absorbance values detected by MTT after HKC-8 cells were stored in preservation solutions containing different concentrations of propyl gallate for 8 days. Compared with the preservation solution without propyl gallate, the OD values of HKC-8 cells detected by MTT increased after being stored in the preservation solution with propyl gallate, indicating high HKC-8 cell activity. The preservation solution containing propyl gallate played a protective role on the cells and could improve the cell activity after reoxygenation. By analysis, the p-value of the HPG preservation solution containing propyl gallate was less than 0.0001 (one-way ANOVA, n = 3), the p-value of the UW preservation solution containing propyl gallate was less than 0.001 (one-way ANOVA, n = 3), and the p-value for comparison between the HPG preservation solution and UW preservation solution containing propyl gallate was 0.4533 (two-way ANOVA).

[0060] Using HPG preservation solution (abbreviated as "HPG"), HPG preservation solution containing 25 μM propyl gallate (abbreviated as "HPG+PG"), UW preservation solution (abbreviated as "UW"), and UW preservation solution containing 25 μM propyl gallate (abbreviated as "UW+PG") as preservation solutions, EA.hy926 cells were preserved at 4°C for 3 days, HepG2 cells were preserved at 4°C for 3 days, and HKC-8 cells were preserved at 4°C for 5 days, and then changed to normal cell culture medium. Among them, DMEM culture medium was used for EA.hy926 cells and HepG2 cells, and K1 + / + medium was used for HKC-8 cells. The cells were cultured at 37°C under the condition of containing 20% oxygen for 48 h. Viable or apoptotic cells were detected by flow cytometry through 7-AAD and Annexin-V staining. The data were expressed as mean ± standard deviation, and the results were as Figures 3a to 3f shown, where Figures 3a to 3f was the detection result of the cell viability after preservation with the preservation solution, Figure 3a was the flow cytometry detection result of EA.hy926 cells after preservation with the preservation solution for 3 days; Figure 3b was the cell survival rate of EA.hy926 cells after preservation with the preservation solution for 3 days. Compared with the preservation solution without propyl gallate, the survival rate of EA.hy926 cells after preservation with the preservation solution containing propyl gallate increased significantly, indicating that the preservation solution containing propyl gallate played a protective role on the cells and improved their survival rate during the process of returning to heat and supplying oxygen; through two-tailed T test analysis, the p value of the comparison between HPG preservation solution and HPG preservation solution containing propyl gallate was less than 0.0001 (n = 7), the p value of the comparison between UW preservation solution and UW preservation solution containing propyl gallate was 0.0001 (n = 7), and the p value of the comparison between HPG preservation solution containing propyl gallate and UW preservation solution was 0.0178 (n = 7); Figure 3c was the flow cytometry detection result of HepG2 cells after preservation with the preservation solution for 3 days, Figure 3d was the cell survival rate of HepG2 cells after preservation with the preservation solution for 3 days; through two-tailed T test analysis, the p value of the comparison between HPG preservation solution and HPG preservation solution containing propyl gallate was 0.0038 (n = 5), the p value of the comparison between UW preservation solution and UW preservation solution containing propyl gallate was 0.4415 (n = 5), and the p value of the comparison between HPG preservation solution containing propyl gallate and UW preservation solution was 0.4374 (n = 5); Figure 3e was the flow cytometry detection result of cells after preservation with the preservation solution for 3 days for HKC-8 cells, Figure 3fCell viability of HKC-8 cells after being stored in the preservation solution for 5 days; the p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was 0.0274 (n = 5) analyzed by two-tailed T-test, the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.6488 (n = 5), and the p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was 0.4782 (n = 5).

[0061] After cells, tissues, and organs are cryopreserved and transplanted into the recipient, the process of reperfusion with warm and oxygenated recipient blood can cause additional damage, namely rewarming-reoxygenation (reperfusion) injury. This rewarming-reoxygenation injury is mainly caused by the mode of apoptosis. The MTT method was used to measure cell viability, and 7-AAD and Annexin-V staining were combined with flow cytometry to detect live or apoptotic cells, so as to examine the effect of cryopreserving cells with the preservation solution containing propyl gallate on cell apoptosis / survival during rewarming-reoxygenation.

[0062] As Figure 2 shown, MTT measurement shows that cryopreserving cells with the preservation solution containing propyl gallate improves the viability or metabolic activity of the three tested cell types during the rewarming-reoxygenation stage. The MTT values of cells cryopreserved with the preservation solution (HPG or UW) containing propyl gallate (10 μM - 50 μM) are higher than those of cells not treated with propyl gallate (HPG or UW group: p < 0.0001, comparison between propyl gallate-treated cells and non-propyl gallate-treated cells, one-way ANOVA). Further analysis shows that there is no significant difference in the effect of propyl gallate in the HPG solution and in the UW among the three types of cells.

[0063] In the case of cell necrosis caused by hypothermic ischemia, rewarming-reoxygenation mainly leads to apoptosis, and this apoptosis can be quantitatively measured by combining 7-AAD and Annexin-V staining with a flow cytometer. Figures 3a to 3f It is shown that in cells not cryopreserved with propyl gallate (such as in the HPG group), apoptosis of EA.hy926 cells is mainly late apoptosis (upper right or double-positive cells) ( Figure 3a ), while apoptosis of HepG2 and HKC-8 cells is mainly early apoptosis (lower right or Annexin-V positive cells) ( Figure 3c and Figure 3e ). In this flow cytometry analysis, the double-negative cells in the lower left corner represent the live cell population. As Figure 3b 、 Figure 3d and Figure 3fAs shown, compared with the HPG group without propyl gallate, the percentage of viable cell population increased in these three types of cells treated with propyl gallate-containing (HPG+PG group), and this protective effect also appeared in the UW+PG group of EA.hy926 cells. However, compared with the UW group, UW with propyl gallate did not show additional beneficial effects on HepG2 and HKC-8 cells. In addition, in EA.hy926 cells, the percentage of double-negative cells in the HPG+PG group was significantly higher than that in the UW+PG group, but there was no difference between the two groups in the protection of HepG2 and HKC-8 cells.

[0064] Example 3: Preservation solution containing propyl gallate inhibits lipid peroxidation of human cells

[0065] Malondialdehyde is one of the final products of polyunsaturated fatty acid peroxidation and is commonly used as a lipid peroxidation marker to detect the effect of preservation solutions containing propyl gallate (UW or HPG preservation solution) on lipid peroxidation in cryopreserved EA.hy926 and HKC-8 cells.

[0066] Using HPG preservation solution (abbreviated as "HPG"), HPG preservation solution containing 50 μM propyl gallate (abbreviated as "HPG+PG"), UW preservation solution (abbreviated as "UW"), and UW preservation solution containing 50 μM propyl gallate (abbreviated as "UW+PG") as preservation solutions, EA.hy926 cells were stored at 4°C for 3 days and HKC-8 cells were stored at 4°C for 5 days, and then the malondialdehyde (MDA) level in the cells was measured by a malondialdehyde assay kit to detect the antioxidant effect of the preservation solution containing propyl gallate on lipid peroxidation. The data were expressed as mean ± standard deviation, and the results are as Figure 4 shown, where Figure 4 is the malondialdehyde content in the cells after preservation with the preservation solution, Figure 4 (a) in is the malondialdehyde content in the cells after preservation of EA.hy926 cells with the preservation solution. The p-value for the comparison between the HPG preservation solution and the HPG preservation solution containing propyl gallate was 0.0093 (two-tailed T-test, n = 4), the p-value for the comparison between the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0106 (two-tailed T-test, n = 4), and the p-value for the comparison between the HPG preservation solution containing propyl gallate and the UW preservation solution was 0.0028 (two-tailed T-test, n = 4); compared with the preservation solution without propyl gallate (UW or HPG preservation solution), the preservation solution containing propyl gallate (UW+PG or HPG+PG preservation solution) reduced the malondialdehyde level in cryopreserved EA.hy926 cells, indicating that the preservation solution containing propyl gallate can reduce the production of cellular malondialdehyde and inhibit cellular lipid peroxidation; Figure 4In (b), the malondialdehyde content of HKC-8 cells after being preserved in the preservation solution was measured. By analyzing, the p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was 0.0191 (two-tailed T-test, n = 4), the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0478 (two-tailed T-test, n = 4), and the p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was 0.0068 (two-tailed T-test, n = 4); compared with the preservation solution without propyl gallate (UW or HPG preservation solution), the preservation solution containing propyl gallate (UW+PG or HPG+PG preservation solution) for cryopreserving HKC-8 cells reduced the malondialdehyde level in the cells, indicating that the preservation solution containing propyl gallate can reduce the production of cellular malondialdehyde and inhibit cellular lipid peroxidation. In HKC-8 cells, the malondialdehyde level in the HPG+PG group was lower than that in UW+PG, but this was not the case in EA.hy926 cells. Generally speaking, supplementing propyl gallate in the HPG or UW preservation solution can significantly reduce lipid peroxidation in these human cells during cold storage.

[0067] Example 4: The preservation solution containing propyl gallate slows down the reduction of intracellular ATP

[0068] Using HPG preservation solution (abbreviated as "HPG"), HPG preservation solution containing 50 μM propyl gallate (abbreviated as "HPG+PG"), UW preservation solution (abbreviated as "UW"), and UW preservation solution containing 50 μM propyl gallate (abbreviated as "UW+PG") as preservation solutions, after preserving EA.hy926 cells at 4°C for 3 days, the effects of propyl gallate on the changes in intracellular ATP levels were detected by measuring intracellular ATP (iATP), extracellular ATP (eATP), and measuring the mitochondrial membrane potential with a JC-1 probe. The data were expressed as mean ± standard deviation, and the results are as Figures 5a to 5d shown, where Figures 5a to 5d the ATP content of EA.hy926 cells after being preserved in the preservation solution, Figure 5a was the iATP content. Low temperature increases the permeability of the cell membrane, resulting in the leakage of intracellular substances including ATP. Compared with the preservation solution without propyl gallate (UW or HPG preservation solution), the preservation solution containing propyl gallate (UW+PG or HPG+PG preservation solution) preserved the intracellular iATP content; by analyzing, the p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was 0.0131 (two-tailed T-test, n = 4), the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0180 (two-tailed T-test, n = 4), and the p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was 0.6953 (two-tailed T-test, n = 4); Figure 5bThe ratio of iATP to eATP. Compared with the preservation solutions (UW or HPG preservation solutions) without propyl gallate, the preservation solutions containing propyl gallate (UW+PG or HPG+PG preservation solutions) increase the ratio of intracellular iATP to extracellular eATP. By analyzing, the p-value for the comparison between the HPG preservation solution and the HPG preservation solution containing propyl gallate is less than 0.0001 (two-tailed T-test, n = 4), the p-value for the comparison between the UW preservation solution and the UW preservation solution containing propyl gallate is 0.0099 (two-tailed T-test, n = 4), and the p-value for the comparison between the HPG preservation solution containing propyl gallate and the UW preservation solution is 0.2108 (two-tailed T-test, n = 4). Figure 5c The flow cytometry JC-1 results of cells after preservation with the preservation solution. The main function of mitochondria is to synthesize ATP, and its functional status can be evaluated using JC-1 staining. The figure shows J aggregates (red) and J monomers (green). Figure 5d The red / green ratio. The mitochondrial membrane potential after different treatments was measured using the red / green ratio of JC-1. By analyzing, the p-value for the comparison between the HPG preservation solution and the HPG preservation solution containing propyl gallate is 0.0078 (two-tailed T-test, n = 4), the p-value for the comparison between the UW preservation solution and the UW preservation solution containing propyl gallate is 0.0101 (two-tailed T-test, n = 4), and the p-value for the comparison between the HPG preservation solution containing propyl gallate and the UW preservation solution is 0.0797 (two-tailed T-test, n = 4). The red / green ratio in the group treated with propyl gallate is significantly higher than that in the group without propyl gallate, indicating that freezing EA.hy926 cells with the HPG or UW preservation solution containing propyl gallate can increase or protect the number of structurally intact mitochondria.

[0069] Using the HPG preservation solution (abbreviated as "HPG"), the HPG preservation solution containing 50 μM propyl gallate (abbreviated as "HPG+PG"), the UW preservation solution (abbreviated as "UW"), and the UW preservation solution containing 50 μM propyl gallate (abbreviated as "UW+PG") as preservation solutions, after preserving HKC-8 cells at 4°C for 5 days, the effect of propyl gallate on the change in intracellular ATP level was detected by measuring intracellular ATP (iATP), extracellular ATP (eATP), and the mitochondrial membrane potential using the JC-1 probe. The data are expressed as mean ± standard deviation, and the results are as Figures 6a to 6d shown, where Figures 6a to 6d The ATP content of cells after preservation with the preservation solution. Figure 6aIt is the content of iATP. Compared with the preservation solutions (UW or HPG preservation solution) without propyl gallate, the preservation solutions containing propyl gallate (UW+PG or HPG+PG preservation solution) increase the intracellular iATP content; by analyzing, the p-value for comparing the HPG preservation solution with the HPG preservation solution containing propyl gallate is 0.0323 (two-tailed T-test, n = 4), the p-value for comparing the UW preservation solution with the UW preservation solution containing propyl gallate is 0.0273 (two-tailed T-test, n = 4), and the p-value for comparing the HPG preservation solution containing propyl gallate with the UW preservation solution is 0.5931 (two-tailed T-test, n = 4); Figure 6b It is the ratio of iATP to eATP. Compared with the preservation solutions (UW or HPG preservation solution) without propyl gallate, the preservation solutions containing propyl gallate (UW+PG or HPG+PG preservation solution) increase the ratio of intracellular iATP to extracellular eATP; by analyzing, the p-value for comparing the HPG preservation solution with the HPG preservation solution containing propyl gallate is 0.0091 (two-tailed T-test, n = 4), the p-value for comparing the UW preservation solution with the UW preservation solution containing propyl gallate is 0.0274 (two-tailed T-test, n = 4), and the p-value for comparing the HPG preservation solution containing propyl gallate with the UW preservation solution is 0.0006 (two-tailed T-test, n = 4); Figure 6c It is the flow cytometry JC-1 result of the cells after being preserved by the preservation solution. The main function of mitochondria is to synthesize ATP, and its functional status can be evaluated by JC-1 staining. The figure shows J aggregates (red) and J monomers (green); Figure 6d It is the red / green ratio. The mitochondrial membrane potential after different treatments is measured by the red / green ratio of JC-1. By analyzing, the p-value for comparing the HPG preservation solution with the HPG preservation solution containing propyl gallate is 0.0003 (two-tailed T-test, n = 4), the p-value for comparing the UW preservation solution with the UW preservation solution containing propyl gallate is 0.0214 (two-tailed T-test, n = 4), and the p-value for comparing the HPG preservation solution containing propyl gallate with the UW preservation solution is 0.1998 (two-tailed T-test, n = 4). Except that the iATP / eATP in HKC-8 cells is significantly higher after HPG+PG treatment than UW+PG treatment, there are no differences in other results of EA.hy926 cells and HKC-8 cells between these two groups (HPG+PG and UW+PG).

[0070] Example 5: The preservation solution containing propyl gallate slows down the reduction of adenosine triphosphate (ATP) in cells

[0071] The effects of propyl gallate-containing preservation solutions on kidney tissue damage during ex vivo cold storage were examined by measuring tissue LDH release and ATP levels, as well as glomerular injury. Mouse kidneys were first perfused with cold preservation solutions with or without propyl gallate and then stored in the same solution. Data were expressed as mean ± standard deviation, and the results are shown as Figure 7 follows, where Figure 7 is the preservation effect of the preservation solution on the kidney, Figure 7 in (a) is the LDH content in the preservation solution. The p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was 0.0026 (two-way ANOVA, n = 8 or 11), the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0011 (two-way ANOVA, n = 6), and the p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was 0.8237 (two-way ANOVA); Figure 7 in (b) is the microscopic image of kidney sections. By processing the kidneys preserved in the preservation solution for histological analysis, it can be seen that after 48 h of cold storage in the preservation solution (HPG or UW preservation solution) with or without propyl gallate, tubulointerstitial dilation and glomerular destruction are the main forms of tissue damage; Figure 7 in (c) is the cumulative score of glomerular injury. The p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was less than 0.0001 (two-tailed T-test, n = 6), the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0003 (two-tailed T-test, n = 6), and the p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was less than 0.0001 (two-tailed T-test); Figure 7 in (d) is the percentage of intact glomeruli. The p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was less than 0.0001 (two-tailed T-test, n = 6), the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0003 (two-tailed T-test, n = 6), and the p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was less than 0.0001 (two-tailed T-test). The destruction of glomeruli in each kidney was scored. Similar to the less glomerular injury in the group with propyl gallate-containing preservation solution, the number of intact glomeruli in the HPG+PG group was higher than that in the HPG group, and the number of intact glomeruli in the UW+PG group was higher than that in the UW group, indicating that adding propyl gallate to the HPG or UW solution can significantly prevent glomerular injury, and the protective effect of adding propyl gallate to the HPG preservation solution on glomeruli is significantly higher than that of adding it to the UW preservation solution; Figure 7In (e), the ATP content. The p-value for comparing the HPG preservation solution and the HPG preservation solution containing propyl gallate was 0.0004 (two-tailed T-test, n = 6), and the p-value for comparing the UW preservation solution and the UW preservation solution containing propyl gallate was 0.0039 (two-tailed T-test, n = 6). The p-value for comparing the HPG preservation solution containing propyl gallate and the UW preservation solution was 0.9021 (two-tailed T-test). Further confirmation was obtained by measuring the residual ATP level in the tissue after freezing that adding propyl gallate to the HPG or UW preservation solution was beneficial to reducing ATP loss in the kidney tissue, and there was no significant difference between the HPG+PG and UW+PG groups. It can be seen from Figure 7 that the HPG or UW preservation solution containing propyl gallate can improve the protection of mouse kidneys and is beneficial for their preservation.

[0072] Example 6: Effects of gallic acid and its alkyl esters on the cryoprotective function of preservation solutions

[0073] EA.hy926 cells and HepG2 cells were respectively placed in 24-well cell culture plates (2.5×10 5 cells per well), adhered and cultured overnight, and then stored in the HPG preservation solution containing different additives (25 μM GA, PG, OG or LG). After being stored at 4 °C for 3 days, the LDH content in the preservation solution was compared with that in the 2% TritonX-100 lysis solution to measure the cell damage degree (%), and the cell protection degree (%) = (damage in the preservation solution without additives - damage in the preservation solution with additives) / damage in the preservation solution without additives × 100% was calculated. The results are as Figure 8 shown, where Figure 8 are the protection effects of different additives. Figure 8 In (a), it is the preservation effect of different additives on EA.hy926 cells. The p-value for comparing the HPG preservation solution added with gallic acid (GA) and the HPG preservation solution without additives was 0.8706 (two-tailed T-test, n = 3), and the p-values for comparing the HPG preservation solutions added with propyl gallate (PG), octyl gallate (OG) or lauryl gallate (LG) and the HPG preservation solution without additives were less than 0.0001 (two-tailed T-test, n = 3); Figure 8Among them, (b) shows the preservation effects of different additives on HepG2 cells. By analyzing the p-values of the comparison between the HPG preservation solution with GA added and the HPG preservation solution without additives, the p-value is 0.6618 (two-tailed T-test, n = 3). The p-value of the comparison between the HPG preservation solution with PG added and the HPG preservation solution without additives is 0.0007 (two-tailed T-test, n = 3). The p-value of the comparison between the HPG preservation solution with OG added and the HPG preservation solution without additives is 0.0011 (two-tailed T-test, n = 3). The p-value of the comparison between the HPG preservation solution with LG added and the HPG preservation solution without additives is 0.0028 (two-tailed T-test, n = 3). It can be seen that the HPG preservation solution with gallic acid added does not play a protective role. The HPG preservation solution with alkyl gallates (such as PG, OG, or LG) added greatly improves the protective effect of the preservation solution on EA.hy926 cells and HepG2 cells, with a significant effect. Moreover, the activity of HepG2 cells in the HPG preservation solutions containing PG, OG, and LG decreases in turn.

[0074] Example 7: Effect of adding propyl gallate to different preservation solutions on their cryoprotective function

[0075] After HepG2 cells were placed in a 24-well cell culture plate (2.5×10 5 cells per well) and cultured adherently for one night, they were then stored in HPG preservation solution, DMEM culture medium (Dulbecco's Modified Eagle Medium) (DMEM), or phosphate-buffered saline (PBS) containing different concentrations (0, 25 μM, 150 μM, 300 μM, 450 μM, or 550 μM) of propyl gallate (PG) at 4°C for 3 days. The cell damage degree (%) was measured by comparing the LDH content in the preservation solution with that in 2% Triton X-100 lysis solution, where the LDH release amount (%) = LDH content in the preservation solution / LDH content in Triton X-100 lysis solution × 100%. The results are as Figure 9 shown. Figure 9To compare the effects of different preservation solutions on the LDH release amount, the p-values for comparing the HPG preservation solution without propyl gallate with the HPG preservation solution containing 25 μM propyl gallate were 0.0003 (two-tailed T-test, n = 4), the p-value for comparing the HPG preservation solution without propyl gallate with the HPG preservation solution containing 150 μM propyl gallate was 0.0059 (two-tailed T-test, n = 4), the p-value for comparing the HPG preservation solution without propyl gallate with the HPG preservation solution containing 300 μM propyl gallate was 0.3092 (two-tailed T-test, n = 4), the p-value for comparing the DMEM cell culture medium containing propyl gallate with the DMEM cell culture medium without propyl gallate was 0.1146 (one-way ANOVA, n = 3), and the p-value for comparing the PBS cell culture medium containing propyl gallate with the PBS cell culture medium without propyl gallate was 0.8970 (one-way ANOVA, n = 3). It can be seen that adding 25 μM or 150 μM of propyl gallate to the HPG preservation solution will further reduce cell damage. When the concentration increases to 300 μM, there is no further effect. When the concentration further increases to 450 μM to 550 μM, it will cause a reverse effect. Moreover, it is also confirmed that adding propyl gallate to DMEM or PBS has no effect on the cryopreservation of HepG2.

[0076] Therefore, the preservation solution containing a colloidal substance and an alkyl gallate provided by this application can be used to preserve cells, tissues, and organs, prevent or reduce ischemic injury, enable the cells, tissues, and organs to have strong vitality, and at the same time maintain a high vitality level during a long preservation time, which is beneficial for its use in the protection of cell, tissue, and organ activities.

[0077] The above is the preferred implementation manner of this application, but it should not be construed as limiting the scope of this application. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of this application, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of this application.

Claims

1. A preservation solution, characterized in that, it contains a colloidal substance and an alkyl gallate, and the concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L.

2. The preservation solution according to claim 1, characterized in that, the concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 150 μmol / L.

3. The preservation solution according to claim 2, characterized in that, the concentration of the alkyl gallate in the preservation solution is 1 μmol / L to 100 μmol / L.

4. The preservation solution according to claim 1, characterized in that, the alkyl gallate includes at least one of propyl gallate, octyl gallate and lauryl gallate.

5. The preservation solution according to claim 1, characterized in that, the colloidal substance includes at least one of polyglycerol, polyethylene glycol and hydroxyethyl starch.

6. The preservation solution according to claim 5, characterized in that, the preservation solution includes at least one of HPG preservation solution, UW preservation solution and IGL-1 preservation solution, the HPG preservation solution includes the polyglycerol, the UW preservation solution includes the hydroxyethyl starch, and the IGL-1 preservation solution includes the polyethylene glycol.

7. The preservation solution according to claim 6, characterized in that, the HPG preservation solution includes lacturonic acid, sodium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, adenosine, reduced glutathione, allopurinol and polyglycerol.

8. The preservation solution according to claim 1, characterized in that, the pH value of the preservation solution is 2 to 9; the osmotic pressure of the preservation solution is 150 mOsm / kg to 1500 mOsm / kg.

9. A preservation method, characterized in that, it includes placing at least one of cells, tissues and organs in the preservation solution according to any one of claims 1 to 8 for preservation.

10. The application of alkyl gallate in a preservation solution, characterized in that, the preservation solution includes a colloidal substance and the alkyl gallate, and the concentration of the alkyl gallate in the preservation solution is 0.1 μmol / L to 299 μmol / L.

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