Application of monounsaturated fatty acid and low-temperature protective agent

Abstract

The invention relates to application of monounsaturated fatty acid and a low-temperature protective agent. Compared with the prior art, the monounsaturated fatty acid is added into the preservation solution, so that the damage problem of cells and tissues in the low-temperature preservation process of 4 DEG C is remarkably reduced, and the monounsaturated fatty acid can protect the integrity of cell membranes of various cells and reduce splitting decomposition caused by low-temperature preservation. And the monounsaturated fatty acid is a natural compound, has low toxicity, and is suitable for wide application.

Description

Technical Field

[0001] This application generally relates to the field of pharmaceutical technology, particularly to the use of monounsaturated fatty acids, or their geometric isomers, tautomers, isotope-labeled compounds, hydrates, solvates or pharmaceutically acceptable salts, and a cryoprotectant. Background Art

[0002] Cryopreservation of cells or tissues is a key technology in modern medicine, biological research and biotechnology, which can effectively extend the storage time of cells and tissues and maintain their biological activities. It is widely used in fields such as organ transplantation and blood storage. However, problems such as cell lysis, death or loss of function often occur during cryopreservation, seriously affecting the preservation quality and clinical application effects. Therefore, optimizing cryopreservation technology and improving the survival rate of cells and tissues have important scientific significance and practical application value. Developing new cryoprotectants with good biocompatibility, high preservation efficiency and high safety is of great significance. Summary of the Invention

[0003] Based on this, the present application provides the use of monounsaturated fatty acids, or their geometric isomers, tautomers, isotope-labeled compounds, hydrates, solvates or pharmaceutically acceptable salts in the preparation of cryoprotectants.

[0004] On the other hand, the present application also provides a cryoprotectant, which comprises a buffer salt solution for maintaining the normal pH value and osmotic pressure of tissues or cells, and monounsaturated fatty acids, or their geometric isomers, tautomers, isotope-labeled compounds, hydrates, solvates or pharmaceutically acceptable salts.

[0005] On the other hand, the present application also provides a method for preventing and / or reducing cryopreservation damage of cells or tissues, which includes adding the cryoprotectant described herein to the external environment of cells or tissues.

[0006] The present application discovers that monounsaturated fatty acids can improve the survival rate of various cells at low temperatures and reduce the level of lipid peroxidation at low temperatures; they can improve the cell survival rate of liver tissue during cryopreservation. The present application discovers that monounsaturated fatty acids can be used as additives for cell and tissue cryoprotectants, and discovers a new use of monounsaturated fatty acids.

[0007] Compared with the prior art, by adding monounsaturated fatty acids to the preservation solution, the present application significantly reduces the damage problems of cells and tissues during cryopreservation at 4°C. Monounsaturated fatty acids can protect the integrity of the cell membranes of various cells and reduce lysis caused by cryopreservation. It improves the protective performance of the preservation solution, extends the cryopreservation period of cells and tissues, and monounsaturated fatty acids are a class of natural compounds with low toxicity and are suitable for wide application.

[0008] Other features and advantages of the present application will be described in the subsequent specification, and in part will become apparent from the specification, or will be understood by implementing the present application. Other advantages of the present application can be achieved and obtained through the solutions described in the specification and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The drawings are used to provide an understanding of the technical solutions of the present application, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present application, and do not constitute a limitation to the technical solutions of the present application.

[0010] Figure 1 Examples of the molecular structures of saturated fatty acids and monounsaturated fatty acids.

[0011] Figure 2 It shows that the monounsaturated fatty acid in Example 1 of the present application improves the survival rate of K562 cells at 4 °C. Among them, (a) is the detection of cell viability by Calcein AM / PI double fluorescence dye staining. Scale bar: 100 μm. (b) is the quantification of the percentage of live cells. Each group has three biological replicates, the error bars represent the standard deviation, and the significance test uses unpaired two-tailed Student's t test, ***P < 0.001, ****P < 0.0001.

[0012] Figure 3 It shows that the monounsaturated fatty acid in Example 2 of the present application improves the survival rate of HeLa cells at 4 °C. Among them, (a) is the detection of cell viability by Calcein AM / PI double fluorescence dye staining. Scale bar: 100 μm. (b) is the quantification of the percentage of live cells. Each group has three biological replicates, the error bars represent the standard deviation, and the significance test uses unpaired two-tailed Student's t test, *P < 0.05.

[0013] Figure 4 It shows that the monounsaturated fatty acid in Example 3 of the present application improves the survival rate of BV2 cells at 4 °C. Among them, (a) is the detection of cell viability by Calcein AM / PI double fluorescence dye staining. Scale bar: 100 μm. (b) is the quantification of the percentage of live cells. Each group has three biological replicates, the error bars represent the standard deviation, and the significance test uses unpaired two-tailed Student's t test, ***P < 0.001.

[0014] Figure 5It shows that monounsaturated fatty acids at different concentrations in Example 4 of the present application increase the survival rate of BV2 cells at 4 °C. Among them, (a) and (c) are the detection of cell viability by Calcein AM / PI double-fluorescent dye staining. Scale bar: 100 μm. (b) and (d) are the quantification of the percentage of live cells. There are three biological replicates in each group, and the error bars represent the standard deviation. The significance test uses unpaired two-tailed Student's t test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0015] Figure 6 It shows that overexpression of desaturase SCD1 in Example 5 of the present application increases the survival rate of cells at 4 °C. Among them, (a) is a schematic diagram of the reaction of desaturase SCD1 converting saturated fatty acids into monounsaturated fatty acids. (b) is the detection of the protein expression level of SCD1 by Western blotting. (c) is the detection of cell viability by Calcein AM / PI double-fluorescent dye staining. Scale bar: 100 μm. (d) is the quantification of the percentage of live cells. There are three biological replicates in each group, and the error bars represent the standard deviation. The significance test uses Two-way ANOVA and Sidak multiple comparisons, ***P<0.001.

[0016] Figure 7 It shows that cis-monounsaturated fatty acids in Example 6 of the present application increase the survival rate of cells at 4 °C. Among them, (a) is the detection of cell viability by Calcein AM / PI double-fluorescent dye staining. The chemical structural formula of the fatty acid used is shown in the figure. Scale bar: 100 μm. (b) is the quantification of the percentage of live cells. There are three biological replicates in each group, and the error bars represent the standard deviation. The significance test uses unpaired two-tailed Student's t test, *P<0.05, **P<0.01, ***P<0.001.

[0017] Figure 8 It shows that monounsaturated fatty acids in Example 7 of the present application reduce the lipid peroxidation level of cells at 4 °C. Among them, the lipid peroxidation level is obtained by measuring its by-product MDA. There are three biological replicates in each group, and the error bars represent the standard deviation. The significance test uses unpaired two-tailed Student's t test, ***P<0.001, ****P<0.0001.

[0018] Figure 9It shows that the monounsaturated fatty acid in Example 8 of the present application increases the survival rate of mouse liver cells at 4°C. Among them, (a) is the detection of cell apoptosis by TUNEL staining, and DAPI is used to label the cell nuclei. Scale bar: 100 μm. (b) is the quantification of the percentage of TUNEL-positive cells. Eight different fields of view were counted in each group, and the error bars represent the standard deviation. The significance test was performed using Two-way ANOVA and Sidak multiple comparisons, *P<0.05, ns: not significant. Detailed implementation mode

[0019] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. When a quantity, concentration, or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper limit of a value and a preferred lower limit of a value, it should be understood that any range formed by combining any upper limit of the range or preferred value with any lower limit of the range or preferred value is specifically disclosed, regardless of whether the range is specifically disclosed. Unless otherwise specified, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0020] The terms "about" and "approximately", when used in combination with numerical variables, generally refer to the value of the variable and all values of the variable within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or within a wider range.

[0021] Expressions such as "comprising" or similar expressions synonymous therewith, such as "including", "containing", and "having", are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consisting of" excludes any unstated element, step, or component. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optionally existing elements, steps, or components that do not substantially affect the basic and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0022] The expression "at least one (piece)" or "one (piece) or more (pieces)" means 1, 2, 3, 4, 5, 6, 7, 8, 9 pieces (pieces) or more.

[0023] Currently, most existing preservation solutions are basic solutions, lacking effective protective additives and unable to comprehensively inhibit cell lysis and functional degradation. Generally, they can only maintain the preservation effect for several hours to several days, and it is difficult to meet the longer-term preservation requirements. Some protectants may bring toxicity or side effects to cells or tissues, further restricting their application.

[0024] Mono-unsaturated fatty acids (MUFA) are a class of fatty acids that contain only one double bond in their carbon chain (see Figure 1 ), and are commonly found in foods such as olive oil, nuts, and avocados. Their main uses include reducing the risk of cardiovascular diseases, controlling blood sugar, alleviating chronic inflammation, assisting in weight control, helping to maintain skin elasticity, delaying aging, etc. Compared with saturated fatty acids, the double bond structure in mono-unsaturated fatty acids gives them higher fluidity in cell membranes, which helps to maintain the integrity and function of cell membranes. In addition, mono-unsaturated fatty acids have certain antioxidant capabilities, which can reduce the generation of free radicals and lower the damage of oxidative stress to cells. Some studies have shown that mono-unsaturated fatty acids can reduce lipid peroxidation and ferroptosis of cells. However, the potential role of mono-unsaturated fatty acids in the cryopreservation of cells and tissues has not been fully studied. This application explored the protective effect of fatty acids on cells and tissues during cryopreservation at 4°C through experiments and found that mono-unsaturated fatty acids can significantly reduce the cell death rate during preservation.

[0025] This application provides the use of mono-unsaturated fatty acids, or their geometric isomers, tautomers, isotope-labeled substances, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of cryoprotectants.

[0026] In some embodiments, the mono-unsaturated fatty acid is a C6-C24 fatty acid, preferably a C12-C20 fatty acid, and contains one unsaturated bond.

[0027] In some embodiments, the mono-unsaturated fatty acid is a cis-mono-unsaturated fatty acid. In some embodiments, the cis-mono-unsaturated fatty acid is palmitoleic acid, oleic acid, or eicosenoic acid.

[0028] As known to those skilled in the art, palmitoleic acid is a naturally occurring mono-unsaturated fatty acid with the chemical name cis-9-hexadecenoic acid, and its molecular formula is C 16 H 30 O 2 . Oleic acid is a mono-unsaturated fatty acid widely present in nature, with the chemical name cis-9-octadecenoic acid, and its molecular formula is C 18 H 34 O 2 . Eicosenoic acid is a mono-unsaturated fatty acid with the chemical name cis-11-eicosenoic acid (Gondoic Acid, C20:1 ω-9), and its molecular formula is C 20 H 38 O 2 .

[0029] In some embodiments, in the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt is about 10 - 250 µM. In the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt is about 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 110 µM, 120 µM, 130 µM, 140 µM, 150 µM, 160 µM, 170 µM, 180 µM, 190 µM, 200 µM, 210 µM, 220 µM, 230 µM, 240 µM or 250 µM. In some embodiments, in the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt is about 250 µM.

[0030] In another aspect, the present application also provides a cryoprotectant, comprising a buffer salt solution for maintaining the normal pH value and osmotic pressure of tissues or cells and a monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt.

[0031] In some embodiments, the monounsaturated fatty acid is a cis-monounsaturated fatty acid. In some embodiments, the cis-monounsaturated fatty acid is palmitoleic acid, oleic acid or eicosenoic acid.

[0032] In some embodiments, in the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt is about 10 - 250 µM. In the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt is about 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 110 µM, 120 µM, 130 µM, 140 µM, 150 µM, 160 µM, 170 µM, 180 µM, 190 µM, 200 µM, 210 µM, 220 µM, 230 µM, 240 µM or 250 µM. In some embodiments, in the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomer, tautomer, isotope-labeled compound, hydrate, solvate or pharmaceutically acceptable salt is about 250 µM.

[0033] In some embodiments, the buffered salt solution is selected from one or more of phosphate buffered saline (PBS) solution, Hank’s balanced salt (HBS) solution, Dulbecco's modified eagle medium (DMEM), Alsever's solution, University of Wisconsin (UW) solution, and histidine-tryptophan-ketoglutarate (HTK) solution.

[0034] In some embodiments, the cryoprotectants described herein can be used for cryopreservation of cells, tissues, and organs.

[0035] In some embodiments, in the cryoprotectants described herein, a compound containing a monounsaturated fatty acid chain can be used to replace the monounsaturated fatty acid, which can achieve a similar effect.

[0036] On the other hand, the present application also provides a method for preventing and / or reducing cryopreservation damage of cells or tissues, including adding the cryoprotectants described herein to the external environment of cells or tissues.

[0037] First, in the cryopreservation models of multiple cell lines (such as K562, HeLa, BV2) in the present application, cryopreservation experiments were carried out using culture media containing different fatty acids, and cell viability was detected after the preservation was completed. The results showed that the cell viability of monounsaturated fatty acids was significantly higher than that of the saturated fatty acid treatment group and the control group.

[0038] Subsequently, the present application overexpressed the key enzyme SCD1 in the synthesis of monounsaturated fatty acid chains in cell lines by genetic engineering means, and carried out cryopreservation experiments using a culture medium containing saturated fatty acids. The results showed that overexpression of SCD1 promoted cryopreserved cell viability.

[0039] In addition, the present application verified the protective effect of monounsaturated fatty acids in the cryopreservation model of mouse liver tissue. Mouse liver tissue samples were placed in a preservation solution containing monounsaturated fatty acids. After preservation at 4°C for 24 hours, it was found by tissue section and immunohistochemistry that the cell mortality rate of the monounsaturated fatty acid treatment group was significantly lower than that of the control group.

[0040] Through the above experiments, the present application confirmed the important role of monounsaturated fatty acids in the cryopreservation of cells and tissues. Compared with the existing preservation solutions, monounsaturated fatty acids are a class of safe and effective additives, which can significantly optimize cryopreservation technology and provide new solutions for cell and tissue preservation.

[0041] Compared with the existing cryopreservation solutions, the present application has the following advantages: Monounsaturated fatty acids (including but not limited to palmitoleic acid, oleic acid, eicosatetraenoic acid) can improve the survival rate of various cells at low temperatures and protect cell morphology. Treatment with monounsaturated fatty acids can reduce apoptosis of cells in liver tissue during cryopreservation and improve cell survival.

[0042] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it will be apparent to those of ordinary skill in the art that there can be more embodiments and implementation solutions within the scope encompassed by the embodiments described in the present application. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically restricted, any feature or element of any embodiment can be combined with any other feature or element in any other embodiment, or can replace any other feature or element in any other embodiment.

[0043] The present application includes and contemplates combinations with features and elements known to those of ordinary skill in the art. The embodiments, features, and elements already disclosed in the present application can also be combined with any conventional features or elements to form a unique inventive solution. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in the present application can be implemented alone or in any suitable combination. Therefore, the embodiments are not subject to other limitations except those made in accordance with the appended claims and their equivalents. In addition, various modifications and changes can be made within the scope of the appended claims.

[0044] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not depend on the specific order of the steps described herein, the method or process should not be limited to the specific order of steps described. As will be understood by those of ordinary skill in the art, other step sequences are possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing their steps in the order written, as those skilled in the art can readily understand that these orders can vary and still remain within the spirit and scope of the embodiments of the present application.

[0045] In the following examples, the experimental methods without specific conditions are usually determined according to national standards. The experimental materials without sources indicated in the following examples are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise defined or stated, all professional and scientific terms used in this application have the same meaning as those familiar to skilled personnel in the art. In addition, any methods and materials similar or equivalent to the recorded content can be applied to the methods of this application.

[0046] Example In this application, monounsaturated fatty acids were added to the cell culture medium, and the cells were placed in a 4°C refrigerator overnight. The next day, by measuring the proportion of live and dead cells, the survival rates of the fatty acid treatment group and the control group at low temperature were compared. Verification was carried out on cell lines from various cell sources such as K562, HeLa, and BV2 to explore the cryoprotective effect of monounsaturated fatty acids. In addition, before the mouse liver was excised, monounsaturated fatty acids were injected via the tail vein, and then an isotonic perfusion solution containing monounsaturated fatty acids was perfused; after the liver was excised, the tissue was placed in a preservation solution containing monounsaturated fatty acids and stored in a 4°C refrigerator. Subsequently, cell viability was measured at different time points to explore the cryoprotective effect of monounsaturated fatty acids on the mouse liver.

[0047] The relevant materials used in the examples are shown below: 1. Reagents, materials Palmitic acid (PA, manufacturer: Chemxyz, product number: K755977), palmitoleic acid (POA, manufacturer: Chemxyz, product number: Q101590), stearic acid (SA, manufacturer: Chemxyz, product number: K755978), oleic acid (OA, manufacturer: Chemxyz, product number: K100268), palmitelaidic acid (PEA, manufacturer: GlpBio, product number: GC33765), gondoic acid (GA, manufacturer: AmBeed, product number: A405460), malondialdehyde (MDA) content detection kit (manufacturer: Solarbio, product number: BC0020), DMEM (manufacturer: Wisent, product number: 319-005-CL), bovine serum albumin (BSA, manufacturer: Beyotime, product number: ST025), fetal bovine serum (FBS, manufacturer: PAN-Biotech, product number: P30-3306), Calcein AM / PI staining (manufacturer: Beyotime, product number: C2015), TUNEL (manufacturer: Servicebio, product number: G1502), DAPI (manufacturer: Servicebio, product number: G1012).

[0048] 2. Instruments Cell culture incubator (manufacturer: Thermo Scientific, model: Heracell 150i), refrigerator (manufacturer: Haier, model: SC-372), fluorescence microscope (manufacturer: Nikon, model: ECLIPSE Ts2).

[0049] Example 1. Monounsaturated fatty acids improve the survival rate of K562 cells at 4°C low temperature Prepare a 250 mM fatty acid (including but not limited to palmitic acid, palmitoleic acid, stearic acid, oleic acid) solution with ethanol (EtOH), i.e., the fatty acid stock solution. Prepare a 10% (v / v) fatty acid-free bovine serum albumin solution with DMEM, i.e., the BSA solution. Add the fatty acid stock solution or an equal volume of ethanol to the BSA solution to prepare a 2.5 mM fatty acid BSA solution or an ethanol BSA control solution. Since the water solubility of fatty acids is low, vortex the fatty acid BSA solution and the control solution for 10 minutes and incubate at 37 °C for 1 hour to allow the fatty acids to fully bind to and dissolve in the BSA. Add the prepared fatty acid BSA solution or the control solution to the cell culture medium (DMEM containing 10% fetal bovine serum) with a final fatty acid concentration of 250 μM. Incubate K562 cells in the cell culture incubator at 37 °C for 6 hours using the fatty acid-containing medium or the control medium. Subsequently, place the cells in the fatty acid-containing medium in a refrigerator at 4 °C. After 16 hours (overnight storage), use Calcein AM / PI staining and fluorescence microscopy imaging to detect cell viability. The results show that monounsaturated fatty acids significantly increase the viability of K562 cells at 4 °C (see Figure 2 ).

[0050] Example 2. Monounsaturated fatty acids increase the viability of HeLa cells at 4 °C The materials and methods used are the same as in Example 1, and the subject of the experiment is HeLa cells. The results show that monounsaturated fatty acids significantly increase the viability of HeLa cells at 4 °C (see Figure 3 ).

[0051] Example 3. Monounsaturated fatty acids increase the viability of BV2 cells at 4 °C The materials and methods used are the same as in Example 1, and the subject of the experiment is BV2 cells. The results show that monounsaturated fatty acids significantly increase the viability of BV2 cells at 4 °C (see Figure 4 ).

[0052] Example 4. Different concentrations of monounsaturated fatty acids increase the viability of BV2 cells at 4 °C The materials and methods used are the same as in Example 1, and the subject of the experiment is BV2 cells. The results show that monounsaturated fatty acids at 10 - 250 μM can significantly increase the viability of BV2 cells at 4 °C to varying degrees (see Figure 5 ).

[0053] Example 5. Overexpression of desaturase SCD1 increases cell viability at 4 °C Desaturase SCD1 (stearoyl-CoA desaturase 1) can convert saturated fatty acids into monounsaturated fatty acids ( Figure 6a). Multiple BV2 cell lines overexpressing SCD1 were obtained by infecting the CRISPRa (clustered regularly interspaced short palindromic repeats activation) BV2 cell line expressing a transcriptional activation element with lentiviruses expressing different targeting SCD1 guide RNAs (gRNAs). The results of Western blotting experiments showed that compared with the wild-type negative control (expressing sgNC) cell line, the three BV2 cell lines overexpressing SCD1 (expressing sgSCD1#1, #2, or #3) had higher SCD1 protein expression levels ( Figure 6 b). To detect the effect of SCD1 overexpression on cell viability at 4 °C, we cultured the SCD1 overexpressing cell line and the negative control cell line in a medium containing saturated fatty acids and stored them in a 4 °C refrigerator. After 16 hours (stored overnight), Calcein AM / PI staining and fluorescence microscopy imaging were used to detect cell viability. The materials and methods used were the same as those in Example 1. The results showed that overexpression of the desaturase SCD1 increased cell viability at 4 °C ( Figure 6 c and d).

[0054] Example 6. Cis-monounsaturated fatty acids increase cell viability at 4 °C To study the universality or specificity of monounsaturated fatty acids in increasing cell viability at 4 °C, we tested the effects of two additional monounsaturated fatty acids, namely trans-palmitoleic acid and eicosatrienoic acid. The other materials and methods used were the same as those in Example 1, and the subject of the experiment was K562 cells. The results showed that trans-palmitoleic acid did not significantly improve the viability of K562 cells at 4 °C, while cis-monounsaturated fatty acids including eicosatrienoic acid increased the viability of K562 cells at 4 °C (see Figure 7 ).

[0055] Example 7. Monounsaturated fatty acids reduce the level of lipid peroxidation in cells at 4 °C To study the effect of monounsaturated fatty acids on the level of lipid peroxidation in cells at 4 °C, we detected the level of malondialdehyde (MDA) in the cells. The other materials and methods used were the same as those in Example 1, and the subject of the experiment was K562 cells. The results showed that storage at 4 °C increased the level of lipid peroxidation in the cells, while monounsaturated fatty acids reduced the level of lipid peroxidation in cells at 4 °C (see Figure 8 ).

[0056] Example 8. Monounsaturated fatty acids increase the viability of mouse liver cells at 4 °C Prepare a 250 mM fatty acid (including but not limited to palmitic acid, palmitoleic acid) solution with ethanol (EtOH), namely the fatty acid stock solution. Prepare a 10% (v / v) fatty acid-free bovine serum albumin solution with DMEM, namely the BSA solution. Add the fatty acid stock solution or an equal volume of ethanol to the BSA solution to prepare a 12.5 mM fatty acid BSA solution or an ethanol BSA control solution. Since the water solubility of fatty acids is low, vortex the fatty SA solution and the control solution for 10 minutes and incubate at 37 °C for 1 hour to allow the fatty acids to fully bind and dissolve with BSA. Inject the fatty acid BSA solution or the ethanol BSA control solution into adult mice via the tail vein at a dose of 80 μg / kg body weight. Meanwhile, add the prepared fatty acid BSA solution or the control solution to the cell culture medium (DMEM containing 10% fetal bovine serum), namely the cryopreservation solution, with a final fatty acid concentration of 250 μM. 6 hours after the tail vein injection, anesthetize the mice and perfuse the mouse liver with 10 mL of the cryopreservation solution via the portal vein. Subsequently, remove the liver tissue intact from the mouse abdominal cavity, place it in the cryopreservation solution, and store it in a 4 °C refrigerator. After 6 hours and 24 hours of storage respectively, isolate the mouse liver tissue, fix it with 4% paraformaldehyde, embed it, section it, and detect the cell viability through TUNEL / DAPI staining and fluorescence microscopy imaging. The results show that monounsaturated fatty acids improve the viability of mouse liver cells at 4 °C (see Figure 9 ).

[0057] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. Use of a monounsaturated fatty acid, or its geometric isomers, tautomers, isotope-labeled substances, hydrates, solvates or pharmaceutically acceptable salts in the preparation of a cryoprotectant.

2. The use according to claim 1, wherein The monounsaturated fatty acid is a cis-monounsaturated fatty acid.

3. The use according to claim 2, wherein The cis-monounsaturated fatty acid is palmitoleic acid, oleic acid or arachidonic acid.

4. The use according to any one of claims 1 to 3, wherein In the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomers, tautomers, isotope labels, hydrates, solvates or pharmaceutically acceptable salts is about 10-250 μM.

5. The use according to claim 4, wherein In the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomers, tautomers, isotope labels, hydrates, solvates or pharmaceutically acceptable salts is about 250 μM.

6. A cryoprotectant comprising a buffered salt solution for maintaining normal pH and osmotic pressure of tissues or cells and a monounsaturated fatty acid, or a geometric isomer, tautomer, isotope label, hydrate, solvate or pharmaceutically acceptable salt thereof.

7. The cryoprotectant according to claim 6, wherein: The monounsaturated fatty acid is a cis-monounsaturated fatty acid.

8. The cryoprotectant according to claim 7, wherein The cis-monounsaturated fatty acid is palmitoleic acid, oleic acid or arachidonic acid.

9. The cryoprotectant according to any one of claims 6 to 8, wherein In the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomers, tautomers, isotope labels, hydrates, solvates or pharmaceutically acceptable salts is about 10-250 μM.

10. The cryoprotectant according to claim 9, wherein In the cryoprotectant, the concentration of the monounsaturated fatty acid, or its geometric isomers, tautomers, isotope labels, hydrates, solvates or pharmaceutically acceptable salts is about 250 μM.

11. The cryoprotectant according to any one of claims 6 to 10, wherein The buffered salt solution is selected from one or more of PBS solution, HBS solution, DMEM, Aldrich solution, UW solution and HTK solution.

12. A method for preventing and / or reducing cryopreservation damage of cells or tissues, comprising adding the cryoprotectant according to any one of claims 6 to 11 to the external environment of the cells or tissues.

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