Freezing solution for improving cell survival rate and application thereof

Abstract

The application discloses a kind of cryopreservation solution for improving cell survival rate and application thereof, and the cryopreservation solution includes the following components by mass fraction: 4-7 parts of propylene glycol, 2-5 parts of glycerol, 1-3 parts of glucose, 4-7 parts of acetamide, 5-17 parts of selenium-rich spirulina extract, 5-12 parts of mannose erythritol lipid, 8-20 parts of compound seaweed polysaccharide, 50-100 parts of basic culture solution.The application uses various osmotic protective agents, propylene glycol, glycerol and acetamide for routine osmotic protection.Then assisted by various sugars to adjust the cryopreservation solution, the concentration and type of sugar have a significant impact on cell freezing, various sugars replace water molecules, form a glass state, reduce the damage of water ice crystals to cells during the recovery process of cells.And increase mannose erythritol lipid, increase the permeability of cell membrane surface, promote the absorption of cells to various sugars, and assist seaweed polysaccharide to increase the antioxidant performance of cells.

Description

Technical Field

[0001] The present invention relates to the field of cell freezing solutions, in particular to a freezing solution for improving cell survival rate and applications thereof. Background Art

[0002] Cryopreservation is the process of placing biological cells under deep low temperature conditions, which puts the cells into a dormant state, reduces the cell metabolic rate, and prolongs the cell preservation time.

[0003] During the warming process of cell recovery, changes in the intracellular and extracellular environments can lead to changes in intracellular pH and ionic strength, protein inactivation, and other factors, reducing cell viability and even causing cell fragmentation and death. Existing cryopreservation solutions primarily employ osmotic pressure regulation and hydrophilic binding to water molecules, mitigating the effect of hydrogen bonds on ice crystal formation, but their effectiveness is relatively limited. Summary of the Invention

[0004] In order to solve the defects in the prior art, the present invention provides a cryopreservation solution for improving cell survival rate and its application, which utilizes multiple amphiphilic compounds to reduce the influence of water molecules and improve the antioxidant and repair functions of cells.

[0005] In order to achieve this object, the present invention adopts the following technical solutions:

[0006] A freezing solution for improving cell survival rate comprises the following components by mass: 4-7 parts of propylene glycol, 2-5 parts of glycerol, 1-3 parts of glucose, 4-7 parts of acetamide, 5-17 parts of selenium-enriched spirulina extract, 5-12 parts of mannose erythritol lipids, 8-20 parts of composite seaweed polysaccharides, and 50-100 parts of basal culture solution.

[0007] The present invention also provides the following preferred embodiment:

[0008] A further preferred formula includes the following components by mass: 5-7 parts of propylene glycol, 2-4 parts of glycerol, 1-2 parts of glucose, 4-6 parts of acetamide, 10-12 parts of selenium-enriched spirulina extract, 10-12 parts of mannose erythritol lipids, 12-16 parts of complex seaweed polysaccharides, and 60-80 parts of basal culture medium.

[0009] The best component ratio in the experiment is, by mass, the following components: 7 parts of propylene glycol, 4 parts of glycerol, 2 parts of glucose, 4 parts of acetamide, 10 parts of selenium-enriched spirulina extract, 10 parts of mannose erythritol lipids, 12 parts of complex seaweed polysaccharides, and 80 parts of basal culture medium.

[0010] Preferably, the cells are hepatocytes.

[0011] Preferably, the composite seaweed polysaccharide is extracted from two or more of blue algae, green algae, red algae and brown algae.

[0012] More preferably, the composite seaweed polysaccharide is extracted from giant kelp and sargassum.

[0013] The basal culture medium adopts an existing basal culture medium, preferably DMEM / F12 culture medium.

[0014] Preferably, the extraction method of the composite seaweed polysaccharide is as follows: the composite seaweed powder is soaked, ultrasonically crushed to obtain a preliminary extract, and a flocculant is added to perform electric field flocculation extraction and centrifugal filtration, and then eluted through a molecular sieve column to obtain the composite seaweed polysaccharide.

[0015] Preferably, the extraction method of the selenium-rich spirulina extract is: adding selenium-rich spirulina to a butanediol solution, performing ultrasonic crushing, then filtering with high-pressure steam, and centrifuging the filtrate to obtain the selenium-rich spirulina extract.

[0016] The invention also discloses an application of a freezing solution for improving cell survival rate, which is mainly used in the preparation of the cell freezing solution.

[0017] The present invention employs a variety of osmotic protective agents, including propylene glycol, glycerol, and acetamide, for conventional osmotic protection. The freezing solution is then adjusted with the addition of various sugars. The concentration and type of sugars significantly influence cell cryopreservation. These sugars replace water molecules, forming a glassy state and reducing damage to cells during cell recovery caused by ice crystals. Mannose and erythritol lipids are also added to increase cell membrane permeability, promoting cellular absorption of the various sugars. Furthermore, seaweed polysaccharides are used to enhance the cells' antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a micrograph of the thawed red blood cells of Comparative Example 2;

[0019] Figure 2 This is a micrograph of the thawed red blood cells of Example 5;

[0020] Figure 3 This is a toluidine blue stained micrograph of the thawed hepatocytes of Example 5;

[0021] Figure 4 This is a toluidine blue staining micrograph of the hepatocytes of Comparative Example 2 after thawing. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0023] The cell survival rate-enhancing freezing solution of the present invention comprises the following components in parts by mass: 4-7 parts of propylene glycol, 2-5 parts of glycerol, 1-3 parts of glucose, 4-7 parts of acetamide, 5-17 parts of selenium-enriched spirulina extract, 5-12 parts of mannose erythritol lipids, 8-20 parts of composite seaweed polysaccharides, and 50-100 parts of basic culture solution.

[0024] In a relatively better embodiment, the following components are included in parts by mass: 5-7 parts of propylene glycol, 2-4 parts of glycerol, 1-2 parts of glucose, 4-6 parts of acetamide, 10-12 parts of selenium-enriched spirulina extract, 10-12 parts of mannose erythritol lipids, 12-16 parts of complex seaweed polysaccharides, and 60-80 parts of basal culture medium.

[0025] According to the experimental results, the optimal component ratio is the following, by mass: 7 parts of propylene glycol, 4 parts of glycerol, 2 parts of glucose, 4 parts of acetamide, 10 parts of selenium-enriched spirulina extract, 10 parts of mannose erythritol lipids, 12 parts of complex seaweed polysaccharides, and 80 parts of basal culture medium.

[0026] The cells are hepatocytes. Hepatocytes are relatively sensitive to antioxidant properties and are much more sensitive to selenium-enriched spirulina than other human cells. Experiments have shown that hepatocytes are most susceptible to the optimization of this freezing solution.

[0027] Compound seaweed polysaccharides are extracted from two or more of the following: blue algae, green algae, red algae, and brown algae. Most of the seaweed species available are blue algae, green algae, red algae, and brown algae, and these are also the most abundant species found in nature.

[0028] Compound seaweed polysaccharides are extracted from giant kelp and sargassum.

[0029] The cell walls of macroalgae are primarily composed of complex, water-soluble, sulfated polysaccharides. These polysaccharides possess unique molecular weight and structural properties (including diverse structures, including branched conformations), as well as sulfation, along with the presence of diverse and unusual sugars (xylose and rhamnose). This contributes to their unique biological activity. These structural characteristics give macroalgae polysaccharides a structure similar to animal glycosaminoglycans (GAGs), such as heparin.

[0030] Sargassum polysaccharides often have a molecular weight exceeding 10,000 and can be divided into branched and branched polysaccharides based on their molecular backbone. Common components in polysaccharide hydrolysates include galactose, xylose, glucose, gulose, uronic acid, and sulfate esters. Both unbranched and branched polysaccharides have β-linked chains (1->4), while branches have 1-2 and 1-3 linkages. The polysaccharides in Sargassum include the outermost skeleton polysaccharide, intercellular mucopolysaccharides, and storage polysaccharides within the protoplasm. Sargassum polysaccharides are a highly soluble, water-soluble, and highly viscous polysaccharide complex extracted from Sargassum.

[0031] The basic culture medium is DMEM / F12 culture medium.

[0032] The extraction method of the composite seaweed polysaccharide comprises the following steps: soaking the composite seaweed powder, ultrasonically crushing the powder to obtain a preliminary extract, adding a flocculant to extract the powder by electric field flocculation, centrifuging the extract, and eluting the extract through a molecular sieve column to obtain the composite seaweed polysaccharide.

[0033] The extraction method of the selenium-rich spirulina extract comprises the following steps: adding the selenium-rich spirulina to a butanediol solution, performing ultrasonic crushing, filtering with high-pressure steam, and centrifuging the filtrate to obtain the selenium-rich spirulina extract.

[0034] The invention also discloses the application of the cell survival rate-enhancing freezing solution in the preparation of the cell freezing solution.

[0035] The above is the content and principle of the present invention. The specific solutions of the present invention are further disclosed and described in combination with embodiments below.

[0036] Unless otherwise specified, the reagents and methods involved in the examples are commonly used reagents and methods in the art. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention shall fall within the scope of protection claimed by the present invention.

[0037] Preparation of cryopreservation solution to improve cell survival rate

[0038] 1. Preparation of Selenium-enriched Spirulina Extract

[0039] A certain amount of selenium-enriched spirulina powder is weighed into an aqueous solution containing 10-15% butanediol, mixed thoroughly, and then placed in an ultrasonic cell disruptor. The ultrasonic cell disruptor is activated to disrupt the cells, producing a cell disrupted liquid. The resulting cell disrupted liquid is subjected to pressurized steam separation to remove the butanediol. The cell disrupted liquid after pressurized steam separation is centrifuged to obtain a supernatant, which is then collected to obtain a selenium-enriched spirulina extract. A small amount of filter aid may be added during the centrifugation process to optimize filtration.

[0040] 2. Preparation of composite seaweed polysaccharides

[0041] The method involves taking powders of giant kelp and sargassum and mixing them in a ratio of 1-5:1:2 (optimally 1:2) of giant kelp to sargassum into an aqueous solution containing 10-15% butanediol. After thorough mixing, the mixture is placed in an ultrasonic cell pulverizer, which is activated to disrupt the cells and obtain a cell pulverization solution. The cell pulverization solution is then added to a flocculation reactor, preferably an electric flocculation reactor. The temperature is adjusted to 30-50°C, the pH to 5-8, the stirring rate to 600-1200 rpm, 100-300 parts of a flocculant are added, and flocculation extraction is performed under an electric field to obtain composite seaweed polysaccharides.

[0042] The obtained composite seaweed polysaccharide is dissolved in a phosphate buffer solution, eluted with a phosphate buffer solution through a molecular sieve column, dialyzed and freeze-dried to obtain the purified composite seaweed polysaccharide.

[0043] 3. Preparation of cryopreservation solution to improve cell survival rate

[0044] Take 50-100 parts of basic culture medium, add 5-17 parts of selenium-enriched spirulina extract, 5-12 parts of mannose erythritol lipids, 8-20 parts of complex seaweed polysaccharides, and 1-3 parts of glucose, mix evenly, and then add 4-7 parts of propylene glycol, 2-5 parts of glycerol, and 4-7 parts of acetamide to obtain the final product of the freezing solution for improving cell survival rate.

[0045] According to Table 1, the ratio of cryopreservation solution to improve cell survival rate

[0046]

[0047] Table 1: Proportions of various embodiments of cryopreservation solutions for improving cell viability. Comparative examples were prepared according to Table 2.

[0048]

[0049]

[0050] Table 2 Comparative proportions

[0051] Freezing and thawing of red blood cells

[0052] Cell cryopreservation: Red blood cells (maintain the cell number at 5-7×10 6 / mL) was added to the cryopreservation tube of the cell survival rate improving cryopreservation solution at a ratio of 1:4, and then the cryopreservation tube was placed in liquid nitrogen to complete the cell freezing.

[0053] Thawing cells: Remove the cryovials from liquid nitrogen and place them in a water bath, gradually heating them from 10°C to 36-38°C for approximately 2 minutes, until the cells are thawed and revived. Wash the cells with saline and centrifuge to remove the supernatant. Repeat this process twice, then resuspend the cells in DMEM / F12 culture medium to complete thawing.

[0054] The survival rates of red blood cells after resuscitation in the examples and comparative examples were statistically analyzed, and the results are shown in Table 3 below.

[0055] Survival rate Example 1 86.3％ Example 2 88.4％ Example 3 92.6％ Example 4 97.1％ Example 5 98.1％ Comparative Example 1 81.1％ Comparative Example 2 76.5％

[0056] Table 3 Red blood cell survival rate

[0057] Figure 1 This is a micrograph of the thawed and cultured red blood cells of Comparative Example 2. Figure 2This is a micrograph of the thawed and cultured red blood cells of Example 5. It can be seen from the figure that the number of surviving cells in Example 5 is significantly higher than that in Comparative Example 2.

[0058] Cryopreservation and thawing of hepatocytes

[0059] Cell cryopreservation: Add hepatocytes to cryopreservation solution at a ratio of 1:4 into a cryopreservation tube containing cryopreservation solution for improving cell viability. Place the cryopreservation tube in liquid nitrogen to complete cell cryopreservation.

[0060] Thawing cells: Remove the cryovials from liquid nitrogen and place them in a water bath, gradually heating them from 10°C to 36-38°C for approximately 2 minutes, until the cells are thawed and revived. Wash the cells with saline and centrifuge to remove the supernatant. Repeat this process twice, then resuspend the cells in DMEM / F12 culture medium to complete thawing.

[0061] The survival rates of the hepatocytes after recovery in the examples and comparative examples were statistically analyzed, and the results were obtained in Table 4 below.

[0062] Survival rate Example 1 88.5％ Example 2 90.2％ Example 3 94.7％ Example 4 98.2％ Example 5 99.2％ Comparative Example 1 76.1％ Comparative Example 2 71.5％

[0063] Table 4 Hepatocyte survival rate

[0064] It can be seen from Tables 3 and 4 that the cell survival rate-enhancing cryopreservation solution of the present invention has a significant effect on improving cell survival rate. The optimization of multiple osmotic protective agents and sugars reduces the cell damage caused by water molecules and ice crystals during cell recovery.

[0065] Figure 3 This is a micrograph of the hepatocytes after thawing and culture in Comparative Example 2. Figure 4 This is a micrograph of the hepatocytes after thawing and culture in Example 5. It can be seen from the figure that the number of surviving cells in Example 5 is significantly higher than that in Comparative Example 2. The number of inactivated cells in Comparative Example 2 is significantly higher than that in Example 5.

[0066] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cryopreservation solution for improving cell survival rate, characterized by: The invention comprises the following components in parts by mass: 4-7 parts of propylene glycol, 2-5 parts of glycerol, 1-3 parts of glucose, 4-7 parts of acetamide, 5-17 parts of selenium-enriched spirulina extract, 5-12 parts of mannose erythritol lipids, 8-20 parts of compound seaweed polysaccharides, and 50-100 parts of basic culture medium; the compound seaweed polysaccharides are extracted from giant kelp and sargassum; the extraction method of the compound seaweed polysaccharides comprises the following steps: infiltrating the compound seaweed powder, ultrasonically crushing the powder to obtain a preliminary extract, adding a flocculant for electric field flocculation extraction, centrifuging and filtering, and eluting through a molecular sieve column to obtain the compound seaweed polysaccharides.

2. The cryopreservation solution for improving cell viability according to claim 1, wherein: The composition includes the following components by mass: 5-7 parts of propylene glycol, 2-4 parts of glycerol, 1-2 parts of glucose, 4-6 parts of acetamide, 10-12 parts of selenium-enriched spirulina extract, 10-12 parts of mannose erythritol lipids, 12-16 parts of complex seaweed polysaccharides and 60-80 parts of basic culture solution.

3. The cryopreservation solution for improving cell viability according to claim 1, wherein: The composition includes the following components in parts by mass: 7 parts of propylene glycol, 4 parts of glycerol, 2 parts of glucose, 4 parts of acetamide, 10 parts of selenium-enriched spirulina extract, 10 parts of mannose erythritol lipids, 12 parts of complex seaweed polysaccharides, and 80 parts of basic culture solution.

4. The cryopreservation solution for improving cell viability according to claim 1, wherein: The cells are hepatocytes.

5. The cryopreservation solution for improving cell viability according to claim 1, wherein: The basic culture medium is DMEM / F12 culture medium.

6. The cryopreservation solution for improving cell viability according to claim 1, wherein: The extraction method of the selenium-rich spirulina extract comprises the following steps: adding the selenium-rich spirulina to a butanediol solution, performing ultrasonic crushing, filtering with high-pressure steam, and centrifuging the filtrate to obtain the selenium-rich spirulina extract.

7. A use of the cryopreservation solution for improving cell viability according to claim 1, characterized in that: Used in the preparation of cell freezing solution.

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