WO2024164853A1 - Vitrification freeze-thawing solution kit - Google Patents

Abstract

A vitrification freeze-thawing solution kit. The vitrification freeze-thawing solution kit comprises a freezing solution, a balance solution, a thawing solution, a diluent and a cleaning solution, wherein the thawing solution comprises hydroxypropyl cellulose, a fatty acid supplement, sucrose and a basic culture medium. The vitrification freeze-thawing solution kit has the advantages of low viscosity, low density, easy defoaming, etc., can maintain the content of lipids in an ovum/embryo cell lipid membrane, can promote a permeable cryoprotectant to enter cells in the freezing process, and can increase the survival rate of an ovum/embryo after thawing in the thawing process, and improve the subsequent development condition of the ovum/embryo.

Description

A vitrification freezing and thawing solution set Technical Field

The present invention belongs to the field of biological tissue cryopreservation, relates to a cell vitrification freezing technology in an assisted reproductive process, and specifically relates to a vitrification freezing and thawing solution set.

Background Art

Oocyte/embryo cryopreservation is one of the important assisted reproductive technologies. For those whose fertility needs to be preserved, their eggs are stored at low temperatures and revived when needed to ensure the vitality of the eggs. In the process of assisted reproduction, vitrification technology is mainly used to preserve oocytes/embryo cells. The basic principle of this technology is: using high concentrations of cryoprotectants, the permeable cryoprotectants are fully introduced into the cells, and the non-permeable cryoprotectants promote the dehydration of the eggs on the outside of the eggs. After that, the eggs are put into liquid nitrogen. As the temperature drops rapidly, the cryoprotectants solidify into a "glassy state", thus crossing the freezing point and preventing ice crystals from forming to damage the structural function of the embryo. At an extremely high freezing rate, the sample is directly transformed from a liquid state to a glassy state without a fixed shape.

At present, the general vitrification and thawing fluid contains the following ingredients: basal culture medium (used to provide energy substances and inorganic ions required for cell growth), permeable cryoprotectants (penetrate into cells to displace water in cells), non-permeable cryoprotectants (outside cells to promote cell dehydration), albumin (provide macromolecular support for cell growth, such as growth factors, lipids, etc.). Vitrification technology has been widely used in clinical practice, but there are still some shortcomings, including: 1. Product usability: most products on the market have high liquid viscosity, and due to the high concentration of protein components, low surface activity, easy to foam during operation, and not conducive to observation under a microscope; 2. Product performance: The permeable cryoprotectant in the vitrification process is a fat-soluble component. The lipid content of the egg is reduced after vitrification, and the phospholipid bilayer of the cell membrane is damaged.

Summary of the invention

In order to solve the problems of high viscosity of vitrification freezing and thawing solution, easy foaming, and easy damage to the phospholipid bilayer of cell membrane, etc., the present invention provides a vitrification freezing and thawing solution set, which contains freezing solution and thawing solution with low liquid viscosity, density and surface tension, and can promote the penetration of permeable cryoprotectants into cells, and at the same time can improve the survival rate and development of cells after thawing during the thawing process.

The technical solution for achieving the purpose of the invention is as follows: a vitrification freezing and thawing solution set, including a balancing solution, a freezing solution, a thawing solution, a diluent, and a cleaning solution. The thawing solution includes hydroxypropyl cellulose, a fatty acid supplement, sucrose, and a basic culture medium. The freezing solution includes hydroxypropyl cellulose, a basic culture medium, ethylene glycol, dimethyl sulfoxide, and sucrose.

In one embodiment, the freezing liquid further comprises a fatty acid supplement.

In one embodiment, in the above-mentioned vitrification freezing and thawing solution set, the fatty acid supplements in the thawing solution and the freezing solution are of the same type, and both include any one or two of unsaturated fatty acids and saturated fatty acids.

Preferably, the unsaturated fatty acids include any one or more of arachidonic acid, linoleic acid, linolenic acid, oleic acid, and palmitoleic acid.

Preferably, the saturated fatty acid includes any one or more of myristic acid, palmitic acid and stearic acid.

In an improved embodiment, the amount of the fatty acid supplement added to the thawing solution is 0.5% to 1.5% of the volume of the thawing solution.

Preferably, the amount of the fatty acid supplement added to the thawing solution is 1.0% of the volume of the thawing solution.

In one embodiment, in the vitrification freezing and thawing solution set, the amount of hydroxypropyl cellulose added to the thawing solution and the freezing solution is the same, and is 0.06-0.12 mg/mL.

In an improved embodiment, the balancing solution, diluent and cleaning solution all include hydroxypropyl cellulose, and the diluent and cleaning solution all include fatty acid supplements.

Preferably, the amount of hydroxypropyl cellulose added to the balancing solution, the diluent, and the cleaning solution is 0.06 to 0.12 mg/mL, and the amount of fatty acid supplement added to the diluent and the cleaning solution is 0.5% to 1.5% of the component volume.

Compared with the prior art, the present invention has the following beneficial effects: the vitrification freezing and thawing solution set provided by the present invention has the advantages of low viscosity, low density, and easy defoaming, and can maintain the lipid content in the cell lipid membrane, and can promote the entry of the permeable cryoprotectant into the cell during the freezing process, and at the same time can increase the survival rate of cells after thawing during the thawing process, and improve the subsequent development of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

FIG1 is a schematic diagram of lipid staining of an egg after being treated with the vitrification freezing and thawing solution kit of the present invention in a specific embodiment;

FIG. 2 is a schematic diagram of lipid staining of eggs after being treated with the existing vitrification and thawing solution set in Table 1 in a specific embodiment.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

The existing vitrification freezing and thawing solution set includes a balancing solution, a freezing solution, a thawing solution, a diluent, a cleaning solution 1, and a cleaning solution 2. See the table below for the composition and function of each component in the existing vitrification freezing and thawing solution set.

Table 1: Available vitrification and thawing solution sets:

When the above-mentioned vitrification freezing and thawing solution set is used, on the one hand, due to the high density and viscosity of each component, the cells often float on the surface of the droplets in each component and cannot fall for a long time, resulting in the cells being unable to be fully wrapped by the balancing solution, uneven pressure around the cells, and poor penetration effect of the cryoprotectant; on the other hand, high concentrations of human serum albumin (such as an added amount of 12 mg/mL) are used in each component, which makes it very easy to foam. During the operation, a large number of bubbles will be generated due to the need to repeatedly blow and suck the embryos with a capillary, which is easy to interfere with cell observation under a microscope; thirdly, during the freezing process, the permeable cryoprotectant is a fat-soluble component, the lipid content of the cells is reduced after vitrification, and the phospholipid bilayer of the cell membrane is damaged.

In view of the above problems, the present invention provides a vitrification freezing and thawing solution set, freezing and thawing The liquid set mainly improves the ingredients and the content of each ingredient in its components to solve the problems of easy foaming and cell membrane damage. The freezing and thawing liquid set includes thawing liquid, freezing liquid, balancing liquid, diluent and cleaning liquid. The freezing liquid set of the present invention is described below through specific embodiments.

Embodiment 1:

The present embodiment provides a vitrification freezing and thawing solution set, including a balancing solution, a freezing solution, a thawing solution, a diluent, and a cleaning solution. The components, their ingredients, and functions of the vitrification freezing and thawing solution set of the present embodiment are shown in Table 2 below.

Table 2: Vitrification Thawing Solution Set in Example 1:

In this embodiment, hydroxypropyl cellulose is used in the balancing liquid, freezing liquid, thawing liquid, diluent, and cleaning liquid (including cleaning liquid 1 and cleaning liquid 2). Hydroxypropyl cellulose can reduce the density of each liquid. The density and viscosity allow cells to fall quickly with less operating resistance. At the same time, it can increase the surface activity of each liquid, making the liquid defoaming faster, which will not hinder the observation of cells under the microscope.

Optionally, in the above-mentioned vitrification freezing and thawing solution set, the amount of hydroxypropyl cellulose added to the balancing solution, freezing solution, thawing solution, diluent and cleaning solution is 0.06-0.12 mg/mL.

In this embodiment, fatty acid supplements are used in the thawing solution, diluent, and cleaning solution (including cleaning solution 1 and cleaning solution 2). Fatty acid supplements can effectively protect the phospholipid bilayer of the cells, prevent the reduction of cell membrane lipid content, and promote cell survival rate and development after thawing.

Optionally, in the same vitrification freezing and thawing solution set, the fatty acid supplements used in each component are of the same type, and any one or both of unsaturated fatty acids and saturated fatty acids can be selected.

Optionally, the unsaturated fatty acids include any one or more of arachidonic acid, linoleic acid, linolenic acid, oleic acid, and palmitoleic acid. The saturated fatty acids include any one or more of myristic acid, palmitic acid, and stearic acid.

Optionally, in the same vitrification freezing and thawing solution set, the amount of the fatty acid supplement added to each of the above components is 0.5% to 1.5% of the volume of each component. Preferably, the amount of the fatty acid supplement added is 1.0% of the volume of each component.

Embodiment 2:

The present embodiment provides a vitrification freezing and thawing solution set, including a balancing solution, a freezing solution, a thawing solution, a diluent, and a cleaning solution. The components, their ingredients, and functions of the vitrification freezing and thawing solution set of the present embodiment are shown in Table 3 below.

Table 3: Vitrification Thawing Solution Set in Example 2:

In this embodiment, hydroxypropyl cellulose is used in the balancing liquid, freezing liquid, thawing liquid, diluent, and cleaning liquid (including cleaning liquid 1 and cleaning liquid 2). Hydroxypropyl cellulose can reduce the density and viscosity of each liquid, so that the cells can fall quickly with less operating resistance. At the same time, it can increase the surface activity of each liquid, making the liquid defoam more quickly, and thus will not hinder the observation of cells under a microscope.

Optionally, in the above-mentioned vitrification freezing and thawing solution set, the amount of hydroxypropyl cellulose added to the balancing solution, freezing solution, thawing solution, diluent and cleaning solution is 0.06-0.12 mg/mL.

In the present embodiment, fatty acid supplements are used in freezing solution, thawing solution, diluent, and cleaning solution (including cleaning solution 1 and cleaning solution 2), and fatty acid supplements can effectively protect the phospholipid bilayer of cells, avoid the reduction of cell membrane lipid content, and promote the survival rate and development of cells after thawing. The difference between the present embodiment and Example 1 is that fatty acid supplements are also added to the freezing solution of the present embodiment to protect cells.

Optionally, in the same vitrification freezing and thawing solution set, the fatty acid supplements used in each component are of the same type, and any one or both of unsaturated fatty acids and saturated fatty acids can be selected.

Optionally, the unsaturated fatty acids include any one or more of arachidonic acid, linoleic acid, linolenic acid, oleic acid, and palmitoleic acid. The saturated fatty acids include any one or more of myristic acid, palmitic acid, and stearic acid.

Optionally, in the same vitrification freezing and thawing solution set, the amount of the fatty acid supplement added to each of the above components is 0.5% to 1.5% of the volume of each component. Preferably, the amount of the fatty acid supplement added is 1.0% of the volume of each component.

The principle of using hydroxypropyl cellulose and fatty acid supplements in the vitrification freezing and thawing solution kits of Examples 1 and 2 is:

First, the amount of hydroxypropyl cellulose added to each component of the freezing and thawing solution set is only 0.06-0.12 mg/mL, which can greatly reduce the density and viscosity of the liquid, so that the cells can be completely wrapped in the liquid, and the cryoprotectant can gradually enter or migrate out of the cells; at the same time, hydroxypropyl cellulose has moderate surface activity. Compared with human serum albumin, it is not easy to foam, and even if bubbles are generated, they can be quickly defoamed. It can reduce the surface tension of the liquid, so that in the actual operation process, dense foaming caused by repeated blowing and suction of the capillary can be avoided, which is convenient for microscopic observation.

Secondly, when using permeable cryoprotectants (dimethyl sulfoxide, ethylene glycol), it may cause the problem of missing some effective ingredients in the cells. Therefore, fatty acid supplements are added to each component, which can alleviate the impact of permeable cryoprotectants on the cell lipid membrane, prevent the decrease of lipid content of cells after freezing, and ensure the subsequent development ability of the cells.

The above-mentioned vitrification freezing and thawing solution set can be used to freeze various cells, such as egg cells, embryonic cells, ordinary cells, etc. The thawing solution set, and the components and ratios of the vitrification thawing solution set shown in Table 4 below, and the components and ratios of the fatty acid supplement in Table 5 illustrate the freezing and thawing of oocytes.

It should be noted that the components and ratios of the vitrification freezing and thawing solution set in Table 4 and the components and ratios of the fatty acid supplement in Table 5 are merely examples of the components in the vitrification freezing and thawing solution set, and do not limit their ratios.

Table 4: Components and ratios of vitrification thawing solution set:

Note: Since sucrose is a solid in some of the above components, it is difficult to measure by volume. However, it will occupy a certain volume after being dissolved in liquid. Therefore, the total volume of some components is not 100%.

Table 5: Components and ratios of fatty acid supplements:

The freezing and thawing process of egg cells is as follows:

Step 1: Vitrification of oocytes

Make a 100uL drop of balancing solution in the culture dish, and use a capillary to place the egg on the center of the balancing solution surface; the egg will sink within 30s, and the egg will be observed to dehydrate and shrink and then gradually recover its original volume. The balancing time is 12min; when there is 1min left in the balancing time, use a pipette to make two 100uL freezing solution drops on the lid of the dish (freezing drop 1, freezing drop 2).

After the balancing operation is completed, the eggs in the balancing solution are sucked to the front end of the pipette, and then moved to the center of the surface of the droplet of freezing solution 1; the timing is 25 seconds, and the eggs are continuously and gently blown and sucked at the bottom of freezing droplet 1; then the eggs are placed in freezing droplet 2, and the timing is 25 seconds, and the eggs are continuously and gently blown and sucked at the bottom of freezing droplet 2; a capillary is used to place the droplet containing the eggs on the slide of the freezing support rod, and it is quickly placed in liquid nitrogen for low-temperature storage to complete the vitrification process of the egg cells.

Step 2: Thawing of egg vitrification:

Before thawing, take out the preheated thawing solution from the 37°C incubator and add 1 mL of thawing solution to the prepared 35 mm small dish; then take 3 35 mm small dishes and add diluent, cleaning solution 1 and cleaning solution 2 equilibrated to room temperature respectively;

Quickly turn the slide end of the carrier rod upside down and immerse it in the thawing solution preheated to 37℃ for 1 minute;

When the time is up, use a Pasteur pipette to suck out the eggs and place them in the center of the bottom of the diluent for 3 minutes;

Use a pipette to suck out the eggs and place them at the bottom of cleaning solution 1, leaving them for 5 minutes; then use a pipette to remove the eggs. The eggs were sucked out through the tube and placed at the bottom of the cleaning solution 2 and placed for 5 minutes. After the time was up, the eggs were taken out and placed in the m16 culture medium for subsequent culture, and the egg development data were recorded.

Another portion of thawed eggs were taken for lipid staining using Nile red, and photographed and observed under a fluorescence microscope, as shown in Figures 1 and 2.

See Tables 6 and 7 below for the development of eggs and the physical properties of the balancing solution and freezing solution.

Table 6: Oocyte development:

Table 7: Physical properties of balance liquid and freezing liquid:

Analysis: From the oocyte development in Table 6, it can be seen that the subsequent blastocyst development of the oocytes frozen and thawed by the freezing and thawing solution set of the present invention is higher than that of the oocytes frozen and thawed by the vitrification freezing and thawing solution set in Table 1; From the measurement of the physical performance parameters, it can be seen that the density, viscosity and surface tension of the balancing solution and the freezing solution in the vitrification freezing and thawing solution set of the present invention are lower than those of the balancing solution and the freezing solution in the vitrification freezing and thawing solution set in Table 1. This characteristic can improve the operating convenience of the vitrification freezing and thawing solution set during the freezing process. In addition, after freezing the eggs treated with different methods, it can be found that the lipid content of the eggs shown in Figure 1 (strong orange-red fluorescent staining part) is higher than that of the eggs shown in Figure 2, indicating that the present invention can protect the phospholipid bilayer of the eggs and maintain the lipid content of the cell membrane during the vitrification freezing and thawing process.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

In addition, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims (5)

A vitrification freezing and thawing solution set, comprising a freezing solution, a balancing solution, a thawing solution, a diluent, and a cleaning solution, characterized in that: the thawing solution comprises hydroxypropyl cellulose, a fatty acid supplement, sucrose, and a basic culture medium; the freezing solution comprises hydroxypropyl cellulose, a basic culture medium, sucrose, ethylene glycol, dimethyl sulfoxide, and a fatty acid supplement for protecting the phospholipid bilayer of cells; The fatty acid supplement in the thawing solution and the freezing solution are of the same type, both comprising unsaturated fatty acids and saturated fatty acids, and the amount of the fatty acid supplement added to the thawing solution is 1.0% of the volume of the thawing solution; The amount of hydroxypropyl cellulose added to the thawing solution and the freezing solution is the same, and is 0.06 to 0.12 mg/mL; The balanced solution comprises hydroxypropyl cellulose, basal culture medium, ethylene glycol and dimethyl sulfoxide. The vitrification freezing and thawing solution kit according to claim 1 is characterized in that the unsaturated fatty acid includes any one or more of arachidonic acid, linoleic acid, linolenic acid, oleic acid, and palmitoleic acid. The vitrification freezing and thawing solution kit according to claim 1 is characterized in that the saturated fatty acid includes any one or more of myristic acid, palmitic acid, and stearic acid. The vitrification freezing and thawing solution kit according to claim 1 is characterized in that: the diluent and the cleaning solution both include hydroxypropyl cellulose, and the diluent and the cleaning solution both include a fatty acid supplement. The vitrification freezing and thawing solution set according to claim 4 is characterized in that the amount of hydroxypropyl cellulose added to the balancing solution, the diluent, and the cleaning solution is 0.06-0.12 mg/mL, and the amount of fatty acid supplement added to the diluent and the cleaning solution is 0.5%-1.5% of the volume of each component.