Mouse embryo cryopreservation and resuscitation method

Abstract

The invention provides a mouse embryo cryopreservation and resuscitation method. The mouse embryo cryopreservation and resuscitation method comprises the following steps that a cryopreservation solution A and a cryopreservation solution B are used for cryopreservation of mouse embryos; a resuscitation solution C and an operation solution D are used for resuscitating the mouse embryo; wherein the cryopreservation solution A comprises the following components: 1-15v / v% of dimethyl sulfoxide and the balance of M2 embryo culture solution; the cryopreservation solution B comprises the following components: 5-30v / v% of dimethyl sulfoxide, 0.01-0.1 g / ml of acetamide, 10-40v / v% of propylene glycol and the balance of an M2 embryo culture solution; the resuscitation solution C comprises the following components: 0.1-0.4 M sucrose and the balance of M2 embryo culture solution; the operating solution D comprises an M2 embryo culture solution. The dimethyl sulfoxide, the acetamide and the propylene glycol are jointly used, so that the toxicity of the dimethyl sulfoxide is reduced, and the freezing efficiency is improved. A permeable cryoprotectant is selected for embryo freezing, the concentration of the cryoprotectant is gradually increased through a two-step balance method, and a non-permeable cryoprotectant is selected for resuscitation, so that permeation shock caused by unfreezing and rewarming is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of mouse genetic resource preservation, and in particular to a mouse embryo cryopreservation and resuscitation method. Background Art

[0002] In the experimental animal industry, model animal resources that are introduced and developed with a large amount of funds mostly face the fate of being discarded after completing scientific research work. This requires corresponding scientific research institutions to have the ability to preserve valuable resources to meet the needs of the development of precision medicine and modern biotechnology industries. This has become a consensus in the experimental animal industry.

[0003] Embryo cryopreservation is a common technology for laboratory animal preservation and biological purification. In actual work, the long-term live preservation of genetically engineered mice not only consumes a lot of space and maintenance costs, but also may cause genetic drift and genetic contamination. The preservation and breeding of mice by cryopreserving embryos can not only save feeding costs, but also ensure genetic stability and prevent genetic drift and genetic contamination within the group. Since Whittingham et al. successfully used liquid nitrogen to cryopreserve mouse in vitro fertilization embryos for the first time in 1972, mammal embryo cryopreservation technology has made great progress. With the widespread application of genetically engineered mice, it is crucial to establish a set of standardized mouse embryo freezing methods to preserve these extremely precious genetic resources.

[0004] At present, freezing of mouse embryos is very costly, for example, freezing rods are used to carry the embryos to be frozen during vitrification. On the other hand, the procedure is cumbersome, for example, the programmed freezing process is complicated and the efficiency after recovery is unstable, which makes many embryos unable to be recovered successfully. Summary of the invention

[0005] The purpose of the present invention is to provide a mouse embryo freezing and resuscitation method in view of the defects of the prior art, comprising the following steps: using freezing solution A and freezing solution B to freeze the mouse embryos; using resuscitation solution C and operating solution D to resuscitate the mouse embryos; wherein the components of the freezing solution A include 1-15v / v% dimethyl sulfoxide, and the remaining components are M2 embryo culture solution; the components of the freezing solution B include 5-30v / v% dimethyl sulfoxide, 0.01-0.1g / ml acetamide, 10-40v / v% propylene glycol, and the remaining components are M2 embryo culture solution; the components of the resuscitation solution C include 0.1-0.4mol / L sucrose, and the remaining components are M2 embryo culture solution; the components of the operating solution D include M2 ​​embryo culture solution.

[0006] Furthermore, the mouse embryo is a closed group ICR mouse embryo, an inbred C57 mouse embryo, or a hybrid F1 generation BDF1 mouse embryo.

[0007] Furthermore, the volume ratio of the freezing solution A to the freezing solution B is 1:(17-21).

[0008] Furthermore, the ratio of the sum of the volumes of the cryopreservation solution A and the cryopreservation solution B to the volume of the resuscitation solution C is 1:(7-12).

[0009] Furthermore, the ingredients of the freezing solution A include 6-8v / v% dimethyl sulfoxide, and the remaining ingredients are M2 embryo culture medium; the ingredients of the freezing solution B include 12-18v / v% dimethyl sulfoxide, 0.03-0.08g / ml acetamide, 18-24v / v% propylene glycol, and the remaining ingredients are M2 embryo culture medium.

[0010] Furthermore, the components of resuscitation solution C include 0.2-0.3 mol / L sucrose, and the remaining components are M2 embryo culture medium.

[0011] Furthermore, the specific method for cryopreserving mouse embryos using cryopreservation solution A and cryopreservation solution B is as follows: firstly, the mouse embryos are transferred into cryopreservation solution A, then cryopreservation solution B is added into cryopreservation solution A, and then cryopreservation is performed using liquid nitrogen.

[0012] Further, the mouse embryos were transferred to cryopreservation solution A and then refrigerated at 0-5°C for 0-10 minutes, cryopreservation solution B was added to cryopreservation solution A and then refrigerated at 0-5°C for 0-10 minutes, and then cryopreserved using liquid nitrogen.

[0013] Furthermore, the specific method for transferring the mouse embryos into the cryopreservation solution A is as follows: prepare a drop of cryopreservation solution A on a culture dish, transfer the mouse embryos into the drop of cryopreservation solution A until the mouse embryo morphology is stable, and then pipette 5-6ul of cryopreservation solution A containing the mouse embryos into a cryopreservation tube.

[0014] Furthermore, the specific method of using resuscitation solution C and operating solution D to resuscitate mouse embryos is as follows: after thawing the mixture of freezing solution A and freezing solution B containing mouse embryos, resuscitation solution C is added to the mixture of freezing solution A and freezing solution B, and after mixing evenly, the mixture of freezing solution A, freezing solution B and resuscitation solution C containing mouse embryos is aspirated onto a culture dish, and the mouse embryos are transferred to the operating solution D for washing to obtain resuscitated mouse embryos.

[0015] The beneficial effects of the present invention are:

[0016] 1. The combined use of acetamide, propylene glycol, and dimethyl sulfoxide further improved the cryopreservation efficiency, while reducing the toxicity of dimethyl sulfoxide, effectively improving the survival rate of mouse embryo resuscitation.

[0017] 2. Cryolytic solution A contains 7.0-7.2 v / v% dimethyl sulfoxide, while cryolytic solution B contains 14.0-14.3 v / v% dimethyl sulfoxide. The concentration of cryolytic solution B is higher than that of cryolytic solution A. By gradually balancing the embryos from a low concentration of protective agent to a high concentration, cryolytic solutions A and B can fully penetrate into the cells, and the embryo water is gradually replaced by the cryolytic solution.

[0018] 3. Due to the low temperature of the cryopreservation state, the purpose of pre-freezing the cryopreservation solution A or the mixture of cryopreservation solutions A and B containing the embryos in an ice-water mixture at 0°C for 5 minutes is to keep the embryos at a lower temperature. In this way, the embryos are at a lower temperature throughout the whole process to prevent large temperature changes from causing damage to the embryos.

[0019] 4. Embryo freezing uses permeable cryoprotectants, which gradually increase the concentration of cryoprotectant through a two-step equilibrium method. During this process, the cryoprotectant solidifies without forming ice crystals. The solid state still maintains the normal molecular and ion distribution of the liquid, which is called the glass state. The resuscitation uses non-permeable cryoprotectants, the main component of which is sucrose, a macromolecular polymer that cannot freely diffuse through the cell membrane when the cell membrane is intact. It can increase the osmotic pressure of the extracellular fluid, control the speed at which water enters the cell, and reduce the osmotic shock caused by thawing and rewarming, thereby avoiding cell rupture and cell swelling. The resuscitation survival rate is further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the cleavage status of closed group ICR mouse embryos, inbred line C57 mouse embryos and hybrid F1 generation BDF1 mouse embryos in the embodiments of the present invention. DETAILED DESCRIPTION

[0021] The drugs used in the following examples are all commercially available products unless otherwise specified, and the methods used are all conventional methods in the art unless otherwise specified.

[0022] The present invention is further illustrated by the following examples and comparative examples.

[0023] Prepare cryopreservation solution A, cryopreservation solution B, resuscitation solution C and operating solution D:

[0024] The composition of cryopreservation solution A is 7.0-7.2 v / v% dimethyl sulfoxide, and the remaining composition is M2 embryo culture medium.

[0025] The ingredients of cryopreservation solution B are 14.0-14.3 v / v% dimethyl sulfoxide, 0.55-0.60 g / ml acetamide, 20-22 v / v% propylene glycol, and the remaining ingredients are M2 embryo culture medium. Dimethyl sulfoxide is the most commonly used freezing solution, which is relatively toxic. Adding acetamide and propylene glycol at the same time can reduce the toxicity of dimethyl sulfoxide. Acetamide and propylene glycol are also used as freezing solutions. However, if dimethyl sulfoxide, acetamide or propylene glycol are used alone, the freezing efficiency is relatively low. Adding acetamide and propylene glycol to dimethyl sulfoxide and using them together can further improve the freezing efficiency and reduce the toxicity of dimethyl sulfoxide.

[0026] The ingredients of resuscitation solution C are 0.25-0.26 mol / L sucrose, and the rest are M2 embryo culture medium. Sucrose improves the resuscitation survival rate through its non-permeability, controlling the speed of water entering and leaving cells.

[0027] The component of operating solution D is M2 embryo culture medium.

[0028] Cryopreservation solution A, preparation of cryopreservation solution B, resuscitation solution C and operating solution D are frozen and stored for future use.

[0029] (1) Preparation of cryopreservation solution A, cryopreservation solution B, resuscitation solution C, and operating solution D: Thaw cryopreservation solution A and cryopreservation solution B in an ice-water mixture (only ice from an ice maker and tap water are needed to maintain a 0°C environment). Thaw solution C in a 37°C CO2 incubator. Thaw solution D in a 37°C CO2 incubator.

[0030] (2) Prepare 3-4 drops of 80-100ul of cryopreservation solution A on a culture dish, place multiple mouse embryos in the drops of cryopreservation solution A and balance them until the mouse embryo morphology is completely stable and unchanged, then move the mouse embryo to 6ul of A solution (the bottom of the culture dish and the pipette tip will lose a little liquid during the transfer process, and the actual liquid volume is about 5ul). Then use a mouth pipette to move the embryo to the bottom of a pre-marked cryopreservation tube and place it in a 0℃ ice-water mixture for pre-freezing for 5 minutes; (the 0℃ here is a low temperature environment maintained at 0℃ by mixing ice from an ice maker with water). Since the temperature of the frozen state is relatively low, the purpose of placing the cryopreservation solution A in a 0℃ ice-water mixture for pre-freezing for 5 minutes is to keep the embryo at a relatively low temperature. In this way, the embryo is at a relatively low temperature throughout the process to prevent damage to the embryo caused by large temperature changes.

[0031] (3) Place the embryo cryopreservation solution B prepared in advance in an ice-water mixture, draw 95ul of cryopreservation solution B and place it at the bottom of the cryopreservation tube containing the mouse embryo, allow cryopreservation solution A and cryopreservation solution B to mix, and continue to pre-freeze at 0°C for 5 minutes; cryopreservation solution A contains 7.0-7.2v / v% dimethyl sulfoxide, while cryopreservation solution B contains 14.0-14.3v / v% dimethyl sulfoxide. Place the embryo in a lower concentration of cryopreservation solution A at room temperature to allow the osmotic protective agent to fully penetrate the cells. At the same time, the embryo is gradually dehydrated. After a certain period of equilibrium, high concentration cryopreservation solution B is added and mixed for a certain period of time, and then the cryopreservation tube is directly placed in liquid nitrogen for cryopreservation.

[0032] (4) Prepare liquid nitrogen in advance, and use tweezers to quickly immerse the cryopreservation tube containing the mouse embryos vertically into the liquid nitrogen until they are completely frozen, thus completing the cryopreservation of the mouse embryos;

[0033] (5) Embryo recovery: Thaw and rewarm the recovery solution C and operating solution D in a 37°C incubator, remove the cryotube containing the mouse embryos from the liquid nitrogen, open the lid of the cryotube, and allow to thaw at room temperature;

[0034] (6) Pipette 900ul of embryo resuscitation solution C to the bottom of the cryopreservation tube and mix it with cryopreservation solution A and cryopreservation solution B. After gently mixing it up and down several times with a 1ml pipette tip, aspirate the mixture of cryopreservation solution A, cryopreservation solution B and resuscitation solution C containing embryos onto the culture dish. Under the stereomicroscope, find the mouse embryos in the mixed solution in the culture dish and transfer them to the pre-prepared operating solution D. Then transfer all the mouse embryos from the operating solution D to the culture dish as soon as possible. Operating solution D is only M2 embryo culture solution, and the basal solution of resuscitation solution C is also M2 embryo culture solution. The purpose of transferring the embryos from resuscitation solution C to operating solution D is to clean the embryos. Make 3-6 drops on the culture dish in advance. Since operating solution D is only the basal solution, the volume only needs to be greater than 20ul.

[0035] (7) After washing the mouse embryos 3-4 times in 3-4 culture dishes containing KSOM culture medium, place them in a KSOM culture dish that has been equilibrated overnight, and then place them in a 37°C incubator for transplantation.

[0036] Experiment 1: Freezing and Thawing of Closed Colony ICR Mouse Embryos

[0037] At 17:00 on the first day, 6 8-week ICR female mice were selected and intraperitoneally injected with 10 IU of PMSG. At 17:00 on the third day, 10 IU of HCG was continued to be intraperitoneally injected. IVF (in vitro fertilization) was performed at 8:00 on the morning of the fourth day. (The male mice were 8-week ICR male mice). At 14:00 in the afternoon of the same day, the embryos were washed and cultured to two cells on the 5th day. After taking pictures, the embryos were frozen in 3 groups on average. After freezing for 24 hours, the embryos were revived on the 6th day. The number of surviving two cells was counted, and the recovery efficiency was calculated. The embryos were continued to be cultured in KSOM to four cells, eight cells, morulas and blastocysts. The embryos at different stages were counted and photographed, and the average efficiency of two cells, four cells, morulas and blastocysts after recovery in the three groups was counted.

[0038] Experiment 2: Freezing and Thawing of Inbred C57 Mouse Embryos

[0039] At 17:00 on the first day, 6 8-week-old C57 female mice were selected for intraperitoneal injection of 10 IU of PMSG, and at 17:00 on the third day, 10 IU of HCG was continued for intraperitoneal injection. IVF was performed at 8:00 on the morning of the fourth day. (The male mice were 8-week-old C57 male mice). The embryos were washed and cultured to two cells on the 5th day at 14:00 in the afternoon of the same day. After taking pictures, the embryos were frozen in 3 groups on average. After freezing for 24 hours, the embryos were revived on the 6th day, and the number of surviving two cells was counted, and the recovery efficiency was calculated. The embryos were continued to be cultured in KSOM to four cells, eight cells, morulas and blastocysts. The embryos at different stages were counted and photographed, and the average efficiency of two cells, four cells, morulas and blastocysts after recovery in the three groups was counted.

[0040] Experiment 3: Embryo freezing and thawing of hybrid F1 generation BDF1 mice.

[0041] At 17:00 on the first day, 6 8-week-old BDF1 female mice were intraperitoneally injected with 10 IU of PMSG. At 17:00 on the third day, 10 IU of HCG was continued to be intraperitoneally injected. IVF was performed at 8:00 on the morning of the fourth day. (The male mice were 8-week-old BDF1 male mice). The embryos were washed and cultured to two cells on the 5th day at 14:00 in the afternoon of the same day. After taking pictures, the embryos were frozen in 3 groups on average. After freezing for 24 hours, the embryos were revived on the 6th day. The number of surviving two cells was counted, and the recovery efficiency was calculated. The embryos were continued to be cultured in KSOM to four cells, eight cells, morulas and blastocysts. The embryos at different stages were counted and photographed, and the average efficiency of two cells, four cells, morulas and blastocysts after recovery in the three groups was counted.

[0042] The statistical summary of the freezing and thawing efficiency of mouse embryos under three different backgrounds is shown in Table 1:

[0043] Table 1

[0044]

[0045] In vitro embryo culture after embryo thawing under three backgrounds Figure 1 As shown, the mouse embryo division is in good condition, especially the efficiency of recovery to 2-cell is stable at 95% and above, and can reach a maximum of 100% recovery efficiency. There is almost no loss compared with unfrozen-C57.

[0046] This embryo freezing method uses a special protective agent (cryopreservation solution) and cooling measures to store the embryo in liquid nitrogen at -196°C and maintain a minimum metabolic level. At -196°C, the activity of cellular enzymes is almost completely inhibited, so that it is in a "dormant" state, thereby achieving long-term preservation. The embryo can be thawed and revived when needed, and the metabolism will be restored after embryo resuscitation. Using a higher concentration of cryoprotectant combined with a rapid cooling method, the protective agent in the embryo is in a glassy state without forming ice crystals, reducing cryoprotection damage to the embryo. The freezing process uses a two-step equilibrium method. First, most of the water in the cell is replaced with cryoprotectant A, and then a higher concentration of cryoprotectant B is used for equilibrium for 5 minutes, and then it is quickly placed in liquid nitrogen. In a low-temperature environment, the cryoprotectant quickly solidifies to form an irregular vitrified solid, which preserves the normal molecular and ion distribution of the cell in the liquid state, protects the cell structure and intracellular components, and achieves low cell damage cryopreservation.

[0047] Since dimethyl sulfoxide is currently the most widely used cryoprotectant, but it has potential toxic effects, acetamide and propylene glycol are additionally added. The base fluid of resuscitation fluid C is M2 embryo culture fluid, so the embryos finally picked out from resuscitation fluid C can be directly moved to operating fluid D (M2 embryo culture fluid) for cleaning. After cleaning, they can be moved to culture fluid and placed in a CO2 incubator for culture.

[0048] Embryo thawing is the opposite of freezing. Thawing is the process of restoring embryos from a relatively low temperature to a normal physiological state. Thawing is mainly divided into two processes: one is rewarming and the other is removing the cryoprotectant, which ultimately achieves the purpose of restoring the physiological activity of the embryo and continuing its development. The embryo is placed in a certain concentration of thawing solution C (non-permeable protective agent) so that the permeable protective agent in the embryo gradually seeps out of the cell, and water gradually seeps into the cell, and the embryo returns to a normal physiological state.

[0049] This method greatly improves the convenience of genetic resource preservation, greatly reduces the economic cost of genetic resource preservation, and better protects precious resources.

[0050] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. A method for freezing and thawing mouse embryos, characterized in that: The following steps are involved: Mouse embryos were cryopreserved using cryopreservation solution A and cryopreservation solution B; Mouse embryos were resuscitated using resuscitation solution C and operating solution D; The components of the cryopreservation solution A include 1-15 v / v% dimethyl sulfoxide, and the remaining components are M2 embryo culture medium; The components of cryopreservation solution B include 5-30 v / v% dimethyl sulfoxide, 0.01-0.1 g / ml acetamide, 10-40 v / v% propylene glycol, and the remaining components are M2 embryo culture medium; The components of resuscitation solution C include 0.1-0.4 mol / L sucrose, and the remaining components are M2 embryo culture medium; The components of the operating solution D include M2 ​​embryo culture medium.

2. The method for freezing and resuscitating mouse embryos according to claim 1, characterized in that: The mouse embryos are closed colony ICR mouse embryos, inbred C57 mouse embryos, or hybrid F1 generation BDF1 mouse embryos.

3. The method for freezing and resuscitating mouse embryos according to claim 1, characterized in that: The volume ratio of the freezing solution A to the freezing solution B is 1:(17-21).

4. The method for freezing and resuscitating mouse embryos according to claim 1, characterized in that: The ratio of the sum of the volumes of the freezing solution A and the freezing solution B to the volume of the resuscitation solution C is 1:(7-12).

5. The method for freezing and resuscitating mouse embryos according to claim 1, characterized in that: The freezing solution A comprises 6-8 v / v% dimethyl sulfoxide, and the remaining components are M2 embryo culture medium; The freezing solution B comprises 12-18 v / v% dimethyl sulfoxide, 0.03-0.08 g / ml acetamide, 18-24 v / v% propylene glycol, and the remaining components are M2 embryo culture medium.

6. The method for freezing and resuscitating mouse embryos according to claim 1, characterized in that: The components of resuscitation solution C include 0.2-0.3 mol / L sucrose, and the remaining components are M2 embryo culture medium.

7. The method for freezing and thawing mouse embryos according to claim 1, characterized in that: The specific method for cryopreservation of mouse embryos using cryopreservation solution A and cryopreservation solution B is as follows: First transfer the mouse embryos to cryopreservation solution A, then add cryopreservation solution B to cryopreservation solution A, and then use liquid nitrogen to freeze.

8. The method for freezing and thawing mouse embryos according to claim 7, characterized in that: The mouse embryos were transferred to cryopreservation solution A and refrigerated at 0-5°C for 0-10 minutes. Cryopreservation solution B was added to cryopreservation solution A and refrigerated at 0-5°C for 0-10 minutes, and then cryopreserved using liquid nitrogen.

9. The method for freezing and thawing mouse embryos according to claim 7, characterized in that: The specific method for transferring mouse embryos into cryopreservation medium A is as follows: Prepare a drop of cryopreservation solution A on a culture dish, transfer the mouse embryos into the drop of cryopreservation solution A until the mouse embryo morphology remains stable, then pipette 5-6ul of cryopreservation solution A containing the mouse embryos into a cryopreservation tube.

10. The method for freezing and thawing mouse embryos according to claim 1, characterized in that: The specific method of resuscitating mouse embryos using resuscitation solution C and operating solution D is as follows: After thawing the mixture of cryopreservation solution A and cryopreservation solution B containing mouse embryos, resuscitation solution C is added to the mixture of cryopreservation solution A and cryopreservation solution B, and after mixing evenly, the mixture of cryopreservation solution A, cryopreservation solution B and resuscitation solution C containing mouse embryos is pipetted onto a culture dish, and the mouse embryos are transferred to operating solution D for washing to obtain resuscitated mouse embryos.

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