A matching rat embryo cryopreservation-thawing system and its application

Abstract

The present invention discloses a rat embryo cryopreservation pretreatment solution, which is composed of a basal culture medium, 95 to 110 mL / L of propylene glycol, 0.8 to 1.5 g / L of polyvinyl alcohol, and an appropriate amount of antibiotics. In addition, the present invention also provides a rat embryo cryopreservation-resuscitation system comprising the aforementioned rat embryo cryopreservation pretreatment solution. The present invention can break through the bottleneck of rat germplasm resource preservation. The application of the supporting rat embryo cryopreservation-resuscitation system can enable the cryopreservation-resuscitation of rat embryos and subsequent artificial assisted reproduction to proceed smoothly, especially for rat resources with homozygous genotypes. The several months required for natural reproduction are greatly reduced to SPF-level offspring within one month, and rat resources with specific genotypes or genetic modifications can be quickly obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of biological seed preservation, and particularly relates to a matching rat embryo cryopreservation-thawing system and application thereof. Background Art

[0002] With the rapid development of various gene-editing technologies, particularly in the laboratory animal industry, a variety of valuable genetically modified strains have emerged. Preservation techniques to preserve these strains have also become widely used. Exploring more efficient preservation techniques warrants further research. Cryogenic techniques primarily include slow freezing and vitrification. The low survival rate of slow freezing limits its application in biological preservation.

[0003] After Rall and Fahy proposed the vitrification technique in 1985, it has been widely used for freezing embryos in various experimental animals. Vitrification involves placing embryos in a high-concentration cryoprotectant at room temperature and rapidly transferring them to liquid nitrogen. The cryoprotectant transforms from a liquid into a non-structured, glass-like state, maintaining a normal ion distribution and minimizing damage to the frozen embryo. High cryoprotectant concentrations and rapid freezing rates are key steps in vitrification. High cryoprotectant concentrations are toxic and may damage embryos. Finding less toxic cryoprotectants is one way to improve freezing efficiency. The frozen embryos are ultimately placed in liquid nitrogen at -196°C, a significant temperature range. This process is responsible for the formation of ice crystals, typically between -5°C and -80°C.

[0004] Rats can be used in the study of a variety of disease models. Combined with the widespread application of genetic modification technologies in scientific research, there is an increasing demand for genetically modified rats for seed preservation. However, unlike mice, due to the unique characteristics of rat sperm, it is difficult to obtain sperm with high motility for assisted reproduction through sperm cryopreservation and thawing. Therefore, the seed preservation of commercial genetically modified rats, especially homozygous genotypes, relies heavily on embryo cryopreservation and thawing methods. However, unlike the EFS20 / 40 or DAP213 methods used for mice, there is currently a lack of commercially available rat embryo cryopreservation and thawing reagents. Therefore, the development of a rat embryo cryopreservation and thawing reagent with low toxicity and minimal ice crystal formation has broad market application value. Summary of the Invention

[0005] The purpose of the present invention is to overcome at least one shortcoming of the prior art and provide a rat embryo cryopreservation-thawing system and its application.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a rat embryo cryopreservation pretreatment solution, which comprises a basal culture medium, 95-110 mL / L of propylene glycol, 0.8-1.5 g / L of polyvinyl alcohol, and an appropriate amount of antibiotics.

[0008] In some examples, the components of the basal culture medium include sodium ions, potassium ions, calcium ions, magnesium ions, chloride ions, dihydrogen phosphate ions, sulfate ions, bicarbonate ions, pyruvate ions, lactate ions, and glucose.

[0009] In some examples, the composition of the basal culture medium is: sodium chloride 95-105 mmol / L, potassium chloride 4-5 mmol / L, calcium chloride 5-6 mmol / L, potassium dihydrogen phosphate 0.3-0.5 mmol / L, magnesium sulfate 0.2-0.3 mmol / L, sodium bicarbonate 24-25 mmol / L, sodium pyruvate 0.3-0.4 mmol / L, sodium lactate 17.5-19.0 mmol / L, and glucose 2-3.5 mmol / L.

[0010] In a second aspect, the present invention provides a rat embryo cryopreservation-thawing system, comprising a rat embryo cryopreservation pretreatment solution, a rat embryo cryopreservation protection solution, and a rat embryo thawing solution, wherein:

[0011] The rat embryo cryopreservation pretreatment solution is as described in the first aspect;

[0012] The rat embryo cryopreservation protection solution is composed of: basal culture medium, sucrose, polyvinyl alcohol, propylene glycol, ethylene glycol, Percoll® culture medium, and antibiotics;

[0013] The rat embryo resuscitation solution is composed of: basic culture medium, polyvinyl alcohol, sucrose and antibiotics.

[0014] In some examples, the rat embryo cryopreservation protection solution contains: 0.3-0.4 mol / L sucrose, 90-150 mL / L propylene glycol, 0.8-1.5 g / L polyvinyl alcohol, 250-400 mL / L ethylene glycol, 120-250 mL / L Percoll® medium, and an appropriate amount of antibiotics.

[0015] In some examples, the rat embryo resuscitation solution contains: 0.3-0.4 mol / L sucrose, 0.8-1.5 g / L polyvinyl alcohol, and an appropriate amount of antibiotics.

[0016] In some examples, the components of the basal culture medium include sodium ions, potassium ions, calcium ions, magnesium ions, chloride ions, dihydrogen phosphate ions, sulfate ions, bicarbonate ions, pyruvate ions, lactate ions, and glucose.

[0017] In some examples, the composition of the basal culture medium is: sodium chloride 95-105 mmol / L, potassium chloride 4-5 mmol / L, calcium chloride 5-6 mmol / L, potassium dihydrogen phosphate 0.3-0.5 mmol / L, magnesium sulfate 0.2-0.3 mmol / L, sodium bicarbonate 24-25 mmol / L, sodium pyruvate 0.3-0.4 mmol / L, sodium lactate 17.5-19.0 mmol / L, and glucose 2-3.5 mmol / L.

[0018] In some examples, the rat embryo cryopreservation pretreatment solution is composed of: basal culture medium, propylene glycol 95-110 mL / L, polyvinyl alcohol 0.8-1.5 g / L, and appropriate amounts of antibiotics;

[0019] The rat embryo cryopreservation protection solution is composed of: basal culture medium, sucrose 0.3-0.4 mol / L, propylene glycol 90-150 mL / L, polyvinyl alcohol 0.8-1.5 g / L, ethylene glycol 250-400 mL / L, Percoll® culture medium 120-250 mL / L, and appropriate amounts of antibiotics;

[0020] The rat embryo resuscitation solution is composed of: basic culture medium, sucrose 0.3-0.4 mol / L, polyvinyl alcohol 0.8-1.5 g / L, and appropriate amount of antibiotics;

[0021] The composition of the basal culture medium is: 95-105 mmol / L sodium chloride, 4-5 mmol / L potassium chloride, 5-6 mmol / L calcium chloride, 0.3-0.5 mmol / L potassium dihydrogen phosphate, 0.2-0.3 mmol / L magnesium sulfate, 24-25 mmol / L sodium bicarbonate, 0.3-0.4 mmol / L sodium pyruvate, 17.5-19.0 mmol / L sodium lactate, and 2-3.5 mmol / L glucose.

[0022] The third aspect is the use of the cryopreservation-thawing system described in the second aspect of the present invention in preserving rat embryos.

[0023] The beneficial effects of the present invention are:

[0024] Due to the important role of rats in various disease models, the present invention breaks through the bottleneck of rat germplasm resource preservation. The application of the accompanying rat embryo cryopreservation-thawing system can enable the cryopreservation-thawing of rat embryos and the subsequent artificial assisted reproduction to be carried out smoothly, especially for homozygous genotype rat resources. The several months required for natural reproduction are greatly reduced to SPF-level offspring within one month, and rat resources with specific genotypes or genetic modifications can be quickly obtained.

[0025] In addition, the rat embryo cryopreservation-thawing system used in conjunction with the present invention enables the realization of rat embryo cryopreservation-thawing technology with high survival efficiency, thereby enabling artificial assisted reproduction after ultra-low temperature cryopreservation of rat germplasm to proceed smoothly, providing a guarantee for the preservation of rat germplasm resources. DETAILED DESCRIPTION

[0026] In a first aspect, the present invention provides a rat embryo cryopreservation pretreatment solution, which comprises a basal culture medium, 95-110 mL / L of propylene glycol, 0.8-1.5 g / L of polyvinyl alcohol, and an appropriate amount of antibiotics.

[0027] In some examples, the components of the basal culture medium include sodium ions, potassium ions, calcium ions, magnesium ions, chloride ions, dihydrogen phosphate ions, sulfate ions, bicarbonate ions, pyruvate ions, lactate ions, and glucose.

[0028] In some examples, the composition of the basal culture medium is: sodium chloride 95-105 mmol / L, potassium chloride 4-5 mmol / L, calcium chloride 5-6 mmol / L, potassium dihydrogen phosphate 0.3-0.5 mmol / L, magnesium sulfate 0.2-0.3 mmol / L, sodium bicarbonate 24-25 mmol / L, sodium pyruvate 0.3-0.4 mmol / L, sodium lactate 17.5-19.0 mmol / L, and glucose 2-3.5 mmol / L.

[0029] In a second aspect, the present invention provides a rat embryo cryopreservation-thawing system, comprising a rat embryo cryopreservation pretreatment solution, a rat embryo cryopreservation protection solution, and a rat embryo thawing solution, wherein:

[0030] The rat embryo cryopreservation pretreatment solution is as described in the first aspect;

[0031] The rat embryo cryopreservation protection solution is composed of: basal culture medium, sucrose, polyvinyl alcohol, propylene glycol, ethylene glycol, Percoll® culture medium, and antibiotics;

[0032] The rat embryo resuscitation solution is composed of: basic culture medium, polyvinyl alcohol, sucrose and antibiotics.

[0033] In some examples, the rat embryo cryopreservation protection solution contains: 0.3-0.4 mol / L sucrose, 90-150 mL / L propylene glycol, 0.8-1.5 g / L polyvinyl alcohol, 250-400 mL / L ethylene glycol, 120-250 mL / L Percoll® medium, and an appropriate amount of antibiotics.

[0034] In some examples, the rat embryo resuscitation solution contains: 0.3-0.4 mol / L sucrose, 0.8-1.5 g / L polyvinyl alcohol, and an appropriate amount of antibiotics.

[0035] In some examples, the components of the basal culture medium include sodium ions, potassium ions, calcium ions, magnesium ions, chloride ions, dihydrogen phosphate ions, sulfate ions, bicarbonate ions, pyruvate ions, lactate ions, and glucose.

[0036] In some examples, the composition of the basal culture medium is: sodium chloride 95-105 mmol / L, potassium chloride 4-5 mmol / L, calcium chloride 5-6 mmol / L, potassium dihydrogen phosphate 0.3-0.5 mmol / L, magnesium sulfate 0.2-0.3 mmol / L, sodium bicarbonate 24-25 mmol / L, sodium pyruvate 0.3-0.4 mmol / L, sodium lactate 17.5-19.0 mmol / L, and glucose 2-3.5 mmol / L.

[0037] In some examples, the rat embryo cryopreservation pretreatment solution is composed of: basal culture medium, propylene glycol 95-110 mL / L, polyvinyl alcohol 0.8-1.5 g / L, and appropriate amounts of antibiotics;

[0038] The rat embryo cryopreservation protection solution is composed of: basal culture medium, sucrose 0.3-0.4 mol / L, propylene glycol 90-150 mL / L, polyvinyl alcohol 0.8-1.5 g / L, ethylene glycol 250-400 mL / L, Percoll® culture medium 120-250 mL / L, and appropriate amounts of antibiotics;

[0039] The rat embryo resuscitation solution is composed of: basic culture medium, sucrose 0.3-0.4 mol / L, polyvinyl alcohol 0.8-1.5 g / L, and appropriate amount of antibiotics;

[0040] The composition of the basal culture medium is: 95-105 mmol / L sodium chloride, 4-5 mmol / L potassium chloride, 5-6 mmol / L calcium chloride, 0.3-0.5 mmol / L potassium dihydrogen phosphate, 0.2-0.3 mmol / L magnesium sulfate, 24-25 mmol / L sodium bicarbonate, 0.3-0.4 mmol / L sodium pyruvate, 17.5-19.0 mmol / L sodium lactate, and 2-3.5 mmol / L glucose.

[0041] In the above-mentioned rat embryo cryopreservation pretreatment solution, rat embryo cryopreservation protection solution and rat embryo thawing solution, the role of antibiotics is to inhibit the unintentional introduction of microorganisms during the operation from adversely affecting the growth of cells. Under strictly sterile conditions, the dosage of antibiotics can be as low as 0. Antibiotics are preferably those with good antibacterial effects and essentially no toxic effects on cells, and their dosage can be determined according to existing literature or textbooks. In some instances, antibiotics are preferably penicillin and streptomycin, and more preferably, the dosage of penicillin is 0.04-0.08 mg / mL and the dosage of streptomycin is 0.04-0.06 mg / mL.

[0042] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0043] The configurations of the rat embryo cryopreservation pretreatment solution, rat embryo cryopreservation protection solution, and rat embryo resuscitation solution used in the embodiments and comparative examples of the present invention are as follows:

[0044] The rat embryo cryopreservation pretreatment solution consists of the following: sodium chloride 101.61 mmol / L, potassium chloride 4.69 mmol / L, calcium chloride 5.14 mmol / L, potassium dihydrogen phosphate 0.40 mmol / L, magnesium sulfate 0.20 mmol / L, sodium bicarbonate 25.00 mmol / L, sodium pyruvate 0.30 mmol / L, glucose 2.78 mmol / L, sodium lactate 18.36 mmol / L, propylene glycol 100 mL / L, polyvinyl alcohol 1 g / L, penicillin 0.06 mg / mL, and streptomycin 0.05 mg / mL. Sodium chloride, potassium chloride, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate, sodium pyruvate, glucose, and sodium lactate are the components of the basal medium, providing a basic living environment for rat embryonic cells. Penicillin and streptomycin are antibiotics that work together to prevent the introduction of microorganisms that could cause embryonic death during cryopreservation. Polyvinyl alcohol can reduce mechanical damage to rat embryos during cryopreservation and promote osmotic pressure balance inside and outside the cells. Propylene glycol can reduce the formation of ice crystals in cells during cryopreservation, reducing the damage caused by ultra-low temperature storage to rat embryos.

[0045] The rat embryo cryoprotectant medium is composed of: sodium chloride 101.61 mmol / L, potassium chloride 4.69 mmol / L, calcium chloride 5.14 mmol / L, potassium dihydrogen phosphate 0.40 mmol / L, magnesium sulfate 0.20 mmol / L, sodium bicarbonate 25.00 mmol / L, sodium pyruvate 0.30 mmol / L, glucose 2.78 mmol / L, sucrose 0.35 mol / L, sodium lactate 18.36 mmol / L, propylene glycol 100 mL / L, polyvinyl alcohol 1 g / L, ethylene glycol 300 mL / L, Percoll® (a commercially available low-viscosity density gradient medium) 200 mL / L, penicillin 0.06 mg / mL, and streptomycin 0.05 mg / mL. Sodium chloride, potassium chloride, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate, sodium pyruvate, glucose, and sodium lactate constitute the basal medium, providing a basic living environment for rat embryonic cells. Penicillin and streptomycin are antibiotics that work together to prevent the introduction of microorganisms during cryopreservation that could lead to rat embryonic death. Polyvinyl alcohol reduces mechanical damage to rat embryos during cryopreservation while promoting osmotic pressure balance inside and outside the cells. Ethylene glycol and propylene glycol reduce the formation of ice crystals within cells during cryopreservation, minimizing damage to rat embryos caused by cryopreservation. Sucrose is used to regulate the osmotic pressure of the cryoprotectant solution. Percoll® is used to reduce ice crystal formation in cryopreservation culture medium.

[0046] The rat embryo thawing medium is composed of: sodium chloride 101.61 mmol / L, potassium chloride 4.69 mmol / L, calcium chloride 5.14 mmol / L, potassium dihydrogen phosphate 0.40 mmol / L, magnesium sulfate 0.20 mmol / L, sodium bicarbonate 25.00 mmol / L, sodium pyruvate 0.30 mmol / L, glucose 2.78 mmol / L, sucrose 0.35 mol / L, sodium lactate 18.36 mmol / L, polyvinyl alcohol 1 g / L, penicillin 0.06 mg / mL, and streptomycin 0.05 mg / mL. Sodium chloride, potassium chloride, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate, sodium pyruvate, glucose, and sodium lactate are the basal components of the culture medium, providing a basic living environment for rat embryonic cells. Penicillin and streptomycin are antibiotics that work together to prevent microbial lethality during manipulation. Polyvinyl alcohol reduces mechanical damage to rat embryos during cryopreservation and promotes osmotic pressure balance inside and outside the cells. Sucrose is used to regulate the osmotic pressure of the resuscitation solution.

[0047] Example 1

[0048] The rat embryo cryopreservation-thawing system of the present invention was used to perform a cryopreservation and thawing experiment on wild-type SD rat embryos. The specific steps are as follows:

[0049] 1) Using ultrapure water as the solvent, prepare rat embryo cryopreservation pretreatment solution, rat embryo cryopreservation protection solution, and rat embryo thawing solution. Mix thoroughly, filter through a 0.22 μm virus filter, and label each solution.

[0050] 2) Place the rat embryos to be frozen into 100 μL of rat embryo cryopreservation pretreatment solution. After 10 minutes, transfer the embryos to a cryovial containing 100 μL of rat embryo cryopreservation protection solution that has been pre-chilled to 0°C. Let the cryovial rest for 5 minutes before storing in liquid nitrogen.

[0051] 3) Remove the cryovial from the liquid nitrogen and place it at room temperature. When a small amount of liquid nitrogen remains in the tube, immediately remove the 37°C preheated rat embryo thawing solution from the CO2 incubator. Open the cryovial cap and immediately add 500 μL of rat embryo thawing solution. Place the cryovial on a 37°C hot plate. After 10 seconds, gently pipette and transfer the solution to a 60 mm culture dish. Then, aspirate 300 μL of rat embryo thawing solution to rinse the walls and bottom of the cryovial to minimize embryo loss. Combine the two rat embryo thawing solutions and remove surviving rat embryos under a microscope for embryo transfer.

[0052] Example 2

[0053] The rat embryo cryopreservation-thawing system of the present invention was used to perform cryopreservation and thawing experiments on SD strain genetically modified rat embryos. The specific experimental procedures were the same as those in Example 1.

[0054] Example 3

[0055] The rat embryo cryopreservation-thawing system of the present invention was used to perform cryopreservation and thawing experiments on Long Evans strain genetically modified rat embryos. The specific experimental procedures were the same as those in Example 1.

[0056] Example 4

[0057] The rat embryo cryopreservation-thawing system of the present invention was used to perform cryopreservation and thawing experiments on F344 strain genetically modified rat embryos. The specific experimental procedures were the same as those in Example 1.

[0058] Comparative Example 1

[0059] In this comparative example, the EFS20 / 40 system commonly used for mouse cryopreservation was used to cryopreserve and thaw SD strain wild-type rat embryos. The specific experimental procedures were the same as those in Example 1.

[0060] Comparative Example 2

[0061] In this comparative example, only the rat embryo cryopreservation protection solution described above was used to cryopreserve and resuscitate SD strain wild-type rat embryos, and the remaining specific experimental procedures were the same as those in Example 1.

[0062] Comparative Example 3

[0063] In this comparative example, only the rat embryo cryopreservation protection solution and rat embryo thawing solution described above were used to cryopreserve and thaw SD strain wild-type rat embryos. The remaining specific experimental procedures were the same as those in Example 1.

[0064] Comparative Example 4

[0065] In this comparative example, propylene glycol in the rat embryo cryopreservation pretreatment solution was replaced with ethylene glycol of the same concentration, and then SD strain wild-type rat embryos were cryopreserved and revived. The remaining specific experimental procedures were the same as in Example 1.

[0066] Comparative Example 5

[0067] In this comparative example, Percoll® in the rat embryo cryopreservation solution was replaced with Ficoll® at the same concentration, and then wild-type SD rat embryos were cryopreserved and revived. The remaining specific experimental procedures were the same as those in Example 1.

[0068] The experimental results of Examples 1 to 4 and Comparative Examples 1 to 5 are shown in Table 1.

[0069] Table 1 Embryo cryopreservation efficiency in each group

[0070]

[0071] From Example 1 in Table 1, the use of the rat embryo cryopreservation pretreatment solution, rat embryo cryopreservation protection solution, and rat embryo resuscitation solution of the present invention can simultaneously improve the survival rate of rat embryos after cryopreservation and the birth rate after transplantation, that is, further significantly improve the efficiency of rat embryo cryopreservation.

[0072] It can be seen from Examples 1-4 that the rat embryo cryopreservation pretreatment solution, rat embryo cryopreservation protection solution and rat embryo thawing solution used in conjunction with the present invention can achieve similar effects of improving the cryopreservation efficiency of rat embryos in different strains of rats.

[0073] Comparative Example 2 Compared with Comparative Example 1, the use of the rat embryo cryopreservation protection solution of the present invention alone can increase the transplantation birth rate of rat embryos after cryopreservation, that is, improve the efficiency of rat embryo cryopreservation;

[0074] Comparative Example 4 Compared with Example 1, the effect of selecting propylene glycol in the rat embryo cryopreservation pretreatment solution is significantly better than that of ethylene glycol, indicating that the key additives selected in the present invention can achieve the effect of improving the efficiency of rat embryo cryopreservation;

[0075] Comparative Example 5 Compared with Example 1, the effect of selecting Percoll® medium in rat embryo cryopreservation protection medium is significantly better than Ficoll®, indicating that the key additives selected in the present invention can achieve the effect of improving the efficiency of rat embryo cryopreservation.

[0076] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A rat embryo cryopreservation pretreatment solution, comprising: a basal culture medium, 95-110 mL / L of propylene glycol, 0.8-1.5 g / L of polyvinyl alcohol, and an appropriate amount of antibiotics, wherein the basal culture medium comprises: 95-105 mmol / L of sodium chloride, 4-5 mmol / L of potassium chloride, 5-6 mmol / L of calcium chloride, 0.3-0.5 mmol / L of potassium dihydrogen phosphate, 0.2-0.3 mmol / L of magnesium sulfate, 24-25 mmol / L of sodium bicarbonate, 0.3-0.4 mmol / L of sodium pyruvate, 17.5-19.0 mmol / L of sodium lactate, and 2-3.5 mmol / L of glucose.

2. A rat embryo cryopreservation-thawing system, characterized in that: The freezing-thawing system comprises a rat embryo freezing pretreatment solution, a rat embryo freezing protection solution and a rat embryo thawing solution, wherein: The rat embryo cryopreservation pretreatment solution is as described in claim 1; The rat embryo cryopreservation protection solution is composed of: basic culture medium, sucrose, polyvinyl alcohol, propylene glycol, ethylene glycol, Percoll ® Culture medium, antibiotics, the rat embryo cryopreservation protection solution contains: sucrose 0.3-0.4 mol / L, propylene glycol 90-150 mL / L, polyvinyl alcohol 0.8-1.5 g / L, ethylene glycol 250-400 mL / L, Percoll ® Culture medium 120-250 mL / L, appropriate amount of antibiotics; The rat embryo resuscitation solution is composed of: basic culture medium, polyvinyl alcohol, sucrose, and antibiotics. The rat embryo resuscitation solution contains: 0.3-0.4 mol / L sucrose, 0.8-1.5 g / L polyvinyl alcohol, and an appropriate amount of antibiotics; The basic culture medium is composed of: 95-105 mmol / L sodium chloride, 4-5 mmol / L potassium chloride, 5-6 mmol / L calcium chloride, 0.3-0.5 mmol / L potassium dihydrogen phosphate, 0.2-0.3 mmol / L magnesium sulfate, 24-25 mmol / L sodium bicarbonate, 0.3-0.4 mmol / L sodium pyruvate, 17.5-19.0 mmol / L sodium lactate, and 2-3.5 mmol / L glucose.

3. Use of the freezing-thawing system according to claim 2 in preserving rat embryos.

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