Freezing carrier device for embryo cryopreservation

Abstract

The invention belongs to the technical field of embryo freezing, and particularly discloses a freezing carrier device for embryo cryopreservation, which comprises a handheld part and a freezing part connected to one end of the handheld part, the freezing part is in a groove shape, and a first polymer hydrogel coating, a second polymer hydrogel coating and a third polymer hydrogel coating are sequentially coated in the groove of the freezing part; the high-molecular hydrogel coating has good liquid absorption capacity and can rapidly absorb redundant refrigerating fluid in the embryo refrigerating fluid, so that the volume of the embryo refrigerating fluid is reduced, the freezing and unfreezing speeds are increased, the damage of ice crystals to embryos is reduced, and the survival rate and conception rate of the embryos are increased.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a freezing carrier device for embryo cryopreservation. The device can be widely used in biomedical research and clinical applications, especially in embryo cryopreservation technology. Background Art

[0002] In the fields of modern animal genetic improvement, reproductive medicine and biotechnology, embryo freezing technology occupies a vital position and is an indispensable key technology for the breeding and preservation of precious and high-value animals.

[0003] Currently, embryo freezing technology mainly covers two mainstream methods: slow freezing and vitrification. The slow freezing method requires the embryo to be placed in a freezing solution, and the freezing purpose is achieved by precisely controlling the cooling rate. This method requires the embryo to be pre-equilibrated in a low-concentration cryoprotectant, and then transferred to the final concentration freezing solution, and gradually cooled according to the preset freezing program. The vitrification freezing method, on the other hand, quickly places the embryo in a high-concentration vitrification solution, using a high-concentration cryoprotectant and an extremely fast freezing rate to instantly transform the cells from a liquid state to an amorphous state similar to glass. During this process, no ice crystals are formed in the cells.

[0004] Vitrification offers significant advantages over slow freezing. Its freezing process is extremely rapid, allowing embryos to be cooled to low temperatures in a short period of time, significantly reducing cell damage during the freezing process. However, despite its clear advantages, vitrification still faces numerous challenges in its practical application and promotion: First, high concentrations of cryoprotectants are required during the vitrification process, which may cause a certain degree of chemical toxicity to the embryos, affecting their normal development and subsequent activity: Secondly, this method has extremely high requirements for operating skills and must rely on professional equipment and experienced operators. Only in this way can the speed and accuracy of the freezing and thawing process be accurately controlled. For example, when using Cryotop, a common freezing carrier, the operator needs to manually aspirate excess liquid. This step is not only complex and delicate, but also requires extremely high technical level and experience of the operator. The slightest mistake may cause damage or loss of embryos, which in turn leads to unstable freezing results and difficulty in ensuring embryo quality: Third, the cost of vitrification remains high, and the high price of freezing carriers is one of the main factors leading to the high cost.

[0005] Cryocarriers are key tools in the embryo freezing and thawing process. Their role is to provide a suitable freezing microenvironment for the embryo and the protective agent, while effectively protecting the embryo from external contamination and physical damage. Currently, the two most common embryo vitrification carriers on the market are open stretched straw (OPS) and Cryotop.

[0006] OPS comes in a 0.25mm diameter tube format, allowing the volume of embryos and protective agent to be controlled within a range of 2-5μL, which helps to increase freezing speed to a certain extent. However, it has a significant drawback: the relatively large volume of freezing fluid limits freezing and thawing speeds, preventing the ideal rapid freezing effect. This leaves embryos vulnerable to ice crystal damage during the freezing and thawing process, which in turn reduces embryo survival and conception rates.

[0007] The Cryotop consists of multiple components, including a tip, a stem, a sleeve, a sleeve balance, a desiccant, and a gas barrier. Its advantage lies in its ability to reduce the volume of embryos and protective agent to approximately 1μL, enabling ultra-fast freezing and thawing. However, its drawbacks are significant. Its high technical requirements and high price make it prohibitively expensive for large-scale use.

[0008] In the field of hydrogel technology, traditional research has focused on the water absorption of substances. The water absorption of a substance is usually closely related to its hydrophilicity, but in the specific application scenario of embryo freezing solution, the performance of liquid absorption is not solely determined by water absorption. The organic solvents contained in embryo freezing solution (such as glycerol and ethylene glycol) are all polar molecules. Although they can combine and mix well with other polar liquids, different substances have significant differences in their absorption of these organic solvents. If the hydrogel only has general water absorption, the concentration of organic solvents in the freezing solution may increase during the embryo freezing process, thereby increasing toxicity to the embryo and seriously affecting the quality and activity of the embryo. Therefore, hydrogels suitable for embryo freezing must have excellent adsorption or solubility properties for the specific liquids in the freezing solution to meet the increasingly high requirements of embryo freezing technology.

[0009] Prior patent CN 113185716 A discloses a thiolated hyaluronic acid polysaccharide hydrogel, which is a polysaccharide hydrogel formed by one or two thiolated hyaluronic acid derivatives and an aqueous solvent; wherein the mass percentage concentration of the thiolated hyaluronic acid derivative is 3-6%, and the thiol substitution degree of the thiolated hyaluronic acid derivative is 3-20%; the thiolated hyaluronic acid derivative is prepared by thiolation reaction of hydroxyethyl hyaluronic acid, hydroxypropyl hyaluronic acid, acetyl hyaluronic acid, butyryl hyaluronic acid, aminoacetic acid hyaluronic acid, aminopropionic acid hyaluronic acid, aminobutyric acid hyaluronic acid, and hydrazide hyaluronic acid; however, although the hydrogel has its own composition and characteristics, some of its components are incompatible with embryonic cells. Therefore, there is an urgent need to provide a freezing carrier device that can effectively improve the quality and success rate of embryo freezing. Summary of the Invention To address the above-mentioned issues, the present invention provides a cryo-carrier device for embryo cryopreservation. The device comprises a handheld portion and a freezing portion connected to one end of the handheld portion. The freezing portion is trough-shaped, and the trough is sequentially coated with a first polymer hydrogel coating, a second polymer hydrogel coating, and a third polymer hydrogel coating. The polymer hydrogel coatings of the present invention have excellent liquid absorption capacity and can quickly absorb excess cryosol in embryo cryopreservation, thereby reducing the volume of cryosol, increasing freezing and thawing speeds, reducing ice crystal damage to embryos, and improving embryo survival and conception rates.

[0010] The technical solutions provided by the present invention are as follows: A freezing carrier device for embryo cryopreservation comprises a handheld portion and a freezing portion connected to one end of the handheld portion; The freezing portion is in a groove shape, and the groove of the freezing portion is coated with a first polymer hydrogel coating, a second polymer hydrogel coating, and a third polymer hydrogel coating in sequence; The material of the first polymer hydrogel coating includes component A1 and component B1; the A1 component includes maleic anhydride-polypropylene-polyethylene glycol and six-arm polyethylene glycol-catechol complexed with trivalent iron ions in a volume ratio of 1:1-1.5; the concentration of maleic anhydride-polypropylene-polyethylene glycol is 5-8wt%; the concentration of six-arm polyethylene glycol-catechol complexed with trivalent iron ions is 5-8wt%; and the B1 component is a 5-8wt% polyvinyl alcohol solution; The material of the second polymer hydrogel coating includes components A2 and B2; the A2 component includes thiolated hyaluronic acid and trivalent iron ion complexed six-arm polyethylene glycol-catechol in a volume ratio of 1:1-1.5, wherein the concentration of the thiolated hyaluronic acid is 5-8wt%; the concentration of the trivalent iron ion complexed six-arm polyethylene glycol-catechol is 5-8wt%; and the B2 component is a 5-8wt% polyvinyl alcohol solution. The materials of the third polymer hydrogel coating include components A3 and B3; the A3 component includes maleic anhydride-polypropylene-polyethylene glycol and thiolated hyaluronic acid in a volume ratio of 1:1-1.5; wherein the concentration of maleic anhydride-polypropylene-polyethylene glycol is 5-8wt%, and the concentration of thiolated hyaluronic acid is 5-8wt%; the B3 component is a 5-8wt% polyvinyl alcohol solution.

[0011] The preparation method of the first polymer hydrogel coating comprises the following steps: 1) Mix the materials of component A1 to form an A1 homogeneous solution, then add component B1 and mix thoroughly to form a second polymer hydrogel composition; 2) applying the first polymer hydrogel composition to the groove of the freezing portion; and freeze-drying to form a second polymer hydrogel coating; the freeze-drying process is as follows: freezing at -25 to -30°C for 25 to 30 minutes, and room temperature for 10 to 15 minutes; then freezing at -25 to -30°C for 25 to 30 minutes, and room temperature for 10 to 15 minutes; drying at 55 to 60°C for 20 to 25 minutes; and freeze-drying at -40 to -45°C for 36 to 48 hours.

[0012] The preparation method of the second polymer hydrogel coating comprises the following steps: 1) Mix the materials of component A2 to form an A2 homogeneous solution, then add component B2 and mix thoroughly to form a second polymer hydrogel composition; 2) applying a second polymer hydrogel composition on the surface of the second polymer hydrogel coating layer of the freezing portion; Freeze drying is performed to form a second polymer hydrogel coating; the freeze drying process is as follows: freezing at -25 ~ -30 ° C for 25-30 minutes, and room temperature for 10-15 minutes; then freezing at -25 ~ -30 ° C for 25-30 minutes, and room temperature for 10-15 minutes; drying at 55-60 ° C for 20-25 minutes; and freeze drying at -40 ~ -45 ° C for 36-48 hours.

[0013] The third method for preparing the polymer hydrogel coating comprises the following steps: 1) Mix the materials of component A3 to form an A3 homogeneous solution, then add component B3 and mix thoroughly to form a third polymer hydrogel composition; 2) applying a third polymer hydrogel composition on the surface of the second polymer hydrogel coating layer in the freezing portion; Freeze drying is performed to form a third polymer hydrogel coating; the freeze drying process is as follows: freezing at -25 ~ -30 ° C for 25-30 minutes, and room temperature for 10-15 minutes; then freezing at -25 ~ -30 ° C for 25-30 minutes, and room temperature for 10-15 minutes; drying at 55-60 ° C for 20-25 minutes; and freeze drying at -40 ~ -45 ° C for 36-48 hours.

[0014] The thickness of the first polymer hydrogel coating is 50-80 μm; the thickness of the second polymer hydrogel coating is 60-70 μm; and the thickness of the third polymer hydrogel coating is 50-60 μm.

[0015] The freezing portion of the present invention can absorb 2-10 μL within 30 seconds.

[0016] The material of the freezing carrier body is a material suitable for freezing, specifically including one or more of plastic, glass, metal, and ceramic.

[0017] The shape of the freezing portion is suitable for loading embryos, specifically including one of cylindrical, U-shaped and V-shaped.

[0018] As one embodiment of the present invention, the length of the frozen carrier is 2-5 cm and the diameter is 2-5 mm.

[0019] The length can be adjusted as needed and the diameter can be adjusted according to the size and number of embryos.

[0020] The application of the freezing carrier in animal breeding also falls within the scope of protection of the present invention, specifically the application in embryo cryopreservation.

[0021] When using the cryotransfer, the embryos and protective agent are thoroughly mixed and transferred to the polymer hydrogel coating. After the excess freezing liquid is absorbed by the polymer hydrogel coating, the cryotransfer is immediately placed in liquid nitrogen for freezing and finally stored in a liquid nitrogen tank for subsequent use. When thawing, the cryotransfer is removed and placed in preheated thawing solution. The embryos and protective agent are then removed from the cryovial using a pipette. Embryos can then be cultured or transplanted.

[0022] The working principle of this freezing carrier is: after mixing the embryo and the protective agent, add them dropwise to the freezing part. The polymer hydrogel coating has good liquid absorption (water and organic solvent) performance. They can quickly absorb the excess high-concentration embryo freezing solution in the embryo freezing solution, thereby reducing the chemical toxicity to the embryo, increasing the freezing and thawing speed, reducing the damage to the embryo by ice crystals, and improving the survival rate and conception rate of the embryo.

[0023] Compared with the prior art, the present invention has the following innovations: (1) The material is transparent. Improving the transparency and optical properties of the material makes it have potential application value in the fields of optical sensing and biological imaging. It is convenient for operators to observe the position and morphology of embryos under a microscope, which is conducive to improving the embryo recovery rate.

[0024] (2) Good safety. The hydrogel is formed by freezing cycles. The method is simple and easier to operate. No additional cross-linking agents are required, which reduces the toxicity of the material and is more environmentally friendly. The material used has good biocompatibility and can be widely used in medical materials and tissue engineering.

[0025] (3) The polymer hydrogel coating of the present invention adopts a layered structure (bottom layer, middle layer, surface layer), and each layer achieves performance complementarity through a gradient combination of different functional components: the bottom layer is composed of maleic anhydride-polypropylene-polyethylene glycol (MAPP-PEG) and six-arm polyethylene glycol-catechol (Fe 3+ -6PEG-CA) as the core, using the dual functional properties of MAPP-PEG "mechanical strength and hydrophilicity" (polypropylene skeleton provides mechanical support, polyethylene glycol segments enhance hydrophilicity) and Fe 3+ -6PEG-CA complex cross-linking (catechol group and Fe 3+ Forming coordination bonds and building a stable three-dimensional network), significantly improving the adhesion between the coating and the frozen part substrate; the middle layer introduces thiolated hyaluronic acid (SH-HA) and Fe 3+ -6PEG-CA, through the oxidation of the sulfhydryl group (-SH) of SH-HA in a neutral environment to form a disulfide bond (-SS-), further bridges the polyvinyl alcohol (PVA) molecular chain to optimize the coating's liquid absorption rate; the surface layer uses a combination of MAPP-PEG and SH-HA, taking advantage of the hydrophilicity of MAPP-PEG and the biocompatibility of SH-HA (hyaluronic acid is naturally similar to the components of the extracellular matrix) to improve the biocompatibility of the coating with the embryo.

[0026] (4) The hydrogel system forms a stable structure through multiple cross-linking mechanisms: PVA acts as a basic skeleton to provide mechanical support; Fe 3+The complexation (coordination bond) of -6PEG-CA and the disulfide bonds (covalent bonds) of SH-HA form a dual cross-linked network, avoiding both the weak stability of single cross-linking (such as coordination bonds alone) and the brittleness caused by covalent cross-linking alone. The maleic anhydride groups (-COOH) of MAPP-PEG form secondary bonds (such as hydrogen bonds) with other components through polar interactions, further enhancing the network density. This complex cross-linking mechanism gives the hydrogel both high elasticity and tear resistance, preventing the coating from shedding or cracking during freeze-thaw processes.

[0027] (5) The numerous polar groups such as hydroxyl (-OH) and carboxyl (-COOH) on the hydrogel molecular chains of the present invention specifically bind to water and organic solvents (such as glycerol and ethylene glycol) molecules through hydrogen bonds (chemical affinity); the porous network structure can physically capture liquid molecules (physical capture). This allows the coating to absorb 2-10 μL of freezing solution within 30 seconds (, significantly reducing the residual volume of freezing solution on the embryo surface, thereby accelerating the freezing rate).

[0028] In summary, the present invention achieves a comprehensive improvement in the adhesion, liquid absorption rate, mechanical properties and biocompatibility of the embryo freezing carrier through a multi-component functional gradient design, a composite cross-linked network construction and a dual-effect liquid absorption mechanism. At the same time, it has both simple operation and cost advantages, effectively solving the problems of low embryo freezing efficiency, complex operation and high cost in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings: Figure 1 It is a schematic structural diagram of the freezing carrier device of the present invention, comprising a freezing tube body and a polymer hydrogel coating; Figure 2 This is a schematic diagram of the use process of the refrigeration device of Example 1. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, provide detailed implementation methods and specific operating procedures, and will help those skilled in the art to further understand the present invention. It should be pointed out that the scope of protection of the present invention is not limited to the following embodiments, and a number of adjustments and improvements made under the premise of the concept of the present invention all fall within the scope of protection of the present invention.

[0031] In the following examples: The preparation method of maleic anhydride-polypropylene-polyethylene glycol includes the following steps: Reference: Zhao Xiuqin, Xiang Qiankun. Research on MAPP-g-PEG Compatibilized Polypropylene Wood Plastic Composites. Hubei Agricultural Sciences. 2013; 52(7): 1641-3. 1.1 Synthesis of Maleic Anhydride Grafted Polypropylene Maleic anhydride and polypropylene (PP) were added to a three-necked flask at a 1:1 mass ratio. 100 mL of xylene was added as the solvent and the mixture was heated in a thermostatic oil bath at 50°C with stirring until completely dissolved. The flask was evacuated and filled with nitrogen. Benzoyl peroxide was then added in three 10 mL portions using a thermostatic dropping funnel. The temperature was raised to 120°C and the mixture was refluxed for 150 minutes at a constant temperature and stirring rate to obtain a yellow, transparent solution. Acetone was used as a precipitant to precipitate the solution, which was then filtered, washed, and dried to obtain maleic anhydride-grafted PP.

[0032] 1.2. Preparation of maleic anhydride-polypropylene-polyethylene glycol Weigh 1 g of maleic anhydride-grafted polypropylene and 0.8 g of polyethylene glycol (molecular weight 8000) and place them in a 250 mL round-bottom flask. Add 50 mL of xylene and heat with an electric heating mantle to dissolve the mixture, maintaining the temperature at around 130 °C. Add 0.05 g of p-toluenesulfonic acid and introduce nitrogen. After constant temperature reaction for 2 h, cool to 90 °C and add ethanol to fully precipitate the mixture. Filter the mixture, vacuum filter the precipitate, and then wash it with a large amount of ethanol. Finally, dry it in an oven at 80 °C to obtain maleic anhydride-polypropylene-polyethylene glycol.

[0033] 1.3. Dissolve 50 mg of maleic anhydride-polypropylene-polyethylene glycol prepared in step 1.2 in 1 mL of ultrapure water to prepare a 5 wt% maleic anhydride-polypropylene-polyethylene glycol solution.

[0034] 2. The synthesis of six-arm polyethylene glycol-catechol complexed with trivalent iron ions comprises the following steps: Reference: Kim, S.; Gim, T.; Jeong, Y.; Ryu, JH; Kang, SM FacileConstruction of Robust Multilayered PEG Films on Polydopamine-Coated SolidSubstrates for Marine Antifouling Applications. ACS Appl. Mater. Interfaces2018, 10 (9), 7626–7631. 2.1 Synthesis of 6-arm polyethylene glycol-catechol: Dissolve 6-arm polyethylene glycol amine (1 g) in 10 mL of N-methylpyrrolidone and allow to dissolve completely at 60°C to prepare a 6-arm polyethylene glycol amine solution. Hydroxycitric acid (182.2 mg), Carter's condensation agent (416.3 mg), 1-hydroxybenzotriazole (122.52 mg), and N,N-diisopropylethylamine (139 μl) were dissolved in 5 mL of N-methylpyrrolidone and added to the 6-arm polyethylene glycol amine solution. The resulting mixture was stirred at room temperature for 12 hours and diluted in 15 mL of 1 M hydrochloric acid. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for 48 hours to remove unreacted coupling reagent and hydroxycitric acid. Dialysis (pH = 5) was continued for 3 days, and the 6-arm polyethylene glycol-catechol was obtained after freeze-drying.

[0035] 2.2. The obtained six-arm polyethylene glycol-catechol was immersed in 10 mM ferric chloride ethanol solution for 5 min, and then incubated in 10 mM phosphate buffer (potassium dihydrogen phosphate-potassium hydrogen phosphate buffer pH 7.4) for 1 h to form a stable catechol-Fe 3+ Afterwards, the substrate was rinsed with deionized water and dried under a nitrogen stream to obtain a six-arm polyethylene glycol-catechol complexed with trivalent iron ions.

[0036] 2.3. Dissolve 50 mg of the six-arm polyethylene glycol-catechol prepared in step 2.2 in 1 ml of ultrapure water to prepare a 5 wt% six-arm polyethylene glycol-catechol solution.

[0037] 3. Synthesis of thiolated hyaluronic acid Reference: Lee H, Dellatore SM, Miller WM, Messersmith PB. Mussel-inspired surface chemistry for multifunctional coatings. Science. 2007 Oct 19;318(5849):426-30. doi: 10.1126 / science.1147241. PMID: 17947576; PMCID: PMC2601629. 3.1 The carboxylic acid groups of hyaluronic acid were chemically activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt). Specifically, 2 g of hyaluronic acid was dissolved in 200 mL of ultrapure water, ensuring complete dissolution. 1 g of EDC·HCl and 0.5 g of HOBt were added, and the mixture was stirred for 2 h. The mixture was then combined with cystamine dihydrochloride at pH 6.8 for 12 h. The reaction mixture was dialyzed against distilled deionized water through a 3500 molecular weight cutoff membrane. Next, dithiothreitol was added to raise the pH to 8.5. After stirring the mixture overnight, the pH of the solution was lowered to 3.5, and NaCl (5% w / v) was added. The thiolated hyaluronic acid was then precipitated with an excess of ethanol, redissolved in distilled deionized water, and freeze-dried to obtain the thiolated hyaluronic acid.

[0038] 3.2. Dissolve 50 mg of the thiolated hyaluronic acid prepared in step 3.1 in 1 ml of ultrapure water to prepare a 5 wt% thiolated hyaluronic acid solution.

[0039] Example 1 1. Frozen carrier structure like Figure 1 As shown, this embodiment provides a freezing carrier, including a handheld portion and a freezing portion connected to one end of the handheld portion; the sizes of the handheld portion and the freezing portion of the freezing carrier can be adjusted as needed; in this embodiment, the handheld portion is a cylindrical plastic rod (length 2 cm); the freezing portion is a U-shaped groove (length 3 cm, diameter 3 mm, made of medical-grade plastic).

[0040] The freezing tank is coated with a first polymer hydrogel composition coating, a second polymer hydrogel composition coating and a third polymer hydrogel composition coating in sequence.

[0041] 2. Preparation of polymer hydrogel composition coating: (1) First polymer hydrogel composition coating (bottom layer): Enhanced adhesion Component A: Maleic anhydride-polypropylene-polyethylene glycol 5wt% (MAPP-PEG), six-arm polyethylene glycol complexed with trivalent iron ions-catechol 5wt% (Fe 3+ -6PEG-CA) volume ratio 1:1; Component B: 5wt% polyvinyl alcohol solution; The substances of component A are mixed to form a homogeneous solution A, and then component B is added and mixed thoroughly to form a first polymer hydrogel composition.

[0042] Coating amount: 40μL (thickness 40μm) Freeze drying: Freeze at -30°C for 25 minutes → room temperature for 10 minutes → repeat once → dry at 55°C for 25 minutes → freeze dry at -45°C for 36 hours.

[0043] (2) Second polymer hydrogel composition coating (middle layer): optimize liquid absorption rate Component A: 5wt% thiolated hyaluronic acid (SH-HA), 5wt% six-arm polyethylene glycol-catechol complexed with trivalent iron ions (Fe 3+ -6PEG-CA), volume ratio 1:1; Component B: 5wt% polyvinyl alcohol solution; The substances of component A are mixed to form a homogeneous solution A, and then component B is added and mixed thoroughly to form a first polymer hydrogel composition.

[0044] Coating amount: 60μL (thickness 60μm); Freeze drying: Freeze at -30°C for 25 minutes → room temperature for 10 minutes → repeat once → dry at 55°C for 25 minutes → freeze dry at -45°C for 36 hours.

[0045] (3) Third polymer hydrogel composition coating (surface layer): Improved biocompatibility Component A: 5 wt% MAPP-PEG, 5 wt% SH-HA, volume ratio 1:1; Component B: 5wt% polyvinyl alcohol solution; The substances of component A are mixed to form a homogeneous solution A, and then component B is added and mixed thoroughly to form a first polymer hydrogel composition.

[0046] Coating amount: 50μL (thickness 50μm) Freeze drying: Freeze at -30°C for 25 minutes → room temperature for 10 minutes → repeat once → dry at 55°C for 25 minutes → freeze dry at -45°C for 36 hours.

[0047] Example 2 1. Frozen carrier structure This embodiment provides a cryo-carrier, comprising a handheld portion and a cryo-carrier connected to one end of the handheld portion; the sizes of the handheld portion and the cryo-carrier can be adjusted as needed; in this embodiment, the handheld portion is a cylindrical plastic rod (2.5 cm in length); the cryo-carrier is a U-shaped groove (3.5 cm in length, 3.5 mm in diameter, and made of medical-grade plastic).

[0048] The freezing tank is coated with a first polymer hydrogel composition coating, a second polymer hydrogel composition coating and a third polymer hydrogel composition coating in sequence.

[0049] 2. Preparation of polymer hydrogel composition coating: (1) First polymer hydrogel composition coating (bottom layer): Component A: Maleic anhydride-polypropylene-polyethylene glycol 8wt% (MAPP-PEG), six-arm polyethylene glycol complexed with trivalent iron ions-catechol 8wt% (Fe 3+ -6PEG-CA) volume ratio 1:1.5; Component B: 8wt% polyvinyl alcohol solution; The substances of component A are mixed to form a homogeneous solution A, and then component B is added and mixed thoroughly to form a first polymer hydrogel composition.

[0050] Coating amount: 40μL (thickness 40μm) Freeze drying: Freeze at -25°C for 30 minutes → room temperature for 15 minutes → repeat once → dry at 60°C for 20 minutes → freeze dry at -40°C for 48 hours.

[0051] (2) Second polymer hydrogel composition coating (middle layer): Component A: 8wt% thiolated hyaluronic acid (SH-HA), 8wt% six-arm polyethylene glycol-catechol complexed with trivalent iron ions (Fe 3+ -6PEG-CA), volume ratio 1:1.5; Component B: 8wt% polyvinyl alcohol solution; The substances of component A are mixed to form a homogeneous solution A, and then component B is added and mixed thoroughly to form a first polymer hydrogel composition.

[0052] Coating amount: 70μL (thickness 70μm); Freeze drying: Freeze at -25°C for 30 minutes → room temperature for 15 minutes → repeat once → dry at 60°C for 20 minutes → freeze dry at -40°C for 48 hours.

[0053] (3) Third polymer hydrogel composition coating (surface layer): Component A: 8 wt% MAPP-PEG, 8 wt% SH-HA, volume ratio 1:1.5; Component B: 8wt% polyvinyl alcohol solution; The substances of component A are mixed to form a homogeneous solution A, and then component B is added and mixed thoroughly to form a first polymer hydrogel composition.

[0054] Coating amount: 60μL (thickness 60μm) Freeze drying: Freeze at -25°C for 30 minutes → room temperature for 15 minutes → repeat once → dry at 60°C for 20 minutes → freeze dry at -40°C for 48 hours.

[0055] Comparative Example 1 The difference between this comparative example and Example 1 is that in the preparation of the first polymer hydrogel composition coating: Component A: Maleic anhydride-polypropylene-polyethylene glycol 5wt% (MAPP-PEG), six-arm polyethylene glycol complexed with trivalent iron ions-catechol 5wt% (Fe 3+ -6PEG-CA) volume ratio was adjusted to 5:1.

[0056] Comparative Example 2 The difference between this comparative example and Example 1 is that in the preparation of the first polymer hydrogel composition coating: Component A: Maleic anhydride-polypropylene-polyethylene glycol 5wt% (MAPP-PEG), six-arm polyethylene glycol complexed with trivalent iron ions-catechol 5wt% (Fe 3+ -6PEG-CA) volume ratio was adjusted to 1:5.

[0057] Comparative Example 3 The difference between this comparative example and Example 1 is that the second polymer hydrogel composition coating is omitted, the coating amount of the first polymer hydrogel composition coating is adjusted to 70 μL (thickness 70 μm), and the coating amount of the third polymer hydrogel composition coating is adjusted to 80 μL (thickness 80 μm).

[0058] Comparative Example 4 The difference between this comparative example and Example 1 is that the polymer hydrogel composition coating is a single layer coating; the specific preparation method is: 1. Coating material: Component A is maleic anhydride-polypropylene-polyethylene glycol (MAPP-PEG, 5wt%), six-arm polyethylene glycol-catechol (Fe 3+ -6PEG-CA, 5wt%), thiolated hyaluronic acid (SH-HA, 5wt%), volume ratio 1:1:1; component B is 5wt% polyvinyl alcohol (PVA) solution.

[0059] Preparation of mixed solution: Mix the solutions of component A at a volume ratio of 1:1:1 to form a homogeneous solution A, and then mix with component B at a volume ratio of 1:1.

[0060] Coating and drying: The mixed solution was applied to the surface of the frozen portion at one time, with a coating amount of 150 μL. The freeze-drying process was the same as in Example 1: freezing at -30°C for 25 min → room temperature for 10 min → freezing at -30°C for 25 min → room temperature for 10 min → drying at 55°C for 25 min → freeze-drying at -45°C for 1.5 days to form a single-layer polymer hydrogel coating (about 150 μm thick).

[0061] Effect test 1. Embryo freezing test Embryo freezing tests were performed on the freezing carriers prepared in Example 1 and Comparative Examples 1-4.

[0062] The test method is as follows: 1. Embryo freezing (vitrification) 1.1 Embryo freezing medium: Table 2

[0063] The unit of percentage is mass percentage.

[0064] Dulbecco's phosphate buffer: 20% calf serum, 0.3 mM sodium pyruvate, 3.3 mM glucose, 0.3% bovine serum albumin, glycerol, ethylene glycol 1.2 Freezing procedure: a) Basal medium: Equilibrate embryos for 5-10 minutes at room temperature (24-25°C) b) Vitrification Solution 1: Equilibrate embryos for 5 minutes at room temperature (24-25°C) c) Vitrification Solution 2: Equilibrate embryos for 5 minutes at room temperature (24-25°C) d) Vitrification Solution 3: Equilibrate embryos for 35-45 seconds at room temperature (24-25°C) e) The embryos treated in step d) (with the freezing solution adsorbed thereon) were transferred to the polymer hydrogel coating of the freezing portion of the freezing carriers prepared in the experimental groups of Example 1 and Comparative Examples 1-3. After the excess freezing solution was absorbed by the polymer hydrogel coating, the freezing carriers were immediately placed in liquid nitrogen for freezing.

[0065] 1.3 Embryo cryopreservation: The liquid nitrogen tanks used to store embryos must comply with the requirements of GB / T 5458.

[0066] 2. Embryo Thawing 2.1 Embryo Thawing Solution: Table 3

[0067] 2.2 Thawing procedure: a) Thawing solution 1: Equilibrate embryos for 1 min at 38°C b) Thawing Solution 2: Equilibrate embryos for 5 minutes at room temperature (24-25°C) c) Basal Medium: Equilibrate embryos for 5 minutes at room temperature (24-25°C).

[0068] 3. Embryo Development: Thawed blastocysts were placed in M16 culture medium (Sigma-M7292-100mL) and cultured in a 37°C, 5% CO2 incubator for 48 hours. After 48 hours, the recovery rate and blastocyst hatching rate were recorded and calculated (see Table 4).

[0069] Recovery rate = number of surviving blastocysts / number of thawed blastocysts × 100%; Blastocyst hatching rate = number of hatched blastocysts / total number of blastocysts × 100%.

[0070] Table 4

[0071] 2. Freeze absorption test The frozen carriers prepared in Examples 1-2 and Comparative Examples 1-43 were subjected to a freezing liquid absorption rate test. The test method is as follows: 5 μL of embryo freezing solution (vitrification solution 1 is shown in Table 2) was added dropwise to the polymer coating of the freezing carrier of each embodiment and comparative example, and the absorption time of the freezing solution by the coating was observed. The results are shown in Table 5.

[0072] Table 5

[0073] Example 1 of the present invention (such as Figure 2 ) and 2 polymer hydrogel coating freezing devices can quickly absorb 2-10µL of freezing solution within 30s, and the absorption effect is significantly improved compared with the comparative and control groups.

[0074] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A freezing carrier device for embryo cryopreservation, characterized in that: The invention comprises a handheld portion and a freezing portion connected to one end of the handheld portion; the freezing portion is groove-shaped, and the groove of the freezing portion is sequentially coated with a first polymer hydrogel coating, a second polymer hydrogel coating, and a third polymer hydrogel coating; The first polymer hydrogel coating comprises an A1 component and a B1 component; the A1 component comprises maleic anhydride-polypropylene-polyethylene glycol and six-arm polyethylene glycol-catechol complexed with trivalent iron ions in a volume ratio of 1:1-1.5; the concentration of maleic anhydride-polypropylene-polyethylene glycol is 5-8wt%; the concentration of six-arm polyethylene glycol-catechol complexed with trivalent iron ions is 5-8wt%; and the B1 component is a 5-8wt% polyvinyl alcohol solution. The material of the second polymer hydrogel coating includes component A2 and component B2; the component A2 includes thiolated hyaluronic acid and six-arm polyethylene glycol-catechol complexed with trivalent iron ions in a volume ratio of 1:1-1.5, wherein the concentration of thiolated hyaluronic acid is 5-8wt%; the concentration of six-arm polyethylene glycol-catechol complexed with trivalent iron ions is 5-8wt%; and the component B2 is a 5-8wt% polyvinyl alcohol solution; The materials of the third polymer hydrogel coating include components A3 and B3; the A3 component includes maleic anhydride-polypropylene-polyethylene glycol and thiolated hyaluronic acid in a volume ratio of 1:1-1.5; wherein the concentration of maleic anhydride-polypropylene-polyethylene glycol is 5-8wt%, and the concentration of thiolated hyaluronic acid is 5-8wt%; the B3 component is a 5-8wt% polyvinyl alcohol solution.

2. The frozen carrier device according to claim 1, characterized in that The preparation method of the first polymer hydrogel coating comprises the following steps: 1) Mix the materials of component A1 to form an A1 homogeneous solution, then add component B1 and mix thoroughly to form a second polymer hydrogel composition; 2) applying the first polymer hydrogel composition to the groove of the freezing portion; and freeze-drying to form a second polymer hydrogel coating; the freeze-drying process is as follows: freezing at -25 to -30°C for 25 to 30 minutes, and room temperature for 10 to 15 minutes; then freezing at -25 to -30°C for 25 to 30 minutes, and room temperature for 10 to 15 minutes; drying at 55 to 60°C for 20 to 25 minutes; and freeze-drying at -40 to -45°C for 36 to 48 hours.

3. The frozen carrier device according to claim 1, characterized in that The preparation method of the second polymer hydrogel coating comprises the following steps: 1) Mix the materials of component A2 to form an A2 homogeneous solution, then add component B2 and mix thoroughly to form a second polymer hydrogel composition; 2) applying a second polymer hydrogel composition on the surface of the second polymer hydrogel coating of the freezing portion; Freeze drying is performed to form a second polymer hydrogel coating; the freeze drying process is as follows: freezing at -25 ~ -30 ° C for 25 - 30 minutes, and room temperature for 10 - 15 minutes; then freezing at -25 ~ -30 ° C for 25 - 30 minutes, and room temperature for 10 - 15 minutes; drying at 55 - 60 ° C for 20 - 25 minutes; and freeze drying at -40 ~ -45 ° C for 36 - 48 hours.

4. The frozen carrier device according to claim 1, characterized in that The third method for preparing the polymer hydrogel coating comprises the following steps: 1) Mix the materials of component A3 to form an A3 homogeneous solution, then add component B3 and mix thoroughly to form a third polymer hydrogel composition; 2) applying a third polymer hydrogel composition on the surface of the second polymer hydrogel coating layer in the freezing portion; Freeze drying is performed to form a third polymer hydrogel coating; the freeze drying process is as follows: freezing at -25 to -30°C for 25 to 30 minutes, and room temperature for 10 to 15 minutes; then freezing at -25 to -30°C for 25 to 30 minutes, and room temperature for 10 to 15 minutes; drying at 55 to 60°C for 20 to 25 minutes; and freeze drying at -40 to -45°C for 36 to 48 hours.

5. The frozen carrier device according to claim 1, characterized in that The thickness of the first polymer hydrogel coating is 50-80 μm; the thickness of the second polymer hydrogel coating is 60-70 μm; and the thickness of the third polymer hydrogel coating is 50-60 μm.

6. The frozen carrier device according to claim 1, characterized in that The shape of the freezing part is one of cylindrical, U-shaped and V-shaped.

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