DMSO-free low-temperature preserving fluid and ovarian tissue cryopreservation method

Abstract

The invention discloses a DMSO (dimethyl sulfoxide)-free low-temperature preserving fluid and an ovarian tissue cryopreservation method, the DMSO-free low-temperature preserving fluid comprises a refrigerating fluid, the refrigerating fluid contains a permeable cryoprotectant, a non-permeable cryoprotectant, a refrigerating base fluid and EGCG (epigallocatechin gallate), and the concentration of the EGCG in the refrigerating fluid is 25-100 [mu] mol / L. The DMSO-free low-temperature preserving fluid can remarkably improve the cryopreservation effect of the ovarian tissue and effectively reduce oxidative damage and mechanical damage of the cryopreserved ovarian tissue. The invention further provides an ovarian tissue cryopreservation method, the DMSO-free low-temperature preservation liquid is combined with hydrogel for packaging, the low-temperature cryopreservation effect of the ovarian tissue is improved, the ovarian function is greatly protected, and the method has practical significance in promoting fertility protection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological material cryopreservation, and in particular relates to a DMSO-free low-temperature preservation solution and an ovarian tissue cryopreservation method. Background Art

[0002] Ovarian tissue cryopreservation is a method of fertility preservation, mainly suitable for prepubertal women and women who cannot delay chemoradiotherapy. This technology can preserve the eggs and the ovarian hormone secretion function by freezing ovarian tissue to avoid irreversible damage to the ovaries caused by disease treatment (such as cancer treatment). Its indications mainly include the protection of fertility and ovarian endocrine function in patients with tumors and non-tumor diseases. In short, ovarian tissue cryopreservation is an important method for preserving fertility and endocrine function.

[0003] Currently, the main methods for cryopreservation of ovarian tissue are slow freezing and vitrification. Rapid freezing (the most ideal state is vitrification) is considered to be more effective than traditional slow freezing due to its advantages such as simple operation. However, in order to prevent osmotic damage, rapid freezing usually requires the use of high-concentration cryoprotective fluid, which can also cause further oxidative stress damage and cryoprotective fluid-mediated toxic damage to the germ cells contained in the ovarian tissue and its cortex.

[0004] Although a variety of cryopreservation solutions have been developed for the cryopreservation of ovarian tissue, oxidative damage and toxic damage to ovarian tissue caused by low temperature and cryoprotectants cannot be avoided, resulting in poor cryopreservation of ovarian tissue. In addition, existing cryopreservation solutions and cryopreservation techniques are mostly for the cryopreservation of partial ovarian tissue or ovarian fragments, while there are few reports on the cryopreservation of complete ovaries (whole ovaries) that are difficult to freeze. Research and development of whole ovary freezing technology is more practical for fertility protection. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a DMSO-free cryopreservation solution, which can improve the antioxidant capacity of frozen ovarian tissue, especially the whole ovary, alleviate the fibrosis process of frozen ovaries, optimize the cryopreservation effect of ovarian tissue, and thus promote the development of low-temperature fertility protection technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides a DMSO-free low-temperature preservation solution, including a freezing solution, wherein the freezing solution contains a permeable cryoprotectant, a non-permeable cryoprotectant, a freezing base solution and EGCG, wherein the concentration of the EGCG in the freezing solution is 25-100 μmol / L.

[0008] In a further embodiment, the permeable cryoprotectant is at least one of ethylene glycol and 1,2-propylene glycol;

[0009] And / or, the non-permeable cryoprotectant is at least one of sucrose and trehalose;

[0010] And / or, the freezing basal liquid is one of DMEM culture medium, TCM199 culture medium and α-MEM culture medium.

[0011] In a further embodiment, in the freezing solution, the volume percentage of the permeable cryoprotectant is 20% to 25%, and the concentration of the non-permeable cryoprotectant is 0.25 to 0.5 mol / L.

[0012] In a further embodiment, the freezing solution contains 10% to 12.5% ​​(v / v) ethylene glycol, 10% to 12.5% ​​(v / v) 1,2-propylene glycol, 0.25 to 0.5 mol / L sucrose and 25 to 100 μmol / L EGCG, and the rest is freezing base solution.

[0013] In a further embodiment, the DMSO-free low-temperature preservation solution also includes a balancing solution.

[0014] In a further embodiment, the balancing solution contains a permeable cryoprotectant, a non-permeable cryoprotectant and a balancing base solution.

[0015] In a further embodiment, the balancing solution contains 5% to 9% (v / v) ethylene glycol, 5% to 9% (v / v) 1,2-propylene glycol and 0.25 to 0.5 mol / L sucrose, and the rest is DMEM culture medium.

[0016] In a further embodiment, the DMSO-free cryopreservation solution also includes several thawing solutions.

[0017] In a further embodiment, the thawing solution contains a non-permeable cryoprotectant and a thawing base solution, and the concentration of the non-permeable cryoprotectant in the thawing solution changes in a gradient.

[0018] The present invention further provides the use of the DMSO-free cryopreservation solution as described above in cryopreservation of ovarian tissue.

[0019] The present invention further provides a method for cryopreservation of ovarian tissue using the DMSO-free cryopreservation solution described above, comprising the following steps:

[0020] S1, loading the ovarian tissue to be frozen with a balancing solution for balancing, and then transferring it into a freezing solution;

[0021] S2, ovarian tissue was then encapsulated using hydrogel;

[0022] S3. Cryopreserving the encapsulated ovarian tissue.

[0023] In a further embodiment, in step S1, the balancing time of the balancing solution is 5 to 8 minutes, and the processing time of the freezing solution is 5 to 8 minutes.

[0024] In a further embodiment, in step S2, the hydrogel is a HA-SA composite hydrogel, and the encapsulation of the ovarian tissue includes the following process:

[0025] S21, immersing the ovarian tissue in a HA-SA solution, wherein the HA-SA solution contains 10-20 mg / mL of SA and 5-10 mg / mL of HA;

[0026] S22, placing the ovarian tissue wrapped with HA-SA solution in a CaCl 2 Cross-linking in solution to achieve encapsulation.

[0027] In a further embodiment, step S3 includes the following process:

[0028] The encapsulated ovarian tissue was directly immersed in liquid nitrogen, rapidly cooled and frozen, and then transferred to a cryopreservation container and stored in liquid nitrogen for at least two weeks.

[0029] In a further embodiment, the ovarian tissue cryopreservation method further includes step S4, thawing, wherein the thawing includes the following process: quickly taking out the cryopreservation container containing the ovarian tissue from the liquid nitrogen, and after taking out the ovarian tissue, transferring it to a thawing solution preheated in a 37°C water bath; soaking the ovarian tissue in multiple steps, each soaking for 5 minutes to 8 minutes, and the concentration of the non-permeable protective agent in the thawing solution used for each soaking gradually decreases until it reaches 0.

[0030] Beneficial effects of the present invention:

[0031] The DMSO-free cryopreservation solution of the present invention has low toxicity and can be used to cryopreserve ovarian tissue, especially whole mouse ovaries, without DMSO. The concentration of the traditional osmotic cryopreservation solution can be reduced to 3.8 mmol / L, thereby improving the antioxidant capacity of the frozen ovaries, avoiding oxidative damage caused by freezing, reducing the degree of fibrosis caused by low temperature, and greatly avoiding cytotoxic damage, thereby improving the effect of low-temperature cryopreservation.

[0032] The ovarian tissue cryopreservation method provided in the present invention adopts a new DMSO-free cryopreservation solution containing EGCG in combination with hydrogel encapsulation technology to achieve cryopreservation of ovarian tissue, especially whole mouse ovaries. The cryopreservation method avoids oxidative damage and mechanical damage, alleviates the fibrosis process of frozen ovarian tissue, and reduces the deposition of extracellular matrix (ECM), improves the cryopreservation effect, and greatly protects ovarian function. The whole mouse ovary preserved by the cryopreservation method can be normally vascularized and restore ovarian endocrine function after freezing and resuscitation. The oocytes obtained from the frozen-thawed ovaries were tested to have mild mitochondrial damage, early apoptosis phenomenon was alleviated, and they had the ability to mature in vitro.

[0033] In addition, the ovarian tissue cryopreservation method of the present invention has the advantages of low toxicity, simple operation, safety and reliability, and can greatly promote the development of fertility preservation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is a schematic diagram of the process of cryopreservation of mouse ovaries using a DMSO-free cryopreservation solution combined with hydrogel encapsulation in a preferred embodiment of the present invention.

[0035] Figure 2 This is a schematic diagram of the cross-linking process and structure of the encapsulated HA-SA hydrogel in Example 2 of the present invention.

[0036] Figure 3 These are the test results of mechanical properties of ovaries after freezing, thawing and rewarming.

[0037] Figure 4 The results of hematoxylin and eosin staining were used to detect the biocompatibility of HA-SA hydrogel.

[0038] Figure 5 To analyze the function of frozen thawed ovarian autologous heterotopic transplantation, Figure 5 A is the result of detecting the follicle density of ovarian transplants using hematoxylin and eosin staining; Figure 5 B: CD31 immunofluorescence staining was used to evaluate the neovascularization of ovarian transplants.

[0039] Figure 6 For the functional analysis of frozen thawed ovarian follicles and oocytes, Figure 6 A is to evaluate the effects of cryopreservation on the ultrastructural morphology of microvilli, granulosa cells and mitochondria under transmission electron microscopy (TEM); Figure 6 B is to evaluate the mitochondrial damage in oocytes isolated from cryopreserved ovaries. The mitochondrial damage indicators selected include ROS, calcium ions, membrane potential and mitochondrial distribution. DETAILED DESCRIPTION

[0040] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] The first aspect of the present invention provides a DMSO-free cryopreservation solution, which includes a freezing solution, a balancing solution and a thawing solution. In the present invention, the composition of the freezing solution is optimized. Specifically, a certain amount of epigallocatechin gallate (EGCG) is added to the freezing solution. EGCG is the main component of green tea extract and has multiple functions such as antibacterial, anti-inflammatory, and anti-tumor. Its special stereochemical structure enables EGCG to quench singlet oxygen and scavenge free radicals. Its strong antioxidant capacity is at least 100 times that of vitamin C, 25 times that of vitamin E, and 2-4 times that of DHA and BHT, and can protect cells and DNA from damage. Therefore, in the present application, the addition of EGCG to the freezing solution reduces the amount of permeable cryoprotectant used, avoids oxidative damage and mechanical damage to ovarian tissue during freezing-thawing, and improves the cryopreservation effect of ovarian tissue.

[0043] In the present invention, the freezing liquid is obtained by adding an appropriate amount of EGCG to the traditional freezing liquid. Specifically, the freezing liquid contains a permeable cryoprotectant, a non-permeable cryoprotectant, a freezing base liquid and EGCG, wherein the concentration of EGCG in the freezing liquid is 25 to 100 μmol / L. When the concentration of EGCG in the freezing liquid is within this range, a significant effect can be exerted. Preferably, the concentration of EGCG in the freezing liquid is 75 μmol / L.

[0044] In a further embodiment, the permeable cryoprotectant, non-permeable cryoprotectant and cryobase solution in the cryoprotectant of the present invention are all common components in the art.

[0045] Among them, the permeable cryoprotectant refers to a class of small molecules that can penetrate the cell membrane and enter the interior of the cell, which can reduce the electrolyte concentration in the unfrozen solution inside and outside the cell during the cell freezing process, thereby reducing the formation of ice crystals and cell damage. The permeable cryoprotectant used in the present invention is a low-toxic small molecule, and specific examples that can be mentioned include at least one of ethylene glycol (EG) and 1,2-propylene glycol (PG), but are not limited to this. It can be understood that the concentration of the permeable cryoprotectant in the freezing solution can be adjusted or optimized according to actual needs. In the present invention, the volume percentage of the permeable cryoprotectant in the freezing solution is 20% to 25%, for example, it can be any value of 20%, 21%, 22%, 24%, and 25%; in some specific embodiments of the present invention, the permeable cryoprotectant used in the freezing solution is a combination of ethylene glycol and 1,2-propylene glycol, and its volume percentage in the freezing solution is 10% to 12.5% ​​ethylene glycol and 10% to 12.5% ​​1,2-propylene glycol, respectively.

[0046] The non-permeable cryoprotectant refers to a type of macromolecular substance that cannot enter the interior of the cell, and is mainly used to adjust the osmotic pressure of the extracellular fluid, thereby protecting the cells from ice crystal formation and dehydration damage during the freezing process. Such macromolecular substances are usually monosaccharides, disaccharides or polymers. In the present invention, specific examples of the non-permeable cryoprotectant include but are not limited to at least one of sucrose and trehalose. It is understood that the concentration of the non-permeable cryoprotectant can be adjusted or optimized according to actual needs. In the present invention, the concentration of the non-permeable cryoprotectant in the freezing solution is 0.25-0.5 mol / L.

[0047] In the present invention, the freezing basal fluid refers to a basal culture fluid that helps maintain cell pH stability and osmotic balance. Specific examples of the freezing basal fluid described in the present invention include one of DMEM culture medium, TCM199 culture medium, and α-MEM culture medium, but are not limited thereto.

[0048] In the present invention, the selection and dosage of conventional components in the freezing solution can be determined by experiments based on the experience of those skilled in the art or known methods, and can be adjusted or optimized as needed. In some specific embodiments of the present invention, the freezing solution is composed of 10% to 12.5% ​​(v / v) ethylene glycol, 10% to 12.5% ​​(v / v) 1,2-propylene glycol, 0.25 to 0.5 mol / L sucrose and 25 to 100 μmol / L EGCG, and the rest are freezing base liquid.

[0049] In a further embodiment, the balancing solution described in the present invention refers to a solution that helps the cells or tissues to be frozen to reach a balanced state, and its composition can adopt the conventional composition in the art. Specifically, the balancing solution in the present invention also adopts a low-toxic composition without DMSO, and the balancing solution contains a permeable cryoprotectant, a non-permeable cryoprotectant and a balanced base solution. The permeable cryoprotectant, the non-permeable cryoprotectant and the balanced base solution here have the same meaning or similar effects as the components in the freezing solution, and are not described one by one here. In some specific embodiments of the present invention, the balancing solution contains 5% to 9% (v / v) ethylene glycol, 5% to 9% (v / v) 1,2-propylene glycol and 0.25 to 0.5 mol / L sucrose, and the rest are DMEM culture medium.

[0050] In a further embodiment, the several thawing solutions described in the present invention refer to solutions for thawing and resuscitating frozen ovarian tissues, and their composition can adopt the conventional composition in the art. Specifically, the thawing solution contains a non-permeable cryoprotectant and a thawing base solution. It is understandable that the concentrations of the non-permeable cryoprotectants in these thawing solutions are different. During specific thawing, the concentrations gradually decrease from a high concentration to 0. It is understandable that the non-permeable cryoprotectants and the thawing base solution in the thawing solution have the same meaning or similar effects as the components in the freezing solution and the balancing solution, and they are not described one by one here. In some specific embodiments of the present invention, the thawing solution includes three types, namely, thawing solution 1 (0.5 mol / L sucrose and DMEM culture medium), thawing solution 2 (0.25 mol / L sucrose and DMEM culture medium) and thawing solution 3 (0 mol / L sucrose and DMEM culture medium).

[0051] The second aspect of the present invention provides the use of the DMSO-free cryopreservation solution as described above in cryopreservation of ovarian tissue. Compared with traditional cryopreservation solutions, the DMSO-free cryopreservation solution can significantly improve the effect of cryopreservation of ovarian tissue.

[0052] The third aspect of the present invention provides a method for cryopreservation of ovarian tissue, using the DMSO-free cryopreservation solution combined with hydrogel encapsulation technology described above. Among them, hydrogel encapsulation is one of the bioengineering technologies for reducing the concentration of protective agents, and can also reduce the formation of extracellular ice, allowing normal material exchange while preventing the entry of immune cells and antibodies, and controlling the rejection problem after ovarian tissue transplantation. Moreover, hydrogels have been proven to be a potential cryoprotective solution. Among the many hydrogels, sodium alginate (SA) is a natural linear polysaccharide extracted from brown algae, which has good biocompatibility, antioxidant properties and other properties, and can be rapidly gelled by divalent cations. SA has also been proven to be a potential cryoprotectant, which can reduce the concentration of traditional protective agents without affecting the vitrification effect; hyaluronic acid (HA) is an anionic glycosaminoglycan, which is an important component of the extracellular matrix in almost all tissues of vertebrates and plays a key role in cell growth, migration and morphogenesis. It initiates and regulates angiogenesis and has been used as a tool to promote angiogenesis in tissue engineering. In the present invention, the cryopreservation effect of ovarian tissue can be significantly improved by combining DMSO-free cryopreservation solution with hydrogel encapsulation technology, which greatly preserves the function of ovarian tissue.

[0053] The method for cryopreservation of ovarian tissue in the present invention comprises the following steps:

[0054] S1, loading the ovarian tissue to be frozen with a balancing solution for balancing, and then transferring it into a freezing solution;

[0055] S2, ovarian tissue was then encapsulated using hydrogel;

[0056] S3. Cryopreserving the encapsulated ovarian tissue.

[0057] In step S1 of the present invention, the balancing time of the balancing solution is 5 to 8 minutes; and the processing time of the freezing solution is 5 to 8 minutes.

[0058] Further, in step S2 of the present invention, the hydrogel is a HA-SA composite hydrogel, and the encapsulation of the ovarian tissue includes the following process:

[0059] S21, immersing the ovarian tissue in a HA-SA solution, wherein the HA-SA solution contains 10-20 mg / mL of SA (for example, any concentration of 10, 12, 14, 15, 16, 18, 20 mg / mL) and 5-10 mg / mL of HA (for example, any concentration of 5, 6, 7, 8, 9, 10 mg / mL);

[0060] S22, placing the ovarian tissue wrapped with HA-SA solution in a CaCl 2 Cross-linking in solution to achieve encapsulation.

[0061] Among them, the HA-SA solution in step S2 can also add an appropriate amount of buffer components and salts that help the hydrogel to be closer to the in vivo environment as needed, such as 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and sodium chloride. In some specific embodiments of the present invention, the HA-SA solution contains 10-25mM 4-hydroxyethylpiperazineethanesulfonic acid and 50-150mM sodium chloride.

[0062] The further step S3 of freezing the encapsulated ovarian tissue comprises the following process:

[0063] The encapsulated ovarian tissue was directly immersed in liquid nitrogen, rapidly cooled and frozen, and then transferred to a cryopreservation container and stored in liquid nitrogen for at least two weeks.

[0064] In a further embodiment, the ovarian tissue cryopreservation method further includes step S4, rewarming; the rewarming includes the following process: quickly taking out the cryopreservation container containing the ovarian tissue from the liquid nitrogen, and after taking out the ovarian tissue, transferring it to a thawing solution preheated in a 37°C water bath; soaking the ovarian tissue in multiple steps, each soaking for 5 minutes to 8 minutes, and the concentration of the non-permeable protective agent in the thawing solution used for each soaking gradually decreases until it is 0.

[0065] It should be noted that the ovarian tissue described herein refers to partial ovarian tissue or ovarian fragments, and can also be a complete ovary, preferably a whole mouse ovary.

[0066] The present invention is described below by means of specific examples. It should be noted that the following specific examples are only for illustrative purposes and do not limit the scope of the present invention in any way. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial channels.

[0067] Example 1 Preparation of DMSO-free cryopreservation solution

[0068] This example provides the preparation and composition of a DMSO-free cryopreservation solution, including a balancing solution, a freezing solution, and a thawing solution, as follows:

[0069] Balanced solution: 7.5% (v / v) ethylene glycol (EG), 7.5% (v / v) 1,2-propylene glycol (PG) and 0.5 mol / L sucrose, and the rest are balanced basic solution DMEM culture medium.

[0070] Freezing medium 1: 12% (v / v) ethylene glycol (EG), 12% (v / v) 1,2-propylene glycol (PG) and 0.5 mol / L sucrose, and the rest are freezing basic liquid DMEM culture medium.

[0071] Freezing medium 2: 12% (v / v) ethylene glycol (EG), 12% (v / v) 1,2-propylene glycol (PG), 0.5 mol / L sucrose and 25 μmol / L ECG, and the rest are freezing basal liquid DMEM culture medium.

[0072] Freezing medium 3: 12% (v / v) ethylene glycol (EG), 12% (v / v) 1,2-propylene glycol (PG), 0.5 mol / L sucrose and 50 μmol / L ECG, and the rest are freezing basal liquid DMEM culture medium.

[0073] Freezing medium 4: 12% (v / v) ethylene glycol (EG), 12% (v / v) 1,2-propylene glycol (PG), 0.5 mol / L sucrose and 75 μmol / L ECG, and the rest are freezing basal liquid DMEM culture medium.

[0074] Freezing medium 5: 12% (v / v) ethylene glycol (EG), 12% (v / v) 1,2-propylene glycol (PG), 0.5 mol / L sucrose and 100 μmol / L ECG, and the rest are freezing basal liquid DMEM culture medium.

[0075] Thawing solution 1: 0.5 mol / L sucrose, and the rest are thawing basic solution DMEM culture medium.

[0076] Thawing solution 2: 0.25 mol / L sucrose, the rest are thawing basic solution DMEM culture medium.

[0077] Thawing medium 3: Thaw only the basal medium DMEM.

[0078] The DMEM culture medium used above is from Gibco, catalog number: C11965500BT.

[0079] Example 2 Cryopreservation of whole mouse ovaries

[0080] In this example, the DMSO-free cryopreservation solution in Example 1 was used to cryopreserve the whole mouse ovary. The specific steps are as follows:

[0081] 1. Ovarian tissue acquisition and preparation

[0082] After removing fat and connective tissue from 6-8 week old mouse ovaries, transfer them to 0.9% saline immediately, rinse twice for about 10 seconds, and then add penicillin (100 μg / mL) and streptomycin (100 μg / mL). After rinsing, immerse the intact mouse ovaries in a 35 mm culture dish containing DMEM medium (Gibco, catalog number: C11965500BT) for subsequent experimental operations.

[0083] 2. Cryopreservation of whole mouse ovaries. For details, please refer to Figure 1 .

[0084] (1) Loading the cryogen

[0085] The whole mouse ovary was loaded with cryoprotective solution in a two-step method. Specifically, the whole mouse ovary was first loaded into the equilibration solution for 5 minutes, and then transferred to the freezing solution for 5 minutes. The excess cryoprotective solution was then removed with sterile medical gauze.

[0086] (2) Hydrogel encapsulation

[0087] The whole mouse ovary was immersed in HA-SA solution, and then the ovary wrapped in HA-SA solution was clamped out with ophthalmic forceps. Gravity caused the whole mouse ovary wrapped in HA-SA solution to fall into 1.5% CaCl 2 Finally, the ovary wrapped with a thin layer of HA-SA hydrogel was taken out and placed on sterile gauze to remove excess CaCl 2 The HA-SA solution was prepared from deionized water and contained 20 mg / mL SA, 5 mg / mL HA, 25 mmol / L HEPES, and 150 mmol / L NaCl. The schematic diagram of the encapsulated HA-SA composite hydrogel can be found in Figure 2 .

[0088] (3) Rapid cryopreservation of ovaries

[0089] Grasp the whole mouse ovary with forceps and immerse the encapsulated and unencapsulated ovaries directly in liquid nitrogen (LN 2 ) to achieve rapid cooling and freezing. Then they were transferred to 1.5 mL cryovials and finally transferred to liquid nitrogen for storage for at least two weeks. Among them, the unencapsulated ovaries were used as controls for subsequent ovarian mechanical properties experiments.

[0090] (4) Thawing of frozen ovaries

[0091] Quickly remove the cryotube containing the ovary from the liquid nitrogen, and transfer the ovary to the thawing solution preheated in a 37°C water bath. Soak the ovary in three steps of thawing solution with a decreasing sucrose concentration, i.e., thawing solution 1, 2, and 3, respectively, for 5 minutes each time to avoid osmotic damage.

[0092] The specific experimental groups are as follows:

[0093] Experimental group 1: balanced solution + freezing solution 2 + thawing solution 1 to 3 + hydrogel encapsulation;

[0094] Experimental group 2: balancing solution + freezing solution 3 + thawing solutions 1 to 3 + hydrogel encapsulation;

[0095] Experimental group 3: balanced solution + freezing solution 4 + thawing solutions 1 to 3 + hydrogel encapsulation;

[0096] Experimental group 4: balanced solution + freezing solution 5 + thawing solutions 1 to 3 + hydrogel encapsulation;

[0097] Control group 1: balanced solution + freezing solution 1 + thawing solutions 1 to 3 + hydrogel encapsulation;

[0098] Control group 2: balanced solution + frozen solution 1 + thawed solutions 1 to 3 + unencapsulated hydrogel;

[0099] Control group 3: balanced solution + frozen solution 2 + thawed solutions 1 to 3 + unencapsulated hydrogel;

[0100] Control group 4: balanced solution + frozen solution 3 + thawed solutions 1 to 3 + unencapsulated hydrogel;

[0101] Control group 5: balanced solution + frozen solution 4 + thawed solutions 1 to 3 + unencapsulated hydrogel;

[0102] Control group 6: balanced solution + frozen solution 5 + thawed solutions 1 to 3 + unencapsulated hydrogel.

[0103] Results and Analysis

[0104] 1. Analysis of ovarian morphology and quality after freezing, thawing and rewarming

[0105] When the ovaries are thawed, some samples are taken for the following tests:

[0106] (1) Mechanical pressure test for mechanical properties analysis [1] (Test conditions: measured at a compression rate of 10 mm / min at 0-90% strain. The strain was calculated based on the deformation generated by the system and the stress-strain curve was plotted. The compression modulus of the ovary was calculated as the compression modulus when the compression strain was 40%).

[0107] The experimental results are shown in Figure 3 , among which, fresh ovaries refer to fresh ovaries that have not been frozen and transplanted in vivo.

[0108] Through the mechanical property test of the ovary, it can be seen that the fresh ovary has the lowest tissue hardness, is the softest, and has the lowest compression elastic modulus. Compared with the fresh group, the mechanical strength of the ovaries treated with frozen resuscitation has increased to varying degrees, which shows that cryopreservation will cause mechanical damage to the ovarian tissue structure, stimulate the deposition of ECM, and increase the hardness of the ovarian tissue.

[0109] Moreover, the mechanical strength of the ovaries was lower when the DMSO-free cryopreservation solution (including the EGCG experimental group) in the present application was used alone than when the traditional cryopreservation solution (excluding the EGCG control group) was used alone, indicating that the DMSO-free cryopreservation solution in the present application has a better protective effect on the ovaries than the traditional cryopreservation solution.

[0110] Furthermore, when the DMSO-free cryopreservation solution of the present invention is combined with the hydrogel encapsulation technology, the mechanical strength of each group of ovaries is lower than the mechanical strength of each group of ovaries when the DMSO-free cryopreservation solution is used alone. This shows that the protective effect of the DMSO-free cryopreservation solution combined with hydrogel encapsulation on the ovaries is better than that of the cryopreservation solution alone. This shows that HA-SA hydrogel encapsulation combined with DMSO-free cryopreservation solution has a better effect on the cryopreservation of ovaries.

[0111] (2) Extracellular matrix analysis by Sirius red staining [2] In this paper, the data of the staining results were quantified and statistically analyzed, and the results are shown in Table 1 - Collagen fibers (%).

[0112] (3) Determination of the content of antioxidant related indicators [1,4] The antioxidant biochemical kits used were from Nanjing Jiancheng Bioengineering Co., Ltd. (T-SOD: A001-3-2, MDA: A003-1-2, CAT: A007-1-1, GSH: A006-2-1, T-AOC: A015-2-1). Immunofluorescence was used to observe the expression of ATP and reactive oxygen species (ROS) in ovarian tissue to evaluate mitochondrial function [1]. TUNEL was used to analyze DNA strand breaks to evaluate cell apoptosis. [1,5] ; Ki-67 proliferation assay [1,5] The test results are shown in Table 1.

[0113] Table 1 Related test results

[0114]

[0115] It can be seen from the test results in Table 1 that cryopreservation will lead to an increase in the content of collagen fibers in the ovaries; excessive production of ROS in the ovaries and destruction of the endogenous antioxidant system (increased MDA content, reduced content or level of T-SOD, CAT, GSH and T-AOC); decreased ATP levels; increased tissue apoptosis and reduced tissue proliferation capacity. The cryopreservation method in this application, that is, EGCG-containing DMSO-free cryopreservation solution combined with hydrogel encapsulation, can significantly reduce the adverse effects of cryopreservation on the ovaries and improve the quality of frozen ovaries. Among them, the ovarian quality of experimental group 3 (75μM EGCG + hydrogel group) is the best, so subsequent experiments will be mainly conducted on experimental group 3.

[0116] 2. Ovarian autotransplantation and functional analysis

[0117] (1) HA-SA composite hydrogel was transplanted into the subcutaneous tissue of 6-8 week-old female KM mice. After 10 days, the main organs (heart, liver, spleen, lung and kidney) were removed and stained with hematoxylin and eosin. [1,3] Analysis of the biocompatibility of the hydrogels.

[0118] Results Figure 4 Group 1 was 6-8 weeks old female KM mice without transplantation treatment, and Group 2 was mice implanted with HA-SA composite hydrogel. It can be seen that there was no inflammatory response in the tissues and organs, indicating that HA-SA composite hydrogel has good biocompatibility and can be used as a good biomaterial in biological experiments.

[0119] (2) After anesthesia, the hair on the back of clean-grade (SPF grade) 6-8 week old female Kunming (KM) mice was shaved and disinfected, and small incisions were made on the skin and peritoneum on both sides of the body at the last third of the spine. After removing the HA-SA hydrogel, the thawed ovaries were immediately transplanted under the renal capsule, and the muscles and skin were sutured. On the 10th and 18th days after transplantation, the ovarian grafts were removed and the blood of the mice was collected by cardiac blood sampling. The ovarian grafts removed on the 18th day after transplantation were fixed with 4% paraformaldehyde overnight at room temperature and embedded in paraffin for the following analysis: Follicle density analysis by hematoxylin and eosin staining [1,6] ; Immunofluorescence staining to observe the expression of CD31 in ovarian transplants and evaluate angiogenesis [1] .

[0120] Results Figure 5 Among them, the fresh group ovaries refer to fresh ovaries that have not been frozen and have not been transplanted in vivo; the fresh transplanted group ovaries refer to fresh ovaries that have not been frozen but have been transplanted in vivo.

[0121] in, Figure 5 The results of hematoxylin and eosin staining in A showed that on the 18th day after transplantation, normal follicles and new small blood vessels were observed in all groups. Compared with the ovaries in the fresh group, the ovaries in the fresh transplant group and the experimental group 3 (75μM EGCG+hydrogel group) had fewer follicles. Although the follicle density of the ovaries in the experimental group 3 was slightly lower than that in the fresh transplant group, there was no statistical difference between the two groups (p>0.05). Figure 5 The results of CD31 immunofluorescence staining in B showed that there was no statistical difference in the number of new blood vessels between the ovaries of the fresh transplantation group and those of the experimental group 3 (p>0.05).

[0122] It can be concluded from the above experimental results that the cryopreservation method provided in the present application can alleviate the loss of follicles in ovarian transplants and reduce ischemia-reperfusion injury (IRI).

[0123] 3. Follicle and oocyte function testing

[0124] (1) Follicles obtained from frozen-thawed ovaries were used to evaluate the ultrastructure of microvilli, granulosa cells, mitochondria, etc. using transmission electron microscopy [1] .

[0125] (2) The oocytes obtained from frozen-thawed ovaries were used to observe the ROS content in the oocytes using fluorescent staining [6] , calcium level [6,8] , membrane potential level [9] The distribution of mitochondria [9] To assess the degree of mitochondrial damage; Analysis of early apoptosis [8] ; In vitro maturation capacity analysis [7] .

[0126] Results Figure 6 and Table 2.

[0127] pass Figure 6 A It can be seen that cryopreservation leads to a decrease in the abundance of microvilli, abnormal shapes of some microvilli, and loose connection structures between oocytes and granulosa cells. Compared with the fresh group, granulosa cells after freezing-thawing were damaged, including: cell membrane damage, nuclear condensation, and the appearance of chromatin clumps. Organelles were unevenly distributed, and more autophagic vesicles were produced. Cryoinjury can also cause mitochondrial morphology swelling, mitochondrial outer membrane rupture, and reduced or even disappearance of mitochondrial cristae. Some mitochondrial cristae expanded and matrix electron density increased. Ovarian ultrastructure showed that the number of microvilli in the ovaries of the fresh group was abundant, and the space around the follicles was normal; the granulosa cells had the most intact morphology, and their cell membranes were intact and smooth; the morphology and distribution of organelles such as mitochondria, Golgi bodies, and smooth endoplasmic reticulum were normal. Compared with the control group 1 (0μM EGCG+hydrogel group), the ultrastructure of microvilli, granulosa cells, and mitochondria in the experimental group 3 (75μM EGCG+hydrogel group) was improved.

[0128] This indicates that the cryopreservation method provided in the present application can reduce the damage caused by cryopreservation to granulosa cells and their mitochondria, thereby protecting the ultrastructure of PAFs.

[0129] By detecting ROS, calcium ion levels, MMP and mitochondrial distribution in oocytes, we determined whether our protocol could reduce the damage caused by cryopreservation to oocyte mitochondria ( Figure 6B). It can be seen that the fluorescence intensity of ROS and calcium ions in the fresh group oocytes is the lowest, and MMP is the highest. Its mitochondria are evenly distributed in the cytoplasm, or aggregated around the nucleus. During the cryopreservation process, the mitochondrial function is damaged, which can also lead to abnormal distribution of mitochondria, showing discontinuous scattered distribution, abnormal aggregation, and missing. The cryopreservation method provided in this application can reduce the accumulation of ROS and reduce Ca 2+ Release, alleviate the decline of MMP and alleviate the abnormal distribution of mitochondria.

[0130] Table 2 Related test results

[0131]

[0132] It can be seen from the test results in Table 2 that cryopreservation will increase the early apoptosis rate of oocytes in the ovaries, and lead to a decrease in the in vitro maturation ability of immature oocytes. However, the cryopreservation method in this application, that is, the DMSO-free cryopreservation solution containing EGCG combined with hydrogel encapsulation, can significantly reduce the adverse effects of cryopreservation on oocytes and improve the quality of immature oocytes retained in frozen ovaries.

[0133] The test, detection and analysis methods involved in the results and analysis are from the following sources:

[0134] 1. Zhang, L., et al., Static magnetic field assisted thawing improves cryopreservation of mouse whole ovaries. Bioeng Transl Med, 2024.9(1):p.e10613.

[0135] 2.Lattouf,R.,et al.,Picrosirius Red Staining:A Useful Tool toAppraise Collagen Networks in Normal and Pathological Tissues.Journal ofHistochemistry&Cytochemistry,2014.62(10):p.751-758.

[0136] 3. Liu, XC, et al., Antioxidative effect of melatonin on cryopreservedovarian tissue in mice. Cryobiology, 2020.96: p.99-105.

[0137] 4.Sun,T.C.,et al.,Melatonin Inhibits Oxidative Stress and Apoptosisin Cryopreserved Ovarian Tissues via Nrf2 / HO-1Signaling Pathway.Front MolBiosci,2020.7:p.163.

[0138] 5.Wang,J.,et al.,Effects of AavLEA1 Protein on Mouse Ovarian TissueCryopreservation by Vitrification.Biopreserv Biobank,2022.20(2):p.168-175.

[0139] 6.Wu,Q.,et al.,Adverse effects of ovarian cryopreservation and auto-transplantation on ovarian grafts and quality of produced oocytes in a mousemodel.Clin Sci(Lond),2023.137(20):p.1577-1591.

[0140] 7.Wang,D.,et al.,Effect of resveratrol on mouse ovarian vitrificationand transplantation.Reprod Biol Endocrinol,2021.19(1):p.54.

[0141] 8.Deng,D.,et al.,Effects of meiotic stage-specific oocytevitrification on mouse oocyte quality and developmental competence.Frontiersin Endocrinology,2023.14.

[0142] 9. Liu, YJ, et al., Melatonin maintains mitochondrial membrane potential and decreases excessive intracellular Ca+ levels in immature humanoocytes. Life Sciences, 2019.235.

[0143] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A DMSO-free cryopreservation solution, comprising a freezing solution, characterized in that: The freezing solution contains a permeable cryoprotectant, a non-permeable cryoprotectant, a freezing base solution and EGCG, wherein the concentration of the EGCG in the freezing solution is 25 to 100 μmol / L.

2. The DMSO-free cryopreservation solution according to claim 1, characterized in that The permeable cryoprotectant is at least one of ethylene glycol and 1,2-propylene glycol; And / or, the non-permeable cryoprotectant is at least one of sucrose and trehalose; And / or, the freezing basal liquid is one of DMEM culture medium, TCM199 culture medium and α-MEM culture medium.

3. The DMSO-free cryopreservation solution according to claim 1, characterized in that In the freezing solution, the volume percentage of the permeable cryoprotectant is 20% to 25%, and the concentration of the non-permeable cryoprotectant is 0.25 to 0.5 mol / L; Preferably, the freezing solution contains 10% to 12.5% ​​(v / v) ethylene glycol, 10% to 12.5% ​​(v / v) 1,2-propylene glycol, 0.25 to 0.5 mol / L sucrose and 25 to 100 μmol / L EGCG, and the rest is freezing base solution.

4. The DMSO-free cryopreservation solution according to claim 1, characterized in that The DMSO-free cryopreservation solution also includes a balance solution; Preferably, the balancing solution contains a permeable cryoprotectant, a non-permeable cryoprotectant and a balancing base solution; Preferably, the balancing solution contains 5% to 9% (v / v) ethylene glycol, 5% to 9% (v / v) 1,2-propylene glycol and 0.25 to 0.5 mol / L sucrose, and the rest is DMEM culture medium.

5. The DMSO-free cryopreservation solution according to claim 1, characterized in that The DMSO-free cryopreservation solution also includes several thawing solutions; Preferably, the thawing solution contains a non-permeable cryoprotectant and a thawing base solution, and the concentration of the non-permeable cryoprotectant in the thawing solution changes in a gradient.

6. Use of the DMSO-free cryopreservation solution as described in any one of claims 1 to 5 in cryopreservation of ovarian tissue.

7. A method for cryopreservation of ovarian tissue, characterized in that: The method is carried out using the DMSO-free cryopreservation solution according to any one of claims 1 to 5, comprising the following steps: S1, loading the ovarian tissue to be frozen with a balancing solution for balancing, and then transferring it into a freezing solution; S2, ovarian tissue was then encapsulated using hydrogel; S3, freezing the encapsulated ovarian tissue; Preferably, in step S1, the balancing time of the balancing solution is 5 to 8 minutes, and the processing time of the freezing solution is 5 to 8 minutes.

8. The method for cryopreservation of ovarian tissue according to claim 7, characterized in that: In step S2, the hydrogel is a HA-SA composite hydrogel, and the encapsulation of the ovarian tissue includes the following process: S21, immersing the ovarian tissue in a HA-SA solution, wherein the HA-SA solution contains 10-20 mg / mL of SA and 5-10 mg / mL of HA; S22. The ovarian tissue wrapped with the HA-SA solution is placed in a CaCl2 solution with a mass content of 1% to 2% for cross-linking to achieve encapsulation.

9. The method for cryopreservation of ovarian tissue according to claim 7, characterized in that: The step S3 includes the following process: The encapsulated ovarian tissue was directly immersed in liquid nitrogen, rapidly cooled and frozen, and then transferred to a cryopreservation container and stored in liquid nitrogen for at least two weeks.

10. The method for cryopreservation of ovarian tissue according to any one of claims 7 to 9, characterized in that: The method further includes step S4, rewarming; the rewarming includes the following process: quickly taking out the cryopreservation container containing the ovarian tissue from the liquid nitrogen, taking out the ovarian tissue, and transferring it to a thawing solution preheated in a 37° C. water bath; The ovarian tissue is immersed in multiple steps, each time for 5 minutes to 8 minutes, and the concentration of the non-permeable protective agent in the thawing solution used for each immersion gradually decreases until it reaches 0.

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