Ovarian tissue cryopreservation solution and use thereof

Abstract

The application discloses an ovary tissue cryopreservation solution and application thereof. 100 mL of the ovary tissue cryopreservation solution is composed of 12-16% dimethyl sulfoxide (v / v), 12-16% ethylene glycol (v / v), 0.5 mol / L sucrose, 10-40 mmol / L L-proline and the rest of a culture medium. The ovary tissue cryopreservation solution in the application adds dimethyl sulfoxide and ethylene glycol as the permeable cryoprotective agent, and adds L-proline and sucrose as the non-permeable cryoprotective agent on the basis, can quickly and efficiently cryopreserve the ovary tissue, effectively improves the survival rate of the follicle after transplantation, reduces the follicle apoptosis, and effectively avoids the possibility of the immune rejection reaction caused by the serum, and is safer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a cryopreservation solution for ovarian tissue and its application in the cryopreservation of ovarian tissue. Background Technology

[0002] Malignant tumors requiring radiotherapy or chemotherapy, hematological diseases requiring radiotherapy, chemotherapy, or bone marrow transplantation, ovarian-related diseases (bilateral ovarian tumors, severe endometriosis, ovarian torsion), and the risk of premature ovarian failure (family history of premature ovarian failure, congenital ovarian hypoplasia, etc.) all have a high potential to impair the fertility of adolescent and reproductive-age women. With the advancement and development of treatments for related benign and malignant diseases, patients' survival times are continuously extending, and the preservation of fertility in female patients before receiving gonadotoxic therapy has become a highly concerned research area. Currently, methods for preserving female fertility mainly include pharmacological ovarian protection (using gonadotropin-releasing hormone analogs), embryo cryopreservation, post-ovarian stimulation oocyte cryopreservation, and ovarian tissue cryopreservation.

[0003] Currently, the American Society for Reproductive Medicine (ASRF) recognizes two clinically applicable methods for female fertility preservation: embryo cryopreservation and post-ovarian stimulation (ROS) oocyte cryopreservation. One condition for performing either embryo cryopreservation or ROS stimulating oocyte cryopreservation is controlled ovarian hyperstimulation followed by oocyte retrieval. Embryo cryopreservation also requires in vitro fertilization and embryo culture. However, for women who cannot delay chemotherapy or radiotherapy, and for pre- or adolescent girls, embryo cryopreservation and ROS stimulating oocyte cryopreservation are difficult to implement. Ovarian tissue cryopreservation is the primary fertility preservation strategy for these groups. Ovarian transplantation after cryopreservation can restore a woman's fertility and allow her to give birth. This strategy has resulted in over 200 live births.

[0004] However, ovarian tissue undergoes permeability and mechanical damage from ice crystals, cytotoxic damage from cryoprotectants, and oxidative stress during cryopreservation and thawing. In recent years, the damage to cells caused by reactive oxygen species (ROS) during cryopreservation has attracted attention. ROS are highly reactive molecules that can oxidize proteins, lipids, and DNA, induce apoptosis and mitochondrial dysfunction, and in ovarian transplantation without surgical vascular anastomosis, over 70% of primordial follicles are lost due to early ischemic injury and free radical oxidative damage between transplantation and revascularization, ultimately affecting the patient's fertility recovery. Summary of the Invention

[0005] In view of this, the present invention needs to provide a cryopreservation solution for ovarian tissue, which can reduce oxidative damage caused during the cryopreservation of ovarian tissue and effectively preserve the reproductive and endocrine functions of ovarian tissue.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a cryopreservation solution for ovarian tissue, wherein each 100 mL of the cryopreservation solution for ovarian tissue consists of 12–16% dimethyl sulfoxide (v / v), 12–16% ethylene glycol (v / v), 0.5 mol / L sucrose, 10–40 mmol / L L-proline, and the remainder of the culture medium.

[0008] This invention innovatively proposes a cryopreservation solution for ovarian tissue. Specifically, it uses methyl sulfoxide and ethylene glycol as permeable cryoprotectants, and adds L-proline and sucrose as non-permeable cryoprotectants. The addition of L-proline, which is safe, non-toxic, and possesses antioxidant properties, is particularly noteworthy. L-proline is a natural amino acid with very high solubility, a neutral pH, and is non-toxic even at high concentrations and high osmotic pressures. Therefore, this application uses L-proline as both a permeable protectant and an antioxidant. Besides providing in vitro permeable protection, it also increases the stability of proteins or membranes during freezing, dehydration, and high temperatures, and possesses the ability to scavenge reactive oxygen species, such as hydroxyl radicals, singlet oxygen, and superoxide anions. Through the above-mentioned formulation optimization, the ovarian cryopreservation solution proposed in this application can effectively reduce oxidative damage during ovarian tissue cryopreservation and improve follicle loss after transplantation.

[0009] Among these, L-proline concentrations of 10–40 mmol / L exhibit significant effects, with an optimal concentration of 20 mmol / L being more preferred.

[0010] In some typical embodiments of the present invention, the cryopreservation solution for ovarian tissue is composed of 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, 20 mmol / L L-proline, and the remainder of the culture medium per 100 mL of ovarian tissue cryopreservation solution.

[0011] It is understood that the culture medium used is not particularly limited and can be any conventional culture medium in the art. In some typical embodiments of the present invention, the culture medium is DMEM medium.

[0012] The present invention further provides the application of the ovarian tissue cryopreservation solution as described above in the cryopreservation of ovarian tissue.

[0013] The present invention further provides a method for cryopreserving ovarian tissue, the method comprising the step of cryopreserving ovarian tissue in the aforementioned ovarian tissue cryopreservation solution.

[0014] This invention also provides a method for cryopreservation of ovarian tissue, comprising the following steps:

[0015] Place the ovarian tissue in a balanced solution for 5–10 minutes;

[0016] Subsequently, the ovarian tissue was placed in the cryopreservation solution for ovarian tissue before transfer for 5 to 10 minutes, then immersed in liquid nitrogen and quickly transferred into a cryopreservation tube.

[0017] The cryopreservation tubes containing ovarian tissue were then stored at -196°C.

[0018] The composition of the equilibration solution can be selected according to the actual situation. In some typical embodiments of the present invention, the composition of the equilibration solution is: each 100 mL of equilibration solution consists of 7.5% dimethyl sulfoxide (v / v), 7.5% ethylene glycol (v / v), 0.5 mol / L sucrose and the remainder of the culture medium.

[0019] In a further embodiment, the cryopreservation method also includes a thawing process, which comprises the following steps:

[0020] Take out the cryopreservation tube containing ovarian tissue, and quickly immerse the ovarian tissue in the thawing solution for 5-10 minutes under a water bath at 37-40°C. Then quickly transfer the ovarian tissue to dilution solution-1 and place it in a water bath at 37-40°C for 5-10 minutes. Then transfer it to dilution solution-2 and washing solution in sequence, for 5-10 minutes each.

[0021] The composition of the resuscitation solution, diluent, and cleaning solution can be selected according to the actual situation. In some typical embodiments of the present invention, the resuscitation solution is composed of 0.5 mol / L sucrose and the remainder of culture medium per 100 mL of resuscitation solution.

[0022] The composition of the diluent-1 is as follows: each 100 mL of diluent-1 consists of 0.25 mol / L sucrose and the remainder of the culture medium;

[0023] The composition of the diluent-2 is as follows: each 100 mL of diluent-2 consists of culture medium.

[0024] In a further embodiment, the washing solution is PBS buffer.

[0025] The beneficial effects of this invention are:

[0026] The ovarian tissue cryopreservation solution of this invention adds dimethyl sulfoxide and ethylene glycol as permeable cryoprotectants, and further adds L-proline and sucrose as non-permeable cryoprotectants. The addition of safe, non-toxic, and antioxidant L-proline reduces reactive oxygen species and free radicals generated during ovarian tissue freezing, mitigating oxidative damage and protecting mitochondrial function. It also reduces the loss of follicles and collagen fibers during ovarian transplantation, thus enabling rapid and efficient cryopreservation of ovarian tissue, effectively improving the survival rate of transplanted follicles and reducing follicle apoptosis.

[0027] Furthermore, the ovarian tissue cryopreservation solution of this invention removes serum or serum substitutes from conventional cryopreservation solutions, thereby effectively avoiding the possibility of serum-induced immune rejection and thus ensuring higher safety. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the ovarian tissue cryopreservation process in a preferred embodiment of the present invention;

[0029] Figure 2 This is an evaluation of the morphology of frozen follicles in Example 2 of the present invention;

[0030] Figure 3 This is a post-implantation follicle morphology assessment in Example 3 of the present invention. Detailed Implementation

[0031] The 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.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Furthermore, unless otherwise specified, methods not specifically describing conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0033] The specific information on the raw materials used in the following examples and comparative examples is as follows:

[0034] The buffer solution was 1×PBS BL302A (Biosharp, China); the culture medium was DMEM basic (1×) 3122701 (Gibco, China); ethylene glycol (Solepro, China); dimethyl sulfoxide (Shanghai Sangon, China); L-proline (Solepro, China); sucrose (Shanghai Sangon, China).

[0035] Example 1: Preparation of cryopreservation solution

[0036] Prepare the cryopreservation solution according to the following formula:

[0037] Equilibration solution: Taking 100mL of equilibration solution as an example, it contains 7.5% dimethyl sulfoxide (v / v), 7.5% ethylene glycol (v / v), 0.5mol / L sucrose, and the balance is DMEM medium.

[0038] Cryopreservation Solution-1: Taking 100 mL of cryopreservation solution-1 as an example, it contains 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, 20 mmol / L L-proline, and the balance is DMEM medium.

[0039] Resuscitation solution: Taking 100mL of resuscitation solution as an example, it contains 0.5mol / L sucrose, and the remainder is DMEM medium.

[0040] Diluent-1: Taking 100mL of Diluent-1 as an example, it contains 0.25mol / L sucrose, and the remainder is DMEM medium.

[0041] Diluent-2: Taking 100mL of diluent-2 as an example, all are DMEM medium.

[0042] Washing solution: PBS buffer.

[0043] Cryopreservation Solution-2: Taking 100 mL of cryopreservation solution-2 as an example, it contains 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, and the balance is DMEM medium.

[0044] Cryopreservation Solution-3: Taking 100 mL of cryopreservation solution-3 as an example, it contains 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, 10 mmol / L L-proline, and the balance is DMEM medium.

[0045] Cryopreservation Solution-4: Taking 100 mL of cryopreservation solution-4 as an example, it contains 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, 30 mmol / L L-proline, and the balance is DMEM medium.

[0046] Cryopreservation Solution-5: Taking 100 mL of cryopreservation solution-5 as an example, it contains 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, 40 mmol / L L-proline, and the balance is DMEM medium.

[0047] Cryopreservation Solution-6: Taking 100 mL of cryopreservation solution-6 as an example, it consists of 12% dimethyl sulfoxide (v / v), 12% ethylene glycol (v / v), 0.5 mol / L sucrose, 20 mmol / L L-proline, and the balance is DMEM medium.

[0048] Cryopreservation Solution-7: Taking 100 mL of Cryopreservation Solution-7 as an example, it consists of 16% dimethyl sulfoxide (v / v), 16% ethylene glycol (v / v), 0.5 mol / L sucrose, 20 mmol / L L-proline, and the remainder is DMEM medium.

[0049] Example 2: Cryopreservation of Ovarian Tissue

[0050] The same ovarian tissue was cryopreserved in cryopreservation solutions -1 to -7 according to the following cryopreservation steps.

[0051] 1. Freezing process

[0052] Ovarian tissue from 6-week-old KM mice (Anhui Medical University Animal Experiment Center) was transferred to a balanced solution and left for 5 minutes. Then, it was transferred to a cryopreservation solution for 5 minutes. Subsequently, the ovarian tissue was immersed in liquid nitrogen and quickly transferred to a cryovial. Finally, the cryovial containing the ovarian tissue was transferred to a liquid nitrogen tank and stored at -196°C.

[0053] 2. Thawing process

[0054] After freezing for one week, the cryovial containing ovarian tissue was removed from the liquid nitrogen tank. The ovarian tissue was quickly immersed in the thawing solution for 5 minutes under a 37°C water bath. Then, the ovarian tissue was quickly transferred to dilution solution-1 and placed in a 37°C water bath for 5 minutes. After that, it was transferred to dilution solution-2 and washing solution for 5 minutes each.

[0055] Example 3: In vitro culture and transplantation of ovaries

[0056] 1. Ovarian in vitro culture

[0057] Prepare complete culture medium (composed of 10% fetal bovine serum + 1% penicillin-streptomycin antibiotics + 89% M2 medium): For example, add 1 mL of fetal bovine serum and 100 μL of penicillin-streptomycin antibiotics to 10 mL of M2 basal culture medium.

[0058] After rewarming, the ovaries were transferred to complete culture medium and cultured in a cell culture incubator at 37°C and 5% CO2 for two hours. The ovaries were then removed in preparation for ovarian transplantation.

[0059] 2. Ovarian transplantation

[0060] Before ovarian transplantation, cultured ovarian tissue was immersed in a penicillin-streptomycin solution for 30 seconds to prevent infection. The ovary was transplanted subrenally into the kidney capsule of mice (female KM mice, 6-8 weeks old, purchased from the Experimental Animal Center of Anhui Medical University), and the skin and muscle were sutured. Pregnant mare serum gonadotropin was injected on day 7 after transplantation, and human chorionic gonadotropin was injected on day 9. The ovary was removed on day 10 after transplantation.

[0061] Example 4: Result Detection and Analysis

[0062] 1. The following functional tests were performed on the frozen and thawed ovarian tissue.

[0063] (1) Lactate dehydrogenase detection: Accurately weigh the thawed ovarian tissue and add nine times the volume of physiological saline. Prepare a 10% tissue homogenate under ice-water bath conditions; centrifuge at 2500 rpm for 10 minutes, collect the supernatant, and dilute it with physiological saline to the optimal sampling concentration. Measure the lactate dehydrogenase content according to the kit (Nanjing Jiancheng) instructions.

[0064] (2) Morphological examination of frozen follicles: Thawed ovarian tissue was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned into 5μm sections, dewaxed with xylene, and dehydrated with graded ethanol. Hematoxylin-eosin staining was performed, and the tissue was observed under a 40x stereomicroscope. Oocytes of normal follicles are spherical with a uniformly distributed granulosa cell layer; abnormal follicles have shrunken oocytes, pyknosis of the nucleus, and separation of granulosa cells from the basement membrane.

[0065] (3) ROS detection in tissue: Dihydroethidium was dissolved in DMSO, the sections were removed, and routinely dewaxed and rehydrated. 100 μL of staining working solution (DHE-1:1000 dilution) was added, and the sections were incubated in a 37℃ incubator in the dark for 20-60 minutes. The nuclei were stained with DAPI for 5 minutes, washed three times with PBS, and observed under a fluorescence microscope (motic, China).

[0066] (4) Antioxidant enzyme content determination: After thawing, the ovarian tissue was prepared into a 10% tissue homogenate using physiological saline and centrifuged at 2500 rpm for 10 min at 4°C. The supernatant was collected for further analysis. The concentrations of superoxide dismutase (T-SOD), catalase (CAT), and malondialdehyde (MDA) in the OTs were determined according to the kit instructions (Nanjing Jiancheng Bioengineering Institute). The absorbance at the corresponding wavelengths was measured using a UV spectrophotometer.

[0067] (5) Post-transfer follicle morphology assessment: On day 10 after transplantation, ovarian tissue was removed from the renal capsule, fixed in 4% paraformaldehyde for 16 hours, embedded in paraffin, and serially sectioned to a thickness of 5 μm. The sections were stained with hematoxylin and eosin (HE) and observed under a stereomicroscope. Follicles with normal morphology were classified as those where the oocyte and nucleus were surrounded by one or more layers of granulosa cells. Abnormal follicles were characterized by cytoplasmic contraction of the oocyte, separation of granulosa cells from the basement membrane, and nuclear pyknosis.

[0068] 2. Results Analysis

[0069] (1) Figure 2 The results show the morphological results of frozen follicles. In the case of cryopreservation solution-2, the follicles in the ovarian tissue were morphologically abnormal, with most granulosa cells separated from the basement membrane and the granulosa cells being scattered. In contrast, although some follicles in the ovarian tissue cryopreservation solution-1 showed morphological abnormalities, most of the oocytes in the follicles were plump, with granulosa cells closely arranged with the oocytes and not separated from the basement membrane.

[0070] (2) Table 1 shows the results of relevant functional tests (ATP (adenosine triphosphate), ROS (reactive oxygen species), LDH (lactate dehydrogenase), MDA (malondialdehyde), CAT (catalase), T-SOD (total superoxide dismutase) content in tissues) (Note: Since the effects of cryopreservation solution-6 and cryopreservation solution-7 are similar to those of cryopreservation solution-1, the test results are not shown in detail).

[0071] Table 1 Results of ovarian tissue function testing

[0072]

[0073] The test results in Table 1 show that the addition of L-proline to cryopreservation solutions 1 through 7 significantly reduced the production of ROS, LDH, and MDA in tissues, while increasing the levels of ATP, CAT, and T-SOD. This indicates that L-proline can significantly reduce the production of reactive oxygen species, alleviate oxidative damage, protect mitochondrial function, and has good biocompatibility. Among these solutions, cryopreservation solution 1, with 20 mM L-proline, showed the most significant effect and represents the optimal addition amount.

[0074] (3) Figure 3 The image shows the morphology of follicles after transplantation. It can be seen that there is no significant difference in the morphology of follicles transplanted from freshly transplanted ovarian tissue and those frozen in cryopreservation solution-1. This indicates that the addition of L-proline can reduce the damage caused by oxidative stress to follicles after ovarian transplantation.

[0075] The above results indicate that adding L-proline, which has good biocompatibility and high antioxidant properties, to the cryopreservation solution for ovarian tissue can not only reduce the production of excessive reactive oxygen species during ovarian tissue freezing, alleviate oxidative damage, and protect mitochondrial function, but also improve follicle loss caused by ischemia-hypoxia damage during ovarian transplantation. Furthermore, it eliminates the need for adding serum or serum substitutes, effectively avoiding immune rejection.

[0076] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0077] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for cryopreservation of ovarian tissue, characterized in that, The cryopreservation method includes the step of cryopreserving ovarian tissue in an ovarian tissue cryopreservation solution; Each 100 mL of ovarian tissue cryopreservation solution consists of 12%–16% dimethyl sulfoxide (v / v), 12%–16% ethylene glycol (v / v), 0.5 mol / L sucrose, 20 mmol / L L-proline, and the remainder of DMEM medium, and does not contain serum or serum substitutes.

2. The cryopreservation method as described in claim 1, characterized in that, Each 100 mL of ovarian tissue cryopreservation solution consists of 15% dimethyl sulfoxide (v / v), 15% ethylene glycol (v / v), 0.5 mol / L sucrose, 20 mmol / L L-proline, and the remainder of DMEM medium, and does not contain serum or serum substitutes.

3. A method for cryopreservation of ovarian tissue, characterized in that, Includes the following steps: Place the ovarian tissue in a balanced solution for 5 to 10 minutes. The ovarian tissue was then transferred to the cryopreservation solution for ovarian tissue as defined in claim 1 or 2 and placed for 5 to 10 minutes. The ovarian tissue was then immersed in liquid nitrogen and quickly transferred to a cryopreservation tube. The cryopreservation tubes containing ovarian tissue were then stored at -196°C.

4. The cryopreservation method for ovarian tissue as described in claim 3, characterized in that, The equilibration solution is composed of 5%-10% dimethyl sulfoxide (v / v), 5%-10% ethylene glycol (v / v), 0.5 mol / L sucrose, and the remainder of the culture medium per 100 mL.

5. The cryopreservation method for ovarian tissue as described in claim 4, characterized in that, Each 100 mL equilibration solution consists of 7.5% dimethyl sulfoxide (v / v), 7.5% ethylene glycol (v / v), 0.5 mol / L sucrose, and the remainder of the culture medium.

6. The cryopreservation method for ovarian tissue as described in claim 3, characterized in that, The cryopreservation method further includes a thawing process, which includes the following steps: Take out the cryopreservation tube containing ovarian tissue, and quickly immerse the ovarian tissue in the thawing solution for 5 min to 10 min under a water bath at 37℃~40℃. Then quickly transfer the ovarian tissue to dilution solution-1 and place it in a water bath at 37℃~40℃ for 5 min to 10 min. Then transfer it to dilution solution-2 and washing solution in sequence, for 5 min to 10 min each.

7. The cryopreservation method for ovarian tissue as described in claim 6, characterized in that, The resuscitation solution is composed of 0.5 mol / L sucrose and the remainder of the culture medium per 100 mL. The composition of the diluent-1 is as follows: each 100 mL of diluent-1 consists of 0.25 mol / L sucrose and the remainder of the culture medium; The composition of the diluent-2 is as follows: each 100 mL of diluent-2 consists of culture medium.

8. The method for cryopreservation of ovarian tissue as described in claim 6, characterized in that, The cleaning solution is PBS buffer.

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