A method for cryopreservation of ovarian tissue and a cutting aid device and application thereof

Abstract

The application discloses a freezing preservation method of ovary tissue, a cutting auxiliary device thereof and application. The application forms multiple open sections by retaining part of medulla tissue and performing regular parallel cutting on the surface of the ovary tissue, increases the exposed area of the tissue without removing the medulla, thereby shortening the diffusion distance of the cryoprotective agent, reducing the penetration deficiency and concentration gradient problems caused by the increase of the tissue thickness, improving the penetration efficiency of the cryoprotective agent, and reducing the freezing damage. The application further provides an auxiliary cutting device. When used, the auxiliary cutting device is covered on the ovary tissue for cutting, so that the cutting process is more standardized and has higher repeatability. The method has the advantages of improving the penetration efficiency of the cryoprotective agent and retaining the integrity of the medulla structure, is helpful to improve the freezing preservation effect of the ovary tissue, and improves the repair of the tissue after thawing and the survival ability after transplantation.

Description

Technical Field

[0001] This invention relates to the field of assisted reproductive technology, and in particular to a method for cryopreservation of ovarian tissue, its cutting aid device, and its application. Background Technology

[0002] Ovarian tissue cryopreservation (OTC) is currently one of the most important technologies for preserving female fertility in clinical practice. It is also the only mature and feasible method for fertility preservation for pre-pubertal girls and women undergoing gonadotoxic treatments such as radiotherapy and chemotherapy. With the improvement of survival rates for cancer patients and the increasing demand for female fertility preservation, the clinical application of ovarian tissue cryopreservation is becoming increasingly widespread.

[0003] Currently, ovarian tissue cryopreservation mainly includes two methods: vitrification and slow-programmed freezing. In existing technologies, to improve the diffusion efficiency of the cryoprotectant (CPA) within the tissue and reduce damage caused by ice crystal formation, the ovarian tissue is typically trimmed into a cortical sheet with a thickness of 1 mm or less before freezing. This method shortens the CPA diffusion path, thereby improving the freezing effect, and is therefore widely used.

[0004] However, this traditional method has significant limitations. First, to prepare thin cortical tissue, most of the ovarian medulla tissue usually needs to be removed. The medulla region is rich in stromal cells, vascular networks, extracellular matrix, and a vascular support microenvironment, playing a crucial role in maintaining tissue structural stability, supporting tissue repair after transplantation, and promoting revascularization. The absence of medulla may affect tissue repair after freezing and long-term survival after transplantation. Second, increasing tissue thickness to preserve the medulla leads to increased diffusion distance of the cryoprotectant, insufficient CPA penetration in the central tissue region, and a significant concentration gradient, thereby increasing the risk of ice crystal formation, osmotic pressure damage, and freeze-thaw damage.

[0005] Therefore, a key technical challenge in the field of ovarian tissue cryopreservation is how to improve the penetration efficiency of cryoprotectants while preserving the medullary structure and its biological functions, thereby balancing cryopreservation effectiveness with the tissue's repair potential after transplantation. Currently, there is no effective solution to this problem. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a method for cryopreservation of ovarian tissue, its cutting auxiliary device, and its application. Compared with the prior art, this invention does not employ the traditional method of preparing simple cortical slices, but instead performs regular parallel cuts on thicker tissue while preserving part of the medullary structure. This allows the tissue to simultaneously possess a higher open exposure area and preserved medullary structure, thereby improving the penetration efficiency of the cryoprotectant.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for cryopreservation of ovarian tissue, the cryopreservation method comprising the following steps: (1) Obtain ovarian tissue containing cortex and medulla; (2) The ovarian tissue is cut in parallel to form multiple incisions, so as to obtain ovarian tissue that retains the cortex and medulla and maintains the overall connection. (3) Place the ovarian tissue processed in step (2) in a cryopreservation solution for cryopreservation.

[0008] In some embodiments of the present invention, the incision includes a deep incision, a shallow incision, and a through incision.

[0009] In some embodiments of the present invention, the penetrating incision completely penetrates the thickness of the ovarian tissue, but the ovarian tissue is not completely severed by the incision and remains connected as a whole.

[0010] In some embodiments of the invention, the deep incision includes an incision that extends from the cortex to the medulla and does not completely penetrate the ovarian tissue.

[0011] In some embodiments of the invention, the shallow incision includes an incision that exists only in the cortical layer.

[0012] In some embodiments of the present invention, the cut is a parallel cut.

[0013] In some embodiments of the present invention, the thickness of the ovarian tissue is 1-5 mm.

[0014] In some embodiments of the present invention, the thickness of the ovarian tissue is 2-4 mm.

[0015] In some embodiments of the present invention, the length of the cut is 1-10 mm.

[0016] In some embodiments of the present invention, the length of the cut is 5-10 mm.

[0017] In some embodiments of the present invention, the distance between two adjacent cuts is 0.5-2 mm.

[0018] In some embodiments of the present invention, the thickness of the medulla in the ovarian tissue is greater than or equal to the thickness of the cortex.

[0019] In some embodiments of the present invention, the thickness of the medulla in the ovarian tissue is 0.5-4 mm.

[0020] In some embodiments of the present invention, the distance between two adjacent incisions is 1 mm. This spacing is designed with reference to the control of tissue thickness during ovarian tissue freezing, that is, to promote the rapid entry of cryoprotectant (CPA) into the tissue by shortening the diffusion distance of the cryoprotectant. The present invention achieves a CPA diffusion advantage similar to that of 1 mm thin tissue by forming parallel through incisions inside thicker tissue, thereby controlling the distance from any point inside the tissue to the nearest open interface to be within about 1 mm, while preserving the medullary structure.

[0021] In some embodiments of the present invention, the cryopreservation solution includes at least one of ethylene glycol, propylene glycol, acetamide, dimethyl sulfoxide, sucrose, and trehalose.

[0022] In some embodiments of the present invention, the cryopreservation solution comprises ethylene glycol and dimethyl sulfoxide.

[0023] In some embodiments of the present invention, the volume percentages of ethylene glycol and dimethyl sulfoxide in the cryopreservation solution are both 10-20%.

[0024] In some embodiments of the present invention, the volume percentages of ethylene glycol and dimethyl sulfoxide in the cryopreservation solution are both 15-20%.

[0025] In some embodiments of the present invention, the cryopreservation includes at least one of vitrification and slow freezing.

[0026] In some embodiments of the present invention, the cutting includes cutting using an ovarian tissue cutting aid; the ovarian tissue cutting aid includes a plate-shaped body with a plurality of parallel slit-shaped grooves on the plate-shaped body; the slit-shaped grooves are used for a cutting blade to pass through, guiding the blade to cut the ovarian tissue placed below the plate-shaped body.

[0027] In some embodiments of the present invention, placing the ovarian tissue treated in step (2) in a cryopreservation solution further includes placing the ovarian tissue treated in step (2) in a balancing solution; the balancing solution includes at least one of ethylene glycol, propylene glycol, acetamide, dimethyl sulfoxide, sucrose and trehalose.

[0028] In some embodiments of the present invention, the equilibration solution includes ethylene glycol and dimethyl sulfoxide.

[0029] In some embodiments of the present invention, the volume percentages of ethylene glycol and dimethyl sulfoxide in the equilibrium solution are both 5-10%.

[0030] A second aspect of the present invention provides an ovarian tissue cutting auxiliary device, the ovarian tissue cutting auxiliary device comprising a plate-shaped body having a plurality of parallel slit-shaped grooves on the plate-shaped body; the slit-shaped grooves are used for a cutting blade to pass through, guiding the blade to cut ovarian tissue placed below the plate-shaped body.

[0031] In some embodiments of the present invention, the length of the slit-shaped channel is 1-10 mm, the width is 0.5-2 mm, and the distance between two adjacent slit-shaped channels is 0.5-2 mm.

[0032] In some embodiments of the present invention, the length of the slit-shaped channel is 1-10 mm, the width is 0.5-2 mm, and the distance between two adjacent slit-shaped channels is 1-2 mm.

[0033] In some embodiments of the present invention, the slit-shaped channels are distributed in an array.

[0034] In some embodiments of the present invention, the slit-shaped channels are arranged in multiple rows and columns in an alternating pattern, and the slits in adjacent rows are staggered in the lateral direction, which is perpendicular to the length direction of the slit-shaped channels.

[0035] A third aspect of the present invention provides the application of the ovarian tissue cutting aid device described above in at least one of ovarian tissue cryopreservation, ovarian tissue culture, and pre-transplantation treatment of ovarian tissue.

[0036] In some embodiments of the invention, the application includes placing the ovarian tissue cutting aid on the ovarian tissue and cutting along a slit-like opening using a blade.

[0037] In some embodiments of the present invention, the application in ovarian tissue cryopreservation includes reducing ovarian tissue cryopreservation damage during ovarian tissue cryopreservation.

[0038] In some embodiments of the present invention, the application includes maintaining the proliferative activity of transplanted tissue cells and the integrity of tissue structure.

[0039] In some embodiments of the invention, the application includes promoting the establishment of a functional vascular network in transplanted tissue.

[0040] The beneficial effects of this invention are: This invention provides a method for cryopreservation of ovarian tissue, along with its cutting aids and applications. By preserving a portion of the medulla, the invention performs regular parallel cuts on the surface of the ovarian tissue, creating multiple open cuts. This increases the exposed tissue area without removing the medulla, thereby shortening the diffusion distance of the cryoprotectant, improving CPA penetration efficiency, and reducing cryoprotective damage.

[0041] This invention provides a medullary-preserving ovarian tissue cutting method that ensures the shortest distance from any point within the tissue to the nearest open cut surface does not exceed approximately 1 mm. This guarantees faster and more uniform penetration of the cryoprotectant into the tissue, reducing inadequate penetration and concentration gradient issues caused by increased tissue thickness, and minimizing tissue damage during freezing and thawing. Furthermore, this invention includes a cutting aid device for performing the aforementioned cutting. In use, the ovarian tissue is placed flat on the operating table, the cutting aid device is placed over the tissue, and the blade is inserted along the pre-set slit of the cutting aid device to complete the cutting, making the cutting process more standardized and reproducible. Further experimental results show that after vitrification, thawing, and in vitro culture for 48 hours, bovine ovarian tissue treated using this method exhibits reduced apoptosis levels and increased stromal cell density. Two weeks after xenografting into nude mice, the expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (ACTA2) in the tissue is enhanced, suggesting that this method helps improve the quality of ovarian tissue cryopreservation and enhances post-transplant tissue viability and repair potential.

[0042] The ovarian tissue cutting method of the present invention, which preserves the medulla, has the advantages of both improving the penetration efficiency of cryoprotectants and preserving the structural integrity of the medulla, which helps to improve the cryopreservation effect of ovarian tissue and improve tissue repair and survival after transplantation. Attached Figure Description

[0043] Figure 1 These are schematic diagrams illustrating the structure and application of the cutting auxiliary device of the present invention. a) is a schematic diagram of the method for cutting medullary ovarian tissue with preservation according to the present invention; b) is a schematic diagram of the slit arrangement structure design of the cutting auxiliary device; c) is a dimensional engineering drawing of the cutting auxiliary device; d) is a physical image of the cutting auxiliary device.

[0044] Figure 2 Schematic diagrams of different ovarian tissue processing methods. a) Ovarian tissue diagram of the 2mm medullary-preserving resection group; b) Comparison of ovarian tissue of the traditional 1mm cortical slice group, the 2mm medullary-preserving group, and the 2mm medullary-preserving resection group; c) Ovarian tissue diagram of the traditional 1mm cortical slice group, the 2mm medullary-preserving group, and the 2mm medullary-preserving resection group after immersion in cryopreservation solution.

[0045] Figure 3Figure 1 shows the results of in vitro culture and functional verification of ovarian tissue after freezing and thawing. Figure 2 shows the TUNEL staining results of bovine ovarian tissue in different treatment groups after vitrification, freezing and thawing and in vitro culture for 48 h, with a scale bar of 200 μm; Figure 3 shows the quantitative analysis of the proportion of TUNEL-positive cells in each group; Figure 4 shows representative HE staining results of different treatment groups after vitrification, freezing and thawing and in vitro culture for 48 h; Figure 5 shows the quantitative statistical analysis of stromal cell density per unit area in each group.

[0046] Figure 4 Images show the results of ACTA2 and PCNA immunofluorescence staining in ovarian tissue before and after transplantation. a) Representative image of ACTA2 immunofluorescence staining in fresh bovine ovarian tissue before transplantation; b) Representative images of PCNA and ACTA2 immunofluorescence staining in nude mice 2 weeks after xenografting of fresh bovine ovarian tissue from different treatment groups (scale bar: 50 μm); c) Quantitative analysis of PCNA fluorescence intensity in the cortical region after transplantation in each treatment group; d) Quantitative analysis of ACTA2 fluorescence intensity in the cortical region after transplantation in each treatment group. Detailed Implementation

[0047] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0048] This invention provides a method for cutting ovarian tissue while preserving the medulla, comprising the following steps: Fresh ovarian tissue was harvested, preserving the cortex and part of the medulla. The tissue was trimmed into regular blocks, approximately 2 mm thick. The trimmed ovarian tissue was then parallelly cut. Cuts were made sequentially along the tissue surface, with adjacent cuts spaced approximately 1 mm apart. The blade was inserted along the tissue thickness, cutting completely through the tissue without preserving the basal connection, thus creating multiple parallel through-cuts. After cutting, the tissue remained intact overall, but multiple open interfaces were created, significantly increasing the contact area between the tissue and the cryoprotectant, and improving the penetration efficiency of CPA within the tissue.

[0049] Traditional ovarian tissue cryopreservation typically controls tissue thickness to within the range of approximately 1-2 mm to reduce the diffusion distance of the cryoprotectant. To further improve cryoprotectant penetration efficiency, some methods tend to prepare thinner cortical tissue. However, excessive thinning may lead to loss of medullary structure. In some embodiments, this invention selects tissue with a thickness of approximately 2 mm as the implementation method, which preserves part of the medullary microenvironment while avoiding excessive tissue thickness that would restrict cryoprotectant diffusion.

[0050] In some embodiments, the incision spacing of the present invention is set to approximately 1 mm. This spacing is designed with reference to the current concept of tissue thickness control in ovarian tissue freezing, which aims to promote rapid CPA entry into the tissue by shortening the diffusion distance of the cryoprotectant. The present invention achieves a CPA diffusion advantage similar to that of 1 mm thin tissue by forming parallel through incisions within thicker tissue, thereby controlling the distance from any point within the tissue to the nearest open interface to within approximately 1 mm, while preserving the medullary structure.

[0051] This invention adjusts the tissue size, number of incisions, incision spacing, cutting depth, and template size according to the size and thickness of the ovarian tissue to be treated, as well as the penetration requirements of the cryoprotectant. Specifically, tissue thickness is primarily used to balance the preservation of medullary structure with CPA diffusion efficiency; incision spacing is mainly used to control the diffusion distance from the tissue interior to the open interface; the number of incisions is determined based on the tissue size and incision spacing; and the template size is adjusted according to the size of the tissue to be treated.

[0052] In some embodiments, the present invention selects a tissue thickness of about 2 mm, an incision spacing of about 1 mm, and a through-cutting method, with the aim of reducing the distance from any location within the tissue to the nearest open interface while preserving the medullary microenvironment, thereby improving the efficiency of CPA entry into the tissue.

[0053] The incision spacing in the method of the present invention is not fixed and can be adjusted according to the tissue thickness. Preferably, the distance from any point inside the tissue to the nearest incision opening surface does not exceed 1 mm. In some embodiments, an incision spacing of about 1 mm is used.

[0054] This invention provides a cutting auxiliary device, such as... Figure 1 As shown, Figure 1 Figure a shows a schematic diagram of a method for cutting ovarian tissue while preserving the medulla. The blue area represents the ovarian tissue, and the yellow lines represent parallel incisions made along the tissue surface. The spacing between these incisions is approximately 1 mm, which is used to increase the open cut surface of the tissue and shorten the penetration distance of the cryoprotectant. Figure 1 Figure b is a schematic diagram of the slit arrangement structure of the cutting auxiliary device. The yellow area represents the through slit opening in the cutting auxiliary device, which allows the blade to be inserted in a preset direction to complete tissue cutting. Adjacent slits are arranged at regular intervals. Figure 1 In the middle, 'c' represents the engineering drawing of the cutting auxiliary device dimensions. Figure 1 The image in the middle (d) shows the actual cutting auxiliary device. This device is a plate-shaped structure, a 50mm × 50mm square structure. The cutting auxiliary device has multiple parallel slit-like openings, which are used to accommodate the blades to pass through.

[0055] The operating method of the cutting auxiliary device is as follows: The ovarian tissue is laid flat on the operating table, and the cutting aid is placed over the tissue, ensuring it adheres to the tissue surface. A blade is then inserted into the tissue along the pre-set slits on the cutting aid to make cuts, ensuring all slits within the covered area are cut, resulting in multiple regularly arranged parallel through-cuts. The processed ovarian tissue can be further used for vitrification, programmed freezing, cryopreservation followed by thawing and culture, xenograft transplantation, or autologous transplantation.

[0056] The present invention will now be described in detail through specific embodiments.

[0057] Example 1 This embodiment provides a method for cutting ovarian tissue while preserving the medulla, and the specific experimental steps are as follows.

[0058] (1) Fresh ovarian tissue was taken from the body. Compared with the traditional method of only preserving the cortical area, this method does not completely remove the medulla. While preserving the ovarian cortical area, it also preserves part of the continuous medullary structure. The ovarian tissue was trimmed into regular tissue blocks with a thickness of about 2 mm, of which the thickness of the cortex and medulla was 1 mm.

[0059] (2) Parallel cutting of the tissue: Place the ovarian tissue flat on the operating table, and then place the cutting aid device above the ovarian tissue so that the cutting aid device is in contact with the surface of the ovarian tissue. Then, use the blade to insert into the tissue one by one along the pre-set slit-like opening on the cutting aid device to complete the cutting. The cutting is carried out sequentially along the surface of the tissue, with an adjacent incision spacing of about 1 mm. The blade cuts along the thickness direction of the ovarian tissue and completely cuts through the tissue thickness without leaving the bottom connection, thereby forming multiple parallel through incisions. All slits in the tissue area are cut, so that the tissue forms multiple regularly arranged parallel through incisions.

[0060] Example 2 This embodiment provides verification of the medullary-preserving ovarian tissue cutting method of the present invention. The bovine ovaries used in the experiment were obtained from healthy adult cows in a slaughterhouse. The specific experimental steps are as follows.

[0061] After extubation, the ovaries were rinsed three times with sterile saline containing penicillin (final concentration 100 U / mL) and streptomycin (final concentration 100 μg / mL), and then immediately transported to the laboratory in Leibovitz's L-15 medium (Thermo Fisher Scientific) at 4°C containing 1% (v / v) penicillin-streptomycin. Tissue processing was completed within 4 hours. Before the experiment, residual fallopian tube tissue was removed, and only the ovarian cortex and part of the medulla were preserved for subsequent experiments.

[0062] Bovine ovarian tissue was prepared into the following groups: Traditional 1mm cortical slice group: Processed ovarian tissue was taken, the cortex was preserved, the medullary structure was removed, and the ovarian tissue was trimmed into regular tissue blocks with a thickness of about 1mm. 2mm medullary preservation group: Processed ovarian tissue was taken, retaining the cortex and part of the medullary structure. The ovarian tissue was trimmed into regular tissue blocks with a thickness of about 2mm, of which the thickness of the cortex and medulla was 1mm each. 2mm medullary-preserving resection group: Processed ovarian tissue was harvested, preserving the cortex and part of the medulla structure. The ovarian tissue was trimmed into regular tissue blocks with a thickness of approximately 2mm, of which the cortex and medulla were both 1mm thick. The medullary-preserving ovarian tissue was resectioned according to the steps in Example 1.

[0063] Subsequently, the ovarian tissues treated in the above ways were vitrified using ethylene glycol (EG) and dimethyl sulfoxide (DMSO) as cryoprotectants, and then cultured in vitro for 48 hours after thawing. The specific steps are as follows: The treated ovarian tissue was placed in a equilibration medium at room temperature for 15 min. The equilibration medium was Leibovitz's L-15 medium containing 7.5% (v / v) ethylene glycol and 7.5% (v / v) dimethyl sulfoxide. Then, it was transferred to a vitrification medium at room temperature for 10 min. The vitrification medium was Leibovitz's L-15 medium containing 15% (v / v) ethylene glycol, 15% (v / v) dimethyl sulfoxide and 0.5 mol / L sucrose. After that, it was quickly placed in liquid nitrogen for preservation. During rewarming, the tissue was placed in the following solutions in sequence: 1.0 mol / L sucrose solution for 10 min; 0.5 mol / L sucrose solution for 10 min; 0.25 mol / L sucrose solution for 10 min; and finally washed twice with basal culture medium (Leibovitz's L-15 medium). After rewarming, the ovarian tissue was placed in a culture medium (DMEM / F12 medium (Thermo Fisher Scientific) supplemented with 1% (v / v) ITS (100×, insulin-transferrin-selenium medium supplement), 10% (v / v) fetal bovine serum (FBS), 100 μg / mL streptomycin and 100 IU / mL penicillin) at 37°C and 5% CO2 for 48 h.

[0064] To evaluate the effects of different treatments on the level of apoptosis in ovarian tissue cells, TUNEL staining was performed on ovarian tissue after 48 h of rewarming culture, and fresh ovarian tissue was used for comparison. The specific steps are as follows: Ovarian tissue was fixed in 4% (w / v) paraformaldehyde, embedded in paraffin, and sectioned. The tissue was then stained using the TUNEL apoptosis detection kit (Shanghai Beyotime Biotechnology Co., Ltd.) according to the manufacturer's instructions: after dewaxing and hydration, sections were permeabilized with proteinase K from the kit, washed three times with PBS buffer, and incubated at 37°C for 60 min in the dark with the TUNEL working solution from the kit. The sections were then washed three more times with PBS buffer, and the cell nuclei were counterstained with DAPI fluorescent dye. After mounting, the sections were observed and images were acquired using a fluorescence microscope. The apoptosis level was evaluated by counting the proportion of TUNEL-positive cells to the total number of cells.

[0065] Simultaneously, ovarian tissue from each group was stained with hematoxylin and eosin (HE) and stromal cell density was calculated. Fresh ovarian tissue was used for comparison. The specific experimental steps were as follows: Ovarian tissue was fixed with 4% (w / v) paraformaldehyde, embedded in paraffin, and serially sectioned (5 μm). The sections were dewaxed with xylene, then hydrated sequentially with 100%, 95%, 85%, and 75% ethanol in a gradient. They were then stained with hematoxylin for 3-10 min and eosin for 30 s-2 min, dehydrated with gradient ethanol, cleared with xylene, mounted with neutral resin, and the entire section was imaged using an optical microscope. ImageJ software was used to analyze the tissue sections, count the total number of nuclei within the tissue area, and calculate the number of nuclei per unit area as the stromal cell density index (cells / mm²). 2 For each tissue sample, multiple intermittent sections were selected for analysis.

[0066] Schematic diagram of ovarian tissue in different group treatments as follows Figure 2 As shown in Figure ac. The TUNEL staining results are as follows. Figure 3 As shown in Figures a and b, the proportion of TUNEL-positive cells in the 2mm medullary-preserving cutting group was significantly lower than that in other cryopreservation groups, demonstrating that the medullary-preserving ovarian tissue cutting method of the present invention can significantly reduce cryopreservation-related damage.

[0067] The results of HE staining experiment are as follows: Figure 3 As shown in C and D, the stromal cell density in the 2 mm medullary-preserving cutting group was higher than that in the other cryopreservation groups, proving that the medullary-preserving ovarian tissue cutting method of the present invention is beneficial to maintaining tissue structural integrity and cell viability.

[0068] Example 3 This embodiment provides xenograft experiments on fresh bovine ovarian tissue from different treatment groups in Example 2. Immunofluorescence was used to detect the expression levels of PCNA (proliferating cell nuclear antigen) and ACTA2 (α-smooth muscle actin). PCNA primarily serves as a marker for detecting the proliferative activity of follicular granulosa cells, used to assess the survival and developmental potential of follicles after cryopreservation or transplantation. ACTA2, on the other hand, serves as a marker for smooth muscle cells, used to label blood vessels and identify membrane cells in ovarian tissue. These two markers provide crucial information for assessing the activity and microenvironment of ovarian tissue from two dimensions: cell proliferation and tissue structure. The specific experimental steps of the xenograft experiment are as follows: Six- to eight-week-old female BALB / c nude mice were selected as recipient animals. Bilateral ovariectomy was performed one week prior to transplantation. After the recipient mice recovered, surgery was performed on the day of transplantation under intraperitoneal anesthesia with sodium pentobarbital. A small incision was made in the lower abdomen to access the peritoneal cavity, and fresh bovine ovarian tissue prepared in different treatment groups as described in Example 2 was implanted into the peritoneal cavity. After two weeks of routine postoperative feeding, the animals were sacrificed and the grafts were removed. The harvested tissues were fixed in 4% (w / v) paraformaldehyde, embedded in paraffin, and serially sectioned for subsequent histological and immunofluorescence analysis to evaluate the effects of different treatments on tissue survival and repair capacity after transplantation. The specific experimental steps for immunofluorescence staining analysis are as follows: After dewaxing and hydration, ovarian tissue sections underwent antigen retrieval. Antigen retrieval was performed using antigen retrieval solution (BioSharp, 50×) diluted to 1× working solution. The sections were placed in the retrieval solution for high-temperature retrieval for 5 min, followed by low-power microwave retrieval for another 5 min, repeated twice. Non-specific binding sites were blocked with 5% (w / v) BSA (bovine serum albumin). Subsequently, PCNA primary antibody (Cell Signaling Technology, 1:200 v / v) and ACTA2 primary antibody (Abcam, 1:100 v / v) were added, and the sections were incubated overnight at 4°C. The next day, the sections were washed three times with PBST buffer (phosphate buffer containing 0.05% (v / v) Tween-20, pH 7.4), and incubated for 1 hour at room temperature in the dark with fluorescently labeled secondary antibodies. The fluorescently labeled secondary antibodies included Alexa Fluor 488-labeled donkey anti-mouse IgG and Alexa Fluor 594-labeled donkey anti-rabbit IgG (both purchased from Abcam, 1:200 v / v). After washing with PBST buffer, the cell nuclei were counterstained with DAPI, and images were acquired and analyzed using a fluorescence microscope.

[0069] Experimental results are as follows Figure 4 As shown, Figure 4 Image a shows a representative image of ACTA2 immunofluorescence staining before transplantation of fresh bovine ovarian tissue. Blue fluorescence represents DAPI staining of cell nuclei, and red fluorescence represents ACTA2. Figure 4 Image a shows a low-power field of view of the whole tissue with a scale bar of 200 μm, and magnified views of the cortex and medulla with a scale bar of 50 μm, used to observe the expression distribution of ACTA2 in different tissue regions; Figure 4 Image b shows representative immunofluorescence staining of PCNA and ACTA2 two weeks after xenografting of fresh bovine ovarian tissue into nude mice from different treatment groups. Blue fluorescence represents DAPI, green fluorescence represents PCNA, and red fluorescence represents ACTA2. The Merge image is a three-channel overlay. Figure 4 In the middle (c), we see the quantitative analysis of PCNA fluorescence intensity in the cortical region after transplantation in each treatment group. Figure 4 In the middle d, it is a quantitative analysis diagram of ACTA2 fluorescence intensity in the cortical region after transplantation in each treatment group.

[0070] The above experimental results show that the expression levels of PCNA and ACTA2 in the 2 mm medullary-preserving resection group in Example 2 were higher than those in the other treatment groups, proving that the medullary-preserving ovarian tissue resection method of the present invention helps to maintain the proliferative activity of transplanted tissue cells and the integrity of tissue structure.

[0071] The above results demonstrate that the medullary-preserving cutting method proposed in this invention not only facilitates cryoprotectant penetration but also improves tissue quality after cryopreservation and thawing, and enhances tissue viability and repair potential after transplantation. This invention shortens the CPA diffusion path by increasing the open cutting surface while preserving part of the medullary structure, thus balancing cryoprotection efficiency with the advantage of medullary preservation.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for cryopreservation of ovarian tissue, characterized in that, The cryopreservation method includes the following steps: (1) Obtain ovarian tissue containing cortex and medulla; (2) The ovarian tissue is cut in parallel to form multiple incisions, so as to obtain ovarian tissue that retains the cortex and medulla and maintains the overall connection. (3) Place the ovarian tissue processed in step (2) in a cryopreservation solution for cryopreservation.

2. The cutting method according to claim 1, characterized in that, The thickness of the ovarian tissue is 1-5 mm.

3. The cutting method according to claim 1, characterized in that, The incision length is 1-10mm.

4. The cutting method according to claim 1, characterized in that, The distance between two adjacent incisions is 0.5-2mm.

5. The cutting method according to claim 1, characterized in that, The cryopreservation solution includes at least one of ethylene glycol, propylene glycol, acetamide, dimethyl sulfoxide, sucrose, and trehalose.

6. The cutting method according to claim 1, characterized in that, The cryopreservation includes at least one of vitrification and slow freezing.

7. The cutting method according to claim 1, characterized in that, The cutting includes cutting using an ovarian tissue cutting aid; the ovarian tissue cutting aid includes a plate-shaped body with multiple parallel slit-shaped grooves on the plate-shaped body; the slit-shaped grooves are used for a cutting blade to pass through, guiding the blade to cut the ovarian tissue placed below the plate-shaped body.

8. An auxiliary device for cutting ovarian tissue, characterized in that, The ovarian tissue cutting auxiliary device includes a plate-shaped body with multiple parallel slit-shaped grooves on the plate-shaped body; the slit-shaped grooves are used for the cutting blade to pass through and guide the blade to cut the ovarian tissue placed below the plate-shaped body.

9. The ovarian tissue cutting auxiliary device according to claim 8, characterized in that, The slit-shaped channel has a length of 1-10 mm, a width of 0.5-2 mm, and a spacing of 0.5-2 mm between two adjacent slit-shaped channels.

10. The use of the ovarian tissue cutting aid device according to any one of claims 8-9 in at least one of ovarian tissue cryopreservation, ovarian tissue culture and ovarian transplantation pretreatment.

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