Culture method for promoting structural maturation of testicular tissues in vitro and application

Abstract

The invention belongs to the field of biological tissue engineering, and discloses a culture method for promoting testicular tissue structural maturation in vitro and application. The method comprises the following steps: cutting a testicular tissue into blocky fragments with the volume of 0.5-1 mm; a gas-liquid interface culture mode is adopted, the lower portion of a pre-balanced 1.5% sepharose gel cubic support is soaked in a basic culture medium, the upper portion of the pre-balanced 1.5% sepharose gel cubic support makes contact with air, and then tissue blocks are placed on the support; the method comprises the following steps: adding a WNT5A pathway agonist Foxy-5 with the final concentration of 200-400 [mu] M into a basic culture medium containing alpha MEM, 10% KSR and 1% P / S, and continuously culturing for 11 days under the conditions of 34 DEG C and 5% COs. According to the method, a WNT5A signal channel is continuously activated by using Foxy-5 through gas-liquid interface culture, so that the polarity of a supporting cell can be effectively maintained, and the integrity of a blood-testis barrier (BTB) and the morphology regularization of a seminiferous tubule are promoted. According to the invention, a standardized and repeatable testicular tissue in-vitro maturation platform is established, and the method can be used for research in fields related to reproductive toxicity evaluation, drug permeability test and spermatogenesis microenvironment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological tissue engineering, and specifically relates to a culture method for promoting structural maturation of testicular tissue in vitro and application thereof. BACKGROUND

[0002] The global incidence of male infertility continues to rise, and has become a key factor affecting about 15% of couples of childbearing age. The latest statistics from the World Health Organization (WHO) show that male infertility is widespread globally, seriously affecting human reproductive health. Tradically, the diagnosis and treatment of male infertility focuses on clinical examination and sperm quality analysis, but with the deepening understanding of the male reproductive system, the in vitro reconstruction of the spermatogenic microenvironment has gradually become an important research direction for solving the mechanism of male infertility.

[0003] One of the core features of the spermatogenic microenvironment is the support cells such as Sertoli cells in the testis, which provide necessary survival support for germ cells by constructing the blood-testis barrier (BTB), secreting nutrients, maintaining immune privilege, etc. The functional maturity of support cells plays a decisive role in the success or failure of spermatogenesis, especially in the establishment of support cell polarity and the formation of tight junctions, which affects the integrity of the blood-testis barrier (BTB) and the stability of the environment in the seminiferous tubule.

[0004] However, the existing in vitro culture system of testicular tissue faces many challenges, especially in maintaining the polarity of support cells and restoring their function. The traditional three-dimensional culture system has significant limitations: within 72 hours of culture, more than 80% of support cells lose their polarity structure, leading to the collapse of the integrity of the blood-testis barrier (BTB), large-scale apoptosis of germ cells due to insufficient secretion of nutrients, and ultimately loss of differentiation potential. This phenomenon directly indicates that the existing culture protocol cannot effectively simulate the in vivo microenvironment, and the function of support cells cannot be fully restored in the ex vivo state, affecting the feasibility of related research and clinical applications.

[0005] In recent years, WNT5A signaling pathway plays an important role in tissue development and cell polarity maintenance, especially in the process of supporting cell polarity establishment and tight junction formation. WNT5A drives the reorganization of supporting cell cytoskeleton and intercellular tight junction in vivo by activating the non-canonical WNT pathway. However, although WNT5A plays an important role in the establishment of supporting cell polarity, its activity in in vitro culture is limited, mainly manifested as the half-life of natural WNT5A protein in the culture medium is less than 6 hours, and the lack of mechanical stimulation to guide the positioning of planar cell polarity complex (PCPs). This makes it difficult for the existing culture system to effectively activate the WNT5A signaling pathway, thereby affecting the polarity of supporting cells and the recovery of the blood-testis barrier (BTB).

[0006] Therefore, how to promote the maturation of supporting cells through effective means, especially by activating the WNT5A pathway and overcoming the time and structural limitations existing in in vitro culture, has become a key problem in current research. Solving this bottleneck not only helps to improve the in vitro culture system of testicular tissue, but also provides an innovative research platform for the field of reproductive toxicity evaluation, drug permeability testing, etc.

[0007] Therefore, the present application provides a culture method for promoting structural maturation of testicular tissue in vitro and application, to solve the problems raised in the above background. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a culture method for promoting structural maturation of testicular tissue in vitro and application, to solve the problems raised in the above background.

[0009] To achieve the above purpose, the present application is realized by the following technical scheme: In a first aspect, the present application provides a culture method for promoting structural maturation of testicular tissue in vitro, comprising: (1) cutting the testicular tissue into block-shaped tissue fragments; (2) placing the block-shaped tissue fragments on an agarose gel scaffold, using air-liquid interfacial culture method, wherein the liquid surface of the culture medium is lower than the upper surface of the scaffold, to ensure that the tissue block is in contact with air above and is immersed in the culture medium below; (3) adding WNT5A pathway agonist Foxy-5 to the culture medium, and maintaining the presence of Foxy-5 during the culture period.

[0010] Further, the volume of the block tissue fragment is 0.5-1 mm3; the mass fraction of agarose in the agarose gel scaffold is 1.5% (w / v); the size of the agarose gel scaffold is a 1*1*3 mm3 cube structure, which needs to be pre-equilibrated before use, and the pre-equilibration time is 12-36 hours; 2-3 block tissue fragments are placed on each agarose gel scaffold during use.

[0011] Further, the gas-liquid interface culture meets the following conditions: the liquid surface of the culture medium is lower than the upper surface of the agarose gel scaffold; the culture conditions meet the following conditions: temperature 34℃, humidity 95%, CO2 concentration 5%; the culture medium is a basic medium, including αMEM, KSR and penicillin and streptomycin complex antibiotic solution (P / S), wherein the volume fraction of KSR is 10%, and the volume fraction of the penicillin and streptomycin complex antibiotic solution (P / S) is 1%.

[0012] Further, the final concentration of Foxy-5 in the culture medium is 200-400 μM; and the culture period is 11 days.

[0013] Further, the determination index of structural maturation includes at least one of the following: (1) The degree of expansion of the seminiferous tubule: the seminiferous tubule morphology is regular and the lumen radius increases after culture; (2) The integrity of the blood-testis barrier (BTB): the expression of the blood-testis barrier (BTB) marker CDH2 is limited between the Sertoli cells; (3) Sertoli cell polarity: the Sertoli cell nucleus marker SOX9 labeled nucleus is limited to the base of the seminiferous tubule.

[0014] In another aspect, the application provides an application of an in vitro culture method for promoting structural maturation of testicular tissue, which includes: The in vitro culture method for promoting structural maturation of testicular tissue can be applied to establish a standardized testicular tissue in vitro maturation platform, and provide a standardized platform for reproductive toxicity testing and research in the field related to spermatogenic microenvironment.

[0015] The application provides an in vitro culture method for promoting structural maturation of testicular tissue and its application, which has the following beneficial effects: 1. Significantly promote the testicular tissue structure and function maturation, break through the existing in vitro culture bottleneck. The present application effectively simulates the mechanical microenvironment and signal pathway activation conditions of in vivo testicular tissue by combining agarose gel scaffold with gas-liquid interface culture and continuously adding WNT5A pathway agonist Foxy-5. Compared with traditional three-dimensional culture system, this method can significantly maintain the polarity of Sertoli cells and avoid the problem of losing polarity and blood-testis barrier (BTB) collapse in a short period. Experiments show that this method can promote the regularity of seminiferous tubule morphology, tubular cavity expansion, and accurate expression and positioning of blood-testis barrier (BTB) marker CDH2, and Sertoli cell nuclear marker SOX9 is positioned at the basal part, so as to be closer to the in vivo mature state in structure.

[0016] 2. Solve the time effectiveness and mechanical guidance problem of WNT5A signal pathway in vitro. The natural WNT5A protein has a half-life of less than 6 hours in the culture medium, and lacks mechanical guidance in vitro, making it difficult to effectively activate the planar cell polarity (PCP) pathway. The present application uses Foxy-5 agonist with high stability and long-lasting activity, and provides appropriate physical support and medium penetration through agarose scaffold in gas-liquid interface culture, which guides the correct positioning of PCP complex, thereby continuously activating WNT5A signal, promoting Sertoli cell cytoskeleton reorganization and tight junction formation.

[0017] 3. Establish a standardized and repeatable in vitro maturation platform for testicular tissue. By optimizing the key parameters such as tissue fragment size, scaffold physicochemical properties, culture conditions and Foxy-5 concentration, the present application forms a stable and controllable culture system. The system supports the testicular tissue to maintain structural and functional integrity for up to 11 days of culture period, providing a highly consistent experimental platform for subsequent research. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The results of the morphological changes of the testicular tissue of the control group and the Foxy-5 treated group of mice in Example 1; Figure 2 The results of the polarity and blood-testis barrier changes of the testicular tissue of the control group and the Foxy-5 treated group of mice in Example 1. DETAILED DESCRIPTION

[0019] In order for those skilled in the art to understand the present application, the technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, other embodiments obtained by those skilled in the art without creative labor should be within the scope of protection of the present application.

[0020] The present invention will now be described in detail with reference to the accompanying drawings: Example 1, referring to Figure 1 and Figure 2 This is a method for promoting the structural maturation of testicular tissue in vitro, and the specific implementation method is as follows: 1. Materials and Reagents (1) Experimental animals: 5-week-old male C57BL / 6J mice, purchased from Beijing Vital River Laboratory Animal Co., Ltd.

[0021] (2) Main reagents: αMEM, KSR, penicillin and streptomycin compound antibiotic solution (P / S), agarose, WNT5A pathway agonist Foxy-5, PBS, 4% paraformaldehyde, paraffin embedding consumables, DAPI.

[0022] (3) Antibodies: SOX9 primary antibody, CDH2 primary antibody and corresponding fluorescent secondary antibody (prepared according to conventional immunofluorescence reagents).

[0023] (4) Equipment: Clean bench, CO2 incubator, confocal microscope, paraffin sectioner.

[0024] 2. Methods 2.1 Group Design Testicular tissue blocks from the same batch of animals were randomly divided into two groups: Control group: No Foxy-5 added; Foxy-5 treatment group: 400 μM Foxy-5 was added to the culture medium.

[0025] Three mice were used in each group, and three testicular tissue fragments were taken from each group at each time point for histological and immunofluorescence detection.

[0026] 2.2 Preparation and pre-equilibration of agarose gel scaffolds (1) Weigh out agarose and add it to ultrapure water at 1.5% (w / v). Heat until completely dissolved and clear.

[0027] (2) Pour the hot agarose solution into a 10 cm petri dish to form a gel layer 3–5 mm thick, and allow it to cool and solidify naturally.

[0028] (3) Under sterile conditions, the gel was cut into 1×1×3 mm³ cubic scaffolds using a sterile scalpel.

[0029] (4) The scaffold is placed in a complete culture medium and pretreated at 34°C for 24 hours. The pre-equilibration allows the interstitial water to be replaced by the culture medium, which stabilizes the gas-liquid interface and provides liquid.

[0030] Note: Agarose "1.5%(w / v)" means 1.5 g agarose per 100 mL solvent.

[0031] 2.3 Obtaining and cutting of testis tissue (1) After the mice are disposed according to the animal ethics, the testes are quickly removed and placed in pre-cooled PBS to remove excess connective tissue and tunica albuginea.

[0032] (2) The testis tissue is cut into 1 mm3 pieces using a sterile scalpel.

[0033] (3) Each testis can be cut into several pieces, and the cutting is immediately followed by the scaffold loading step to avoid tissue inactivation.

[0034] 2.4 Construction of gas-liquid interface culture system and culture conditions (1) Culture plate selection: a 6-well plate is used.

[0035] (2) 1-1.5 mL of basal medium is added to each well, with the specific liquid level being stable below the upper surface of the scaffold.

[0036] The basal medium formula is: aMEM + 10%(v / v) KSR + 1%(v / v) penicillin and streptomycin complex antibiotic solution (P / S), and the above percentages are all volume fractions. The "complete medium" is the basal medium with the addition of Foxy-5 to promote the structural maturation of the testis tissue.

[0037] (3) The pre-equilibrated agarose scaffold is placed in the medium in each well, and the medium level is adjusted to be lower than the upper surface of the scaffold, so that the top of the scaffold is not submerged in the culture solution.

[0038] (4) Three testis tissue pieces are placed on the top of a single scaffold, ensuring that the tissue pieces are in a gas-liquid interface state, i.e., the tissue is in contact with the gas phase above and obtains medium infiltration through the scaffold below.

[0039] (5) Culture conditions: culture in a CO2 incubator with conditions of 34°C, 95% humidity, and 5% CO2.

[0040] 2.5 Foxy-5 addition method and medium replacement strategy (1) Foxy-5 is prepared as a stock solution, and when used, it is added to the medium to a final concentration of 400 μM.

[0041] (2) Foxy-5 is maintained throughout the culture period: Foxy-5 is added to a final concentration of 400 μM each time the medium is replaced.

[0042] (3) Medium replacement frequency: medium is replaced every 48 hours; to ensure consistency with the sampling point, medium can also be replaced on days 2, 5, 8, and 11, and samples can be taken simultaneously.

[0043] 2.6 Sampling time points and sample processing (1) Sampling time points: samples were taken on days 2, 5, 8, and 11 of culture, with 3 tissue pieces taken at each time point for each group.

[0044] (2) Fixation: the removed tissue pieces were fixed with 4% (w / v) paraformaldehyde at 4°C overnight, and then dehydrated, transparentized, and embedded in paraffin after PBS washing.

[0045] 4% (w / v) means that 4 g of paraformaldehyde is contained in 100 mL of solution.

[0046] (3) Sectioning: the paraffin sections were 6 μm thick.

[0047] 2.7 H&E staining for detection of seminiferous tubule morphology (1) Paraffin sections were deparaffinized to water.

[0048] (2) Hematoxylin-eosin (H&E) staining.

[0049] (3) The arrangement of seminiferous tubules, regularity of lumen morphology, and lumen dilation were observed and recorded under a microscope.

[0050] 2.8 Immunofluorescence detection of Sertoli cell polarity and blood-testis barrier (BTB) (1) After deparaffinization to water, the paraffin sections were subjected to heat-induced antigen retrieval in citrate buffer (pH = 6) for 15 minutes.

[0051] (2) Blocking with 5% fetal bovine serum for 1 hour.

[0052] (3) Addition of primary antibodies: Sertoli cell nuclear marker SOX9 and blood-testis barrier (BTB) related marker CDH2, incubation at 4°C overnight.

[0053] (4) Addition of fluorescent secondary antibodies for incubation, and DAPI staining of nuclei.

[0054] (5) Images were taken under a confocal microscope under the same exposure parameters for comparative analysis.

[0055] 2.9 Judgment index and recommended quantification method Seminiferous tubule dilation: the lumen radius was measured by image software, and multiple fields and multiple tubular cross-sections were selected for each tissue piece to obtain an average value.

[0056] CDH2 localization: whether the CDH2 fluorescent signal was localized to the basal region between Sertoli cells was counted, and the ratio of "basal signal to total signal" was calculated as the localization index.

[0057] SOX9 polarity: whether SOX9 positive nuclei are concentrated at the base of the seminiferous tubule, which can be characterized by the normalized distance of "nucleus-to-basement membrane distance and tubule radius".

[0058] 3. Experimental results 3.1 Changes in seminiferous tubule morphology (H&E) Compared with the control group, the seminiferous tubule lumen gradually expanded in the Foxy-5 treatment group during the culture process, and the seminiferous tubule morphology was more regular and the lumen radius increased on the 11th day of culture (see Figure 1 ).

[0059] By adding Foxy-5, the histological performance of the seminiferous tubule structure is more regular and the lumen is expanded, achieving the effect of promoting the development of the seminiferous tubule structure.

[0060] 3.2 Changes in Sertoli cell polarity and blood-testis barrier (BTB) (immunofluorescence) The immunofluorescence results show (see Figure 2 ): The SOX9 positive Sertoli cell nuclei in the control group are relatively irregularly distributed; the SOX9 positive cell nuclei in the Foxy-5 treatment group are mainly localized at the base of the seminiferous tubule and arranged regularly; The CDH2 signal in the control group is diffusely distributed; the CDH2 signal in the Foxy-5 treatment group is mainly localized in the Sertoli cell intercellular space at the base of the seminiferous tubule.

[0061] By continuously adding Foxy-5, the Sertoli cell nuclei are located closer to the base, and the CDH2 localization is more concentrated in the Sertoli cell junction area, achieving the effect of promoting the establishment of Sertoli cell polarity and the assembly of the blood-testis barrier (BTB).

[0062] 4. Conclusion In summary, using the gas-liquid interface culture system of the present embodiment, continuously maintaining 400 μM Foxy-5 in the basic culture medium can significantly promote the structural maturation of mouse testicular tissue in vitro, manifested as the development of seminiferous tubule structure, the establishment of Sertoli cell polarity, and the assembly of the blood-testis barrier (BTB). The system can be used for non-therapeutic reproductive toxicity evaluation and research in the field related to spermatogenic microenvironment.

[0063] Example 2, which detects the effects of different concentrations of Foxy-5 and culture periods on the structural maturation of mouse testicular tissue, is as follows: 1. Materials and reagents The experimental animals, main reagents, antibodies, and equipment are the same as in Example 1.

[0064] 2. Method 2.1 Grouping design Using 5-week-old C57BL / 6J male mice, testicular tissue pieces from the same batch of animals were randomly divided into three groups: Group 1: 200 μM Foxy-5 was added to the culture medium; Group 2: 300 μM Foxy-5 was added to the culture medium; Group 3: 400 μM Foxy-5 was added to the culture medium.

[0065] Three mice were used in each group, and three testicular tissue pieces were taken from each group at each time point for histological and immunofluorescence detection.

[0066] 2.2 Preparation and pre-equilibration of agarose gel scaffolds The same as Example 1.

[0067] 2.3 Acquisition and cutting of testicular tissue The same as Example 1.

[0068] 2.4 Construction of gas-liquid interface culture system and culture conditions The same as Example 1.

[0069] 2.5 Foxy-5 addition method and medium replacement strategy The same as Example 1.

[0070] 2.6 Sampling time points and sample processing The same as Example 1.

[0071] 2.7 H&E staining detection of seminiferous tubule morphology The same as Example 1.

[0072] 2.8 Immunofluorescence detection of Sertoli cell polarity and blood-testis barrier (BTB) The same as Example 1.

[0073] 2.9 Judgment index and recommended quantification method Seminiferous tubule dilation: the radius of the transverse lumen was measured by image analysis software, and the average value was taken from multiple fields and multiple transverse sections of each tissue.

[0074] CDH2 localization: whether the CDH2 fluorescence signal is limited to the basal region between Sertoli cells was counted, and the ratio of basal signal to total signal was calculated.

[0075] SOX9 polarity: whether SOX9 positive nuclei are concentrated in the seminiferous tubule basal part was counted, which can be characterized by the normalized distance of the nucleus to the basal membrane and the radius of the tube.

[0076] 3. Experimental results Group 3 showed the best support for cell polarity establishment and blood-testis barrier (BTB) recovery, group 2 showed a certain degree of effect, and group 1 showed relatively low maturity. The above results show that Foxy-5 can promote in the range of 200-400 μΜ.

[0077] 4. Conclusion In summary, using the gas-liquid interface culture system combined with Foxy-5 in the basic culture medium, under different concentrations, can significantly promote the structural maturation of mouse testicular tissue in vitro, including the development of seminiferous tubule structure, the establishment of Sertoli cell polarity, and the assembly of blood-testis barrier (BTB), with 400 μΜ Foxy-5 being the most effective. This system not only provides an effective solution for in vitro testicular tissue culture, but also opens up new paths for reproductive toxicity evaluation and research related to the spermatogenic microenvironment.

[0078] Example 3, which provides a comparison of the gas-liquid interface and complete immersion culture method for the maturation of mouse testicular tissue.

[0079] 1. Materials and reagents The experimental animals, main reagents, antibodies, and equipment are the same as in Example 1.

[0080] 2. Methods 2.1 Grouping design Use 5-week-old C57BL / 6J male mice, and randomly divide the testicular tissue pieces from the same batch of animals into two groups.

[0081] Group 1: gas-liquid interface culture under the conditions in Example 1; Group 2: complete immersion culture, i.e., the tissue is completely immersed in the culture solution; the culture medium composition is the same as in Example 1, and Foxy-5 is also added.

[0082] Each group uses 3 mice, and 3 testicular tissue pieces are taken from each group at each time point for histological and immunofluorescence detection.

[0083] 2.2 Preparation and pre-equilibration of agarose gel scaffold The same as in Example 1.

[0084] 2.3 Acquisition and cutting of testicular tissue The same as in Example 1.

[0085] 2.4 Construction of gas-liquid interface culture system and culture conditions The same as in Example 1.

[0086] 2.5 Foxy-5 addition method and medium exchange strategy The same as Example 1.

[0087] 2.6 Sampling time points and sample processing The same as Example 1.

[0088] 2.7 H&E staining to detect seminiferous tubule morphology The same as Example 1.

[0089] 2.8 Immunofluorescence to detect Sertoli cell polarity and blood-testis barrier (BTB) The same as Example 1.

[0090] 2.9 Judgment index and recommended quantification method Seminiferous tubule expansion: Measure the cross-sectional lumen radius by image analysis software, and take the average value of multiple fields and multiple tube cross-sections for each tissue.

[0091] CDH2 localization: Statistics whether the CDH2 fluorescence signal is limited to the basal region between Sertoli cells, and calculate the ratio of basal signal to total signal.

[0092] SOX9 polarity: Statistics whether SOX9 positive nuclei are concentrated in the seminiferous tubule basal part, which can be characterized by the normalized distance of the nucleus to the basement membrane and the tube radius.

[0093] 3. Experimental results The gas-liquid interface group showed better Sertoli cell polarity and blood-testis barrier (BTB), and the seminiferous tubule morphology was more regular, while the immersion group showed loss of Sertoli cell polarity, incomplete blood-testis barrier (BTB), etc.

[0094] 4. Conclusion Gas-liquid interface culture has obvious advantages compared with complete immersion culture, which can better maintain the polarity of Sertoli cells and the function of blood-testis barrier (BTB), and provides a more effective solution for the structural maturation of in vitro testicular tissue.

[0095] It should be particularly pointed out that the various embodiments listed in the specification and drawings are intended to illustrate the technical solutions of the present application and their advantages, and are not intended to limit the protection scope of the present application. Without departing from the core idea and technical effects of the present application, those skilled in the art can make any form of improvement, replacement, combination or deformation on the structure arrangement, process parameters, material selection, etc. of the embodiments; any obvious changes based on the same concept should be regarded as equivalent solutions of the present application, and should be included in the protection scope defined by the claims of the present application. The actual protection scope of the present application is subject to the appended claims, and should be correctly understood in combination with the specification and drawings.

Claims

1. A culture method for promoting structural maturation of testicular tissue in vitro, characterized by, The method comprises the following steps: (1) cutting testicular tissue into block-shaped tissue fragments; (2) placing the block-shaped tissue fragments on an agarose gel support in an air-liquid interface culture mode, wherein the liquid surface of the culture medium is lower than the upper surface of the support to ensure that the tissue block is in contact with air above and is immersed in the culture medium below; (3) adding a WNT5A pathway agonist Foxy-5 to the culture medium and maintaining the presence of Foxy-5 during the culture period.

2. The culture method of claim 1, wherein the culture method is a method of promoting structural maturation of testicular tissue in vitro. The volume of the block-shaped tissue fragments is 0.5-1 mm3; the mass fraction of agarose in the agarose gel support is 1.5% (w / v); the agarose gel support has a size of 1x1x3 mm3 cubic structure and needs to be pre-equilibrated before use, the pre-equilibration time is 12-36 hours; 2-3 block-shaped tissue fragments are placed on each agarose gel support during use.

3. The culture method of claim 1, wherein the culture medium is a medium for promoting structural maturation of testicular tissue in vitro. The air-liquid interface culture satisfies: the liquid surface of the culture medium is lower than the upper surface of the agarose gel support; the culture conditions satisfy: temperature 34℃, humidity 95%, CO2 concentration 5%; the culture medium is a basic culture medium, including αMEM, KSR and a penicillin and streptomycin complex antibiotic solution (P / S), wherein the volume fraction of KSR is 10%, and the volume fraction of the penicillin and streptomycin complex antibiotic solution (P / S) is 1%.

4. The culture method of claim 1, wherein the culture medium is a medium for promoting structural maturation of testicular tissue in vitro. The final concentration of Foxy-5 in the culture medium is 200-400 μM.

5. The culture method of claim 1, wherein the culture medium is a medium for promoting structural maturation of testicular tissue in vitro. The culture period is 11 days.

6. The culture method of claim 1, wherein the culture medium is a medium for promoting structural maturation of testicular tissue in vitro. The determination index of structural maturation includes: (1) curvature of the seminiferous tubule: the morphology of the seminiferous tubule is regular and the lumen radius increases after culture; (2) blood-testis barrier (BTB) integrity: the expression of BTB marker CDH2 is limited between Sertoli cells; (3) Sertoli cell polarity: the Sertoli cell nucleus marker SOX9 labeled nucleus is limited to the base of the seminiferous tubule.

7. The culture method of claim 1, wherein the culture medium is a medium for promoting structural maturation of testicular tissue in vitro. The culture method is used for in vitro maturation of testicular tissue for reproductive toxicity testing or spermatogenic microenvironment research.

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