A method for efficiently constructing pig testis organoids in vitro

Abstract

The application relates to the technical field of reproductive biology, and particularly relates to a method for efficiently constructing a pig testis organoid in vitro. The method comprises the following steps: S1, zygote testis tissue enzymolysis; S2, testis cell resuspension counting: after the testis cells obtained by the enzymolysis in S1 are uniformly mixed, the cells are collected into U-bottom 96-hole plates in sequence, and DMEM basic culture medium containing 5% FBS, 10% KSR and 1% P / S is added into each hole to complete resuspension culture of the cells. S3, testis organoid in-vitro culture: testis cell groups are separated, and the separated cell groups are transferred into U-bottom 96-hole plates and cultured in DMEM basic culture medium containing 5% FBS, 10% KSR and 1% P / S; the cell amount in the U-bottom 96-hole plates is 5*10 4 6 cells per hole. After the culture system is optimized, the application simplifies the experimental process, can directly culture the testis organoid in the U-bottom 96-hole plates, reduces the replacement frequency, reduces the pollution risk, significantly reduces the cell amount, and improves the culture efficiency.

Description

Technical Field

[0001] This invention relates to the field of reproductive biology technology, specifically to a method for efficiently constructing porcine testicular organoids in vitro. Background Technology

[0002] In recent years, approximately 15%-20% of couples worldwide have faced the challenge of infertility, with male factors accounting for about half of these cases. The causes of infertility are diverse. In men, testicular developmental or functional disorders lead to various conditions, including sexual developmental disorders (DSD) and infertility. Male infertility has complex and diverse causes, clinically manifesting as oligospermia, asthenospermia, teratospermia, or azoospermia. Azoospermia is the most severe form of male infertility, further classified into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) based on the presence or absence of sperm duct obstruction. Because NOA patients lack functional sperm, assisted reproductive technologies cannot be an effective treatment. Furthermore, male cancer patients can preserve fertility by cryopreserving sperm before undergoing reproductively toxic chemotherapy and radiotherapy; however, this method is not feasible for pre-pubescent boys whose spermatogenesis has not yet begun. Therefore, constructing testicular organoids from cryopreserved testicular cells to produce sperm in vitro is of great significance for preserving or restoring male fertility.

[0003] The testis is a structurally complex organ in the male reproductive system, possessing the dual functions of producing germ cells and secreting male hormones. In mice, the testis begins development in the gonads at embryonic day E11.5. This stage is jointly regulated by the sex-determining gene Sry and its downstream target gene Sox9. During development, the testis ultimately forms tubular and interstitial compartments. The tubular compartment consists of the immature testicular cords of the newborn and the mature seminiferous tubules (containing the lumen) of the adult male. The tubular compartment is mainly composed of peritubular myoid cells (PTMCs) and Sertoli cells, which contain various developing germ cells. The interstitial compartment consists of connective tissue, blood vessels, and interstitial cells capable of producing androgens. Therefore, testicular organogenesis is considered a crucial process in embryonic development and has long-term effects on reproductive potential. In conclusion, developing an organoid system that can recreate in vivo testicular tissue and can be applied to large animal models is of great significance.

[0004] As early as 2011, Takehiko Ogawa's team established conditions for in vitro organ culture and successfully obtained spermatogonia from the testes of newborn mice, producing fertile sperm under in vitro culture conditions. In 2017, Jan-Bernd Stukenborg's team first proposed the three-dimensional gradient model (3-LGS), which can culture rat testicular organoids with a functional blood-testis barrier and germ cell proliferation. This model more closely approximates the relationship between germ cells and somatic cells in vivo. In 2024, Nitzan Gonen's team successfully cultured organoids from neonatal and embryonic testicular cells of mice. These organoids maintained their structure and gene expression profile for up to 9 weeks and exhibited cell types similar to those in vivo, including major cell types such as Sertoli cells and Leydig cells.

[0005] However, traditional studies on testicular organoids mostly use rodent cells, which has limitations in model design. Furthermore, traditional testicular organoid culture methods suffer from low efficiency due to the large number of cells required and the cumbersome procedures involved. Therefore, there is an urgent need to improve existing methods for constructing testicular organoids in vitro. Summary of the Invention

[0006] To address the shortcomings of existing technologies, pigs, due to their anatomical and physiological similarities to humans, have gradually become an important model in biomedical research. This application uses piglet testicular cells as starting material and, through optimization of cell cluster culture and culture systems, successfully cultured testicular organoids with distinct seminiferous tubule structures. A complete model of testicular organoid development and in vitro spermatogenesis was constructed, which can provide direction for solving human clinical infertility problems.

[0007] The technical solution of this invention is as follows:

[0008] A method for efficiently constructing porcine testicular organoids in vitro, comprising the following steps:

[0009] S1: Enzymatic hydrolysis of piglet testicular tissue: The piglets referred to are newborn piglets to 4-week-old piglets.

[0010] S2: Testicular cell resuspension and counting: After the testicular cells obtained from S1 enzymatic digestion were uniformly resuspended, they were collected sequentially into U-bottom 96-well plates. DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S was added to each well to complete the cell resuspension culture.

[0011] S3: In vitro culture of testicular organoids: Testicular cell clusters were isolated and transferred to U-bottom 96-well plates. The cells were cultured in DMEM basic medium containing 5% FBS, 10% KSR, and 1% P / S. The cell density in each U-bottom 96-well plate was 5 × 10⁶ cells / well.4 1,000 cells; place a U-bottom 96-well plate in an incubator containing 5% carbon dioxide for suspension culture. During the culture, change the culture medium every 24 hours. Testicular organoids can be cultured in vitro for 3 weeks.

[0012] Preferably, S1 involves rinsing the testicular tissue of 4-week-old piglets with physiological saline, followed by disinfection with alcohol, and placing it in a sterile beaker. This is then rinsed with physiological saline containing 1% P / S. After rinsing, the testicular tissue is transferred to a culture dish lined with PBS for soaking; excess testicular tissue is removed, and the separated testicular tissue is transferred to a new culture dish and rinsed again with PBS containing 1% P / S. After rinsing, the testicular tissue is cut into small pieces, the white connective tissue is removed, and the tissue is transferred to centrifuge tubes. PBS and collagenase IV are added to the minced tissue, and digestion is performed in a 37°C water bath for 15-20 minutes. After digestion, the supernatant is collected and centrifuged. After centrifugation, the supernatant is discarded, and TrypLE is added to the centrifuge tube. TM The testicular tissue was digested using Expression enzyme; after the tissue blocks were digested into single cells, they were immediately centrifuged. After centrifugation, the supernatant was discarded, and DMEM / F12 containing 10% FBS was added to terminate the digestion.

[0013] Preferably, S2 involves filtering the enzymatically digested testicular cells through a 40 μm filter membrane. The filtered cell suspension is centrifuged, and the supernatant is discarded. The cells are resuspended in DMEM / F12 medium containing 1% P / S and centrifuged again. After centrifugation, the supernatant is collected, and the cells are counted using a hemocytometer. The cells are then resuspended in DMEM / F12 medium. After centrifugation again, the supernatant is discarded, and the uniformly resuspended cells are collected sequentially into U-bottom 96-well plates, with 5 × 10⁶ cells per well. 4 100 cells; add 200 μL of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S to each well to complete the cell resuspension culture.

[0014] Preferably, S3 is as follows: First, prepare three 200 μL droplets of DMEM basic medium containing 5% FBS, 10% KSR, and 1% P / S in a culture dish; wash the testicular cell clusters three times sequentially in droplets of DMEM basic medium containing 5% FBS, 10% KSR, and 1% P / S. Then, transfer the separated cell clusters to U-bottom 96-well plates for culture. Add 5 × 10⁵ cells to each well of the U-bottom 96-well plate. 4Add 200 μL of DMEM basic medium containing 5% FBS, 10% KSR, and 1% P / S to each cell. Finally, place the U-bottom 96-well plate in an incubator containing 5% CO2 for suspension culture to allow gas and nutrients to pass through simultaneously; change the medium every 24 hours during the culture process.

[0015] Preferably, the testicular organoids cultured by this method for 21 days and the testicular tissue cultured in vivo for 28 days both clearly expressed β-catenin positive signals.

[0016] Preferably, the volume of the testicular organoid cultured by this method for 21 days is 649*301μm.

[0017] The beneficial effects achieved by this invention are as follows:

[0018] 1. By optimizing the culture system, this invention simplifies the experimental process, allowing testicular organoids to be cultured directly in a U-shaped 96-well plate without first culturing cell clusters on agar blocks. This reduces the number of cell replacements, the risk of contamination, and improves culture efficiency.

[0019] 2. Based on the DMEM basic culture medium containing 5% FBS, 10% KSR, and 1% P / S, this invention requires only 5 × 10⁻⁶ cells per testicular organoid. 4 10 cells, compared to the previous 1×10 6 Compared to single-cell approaches, the number of cells used is significantly reduced, which can increase cell utilization.

[0020] 3. The present invention can culture testicular organoids in vitro for up to 3 weeks. Compared with the testicular volume cultured by existing methods, the testicular organoids of the present invention grow to a larger and more similar volume, and are more uniform in size, which facilitates subsequent observation and experimentation.

[0021] 4. This invention can cultivate testicular organoids with seminiferous tubules, providing a foundation for the construction of a spermatogenic microenvironment, the provision of an immune barrier, and the maintenance of normal spermatogenesis. Attached Figure Description

[0022] Figure 1 This is a flowchart of the in vitro culture of testicular organoids according to the present invention.

[0023] Figure 2 These are bright-field images of testicular organoids cultured in vitro according to the present invention: where A is a bright-field image of testicular organoids cultured in vitro for 7 days; B is a bright-field image of testicular organoids cultured in vitro for 14 days; and C is a bright-field image of testicular organoids cultured in vitro for 21 days.

[0024] Figure 3These are immunofluorescence staining images of β-catenin in vivo testicular tissue after 28 days and testicular organoids cultured in vitro for 21 days according to this invention.

[0025] Figure 4 This is an HE staining image of testicular organoids cultured in vitro for 21 days in Example 1 of this invention.

[0026] Figure 5 This is an HE staining image of testicular organoids cultured in vitro for 21 days in Example 2 of this invention.

[0027] Figure 6 This is an HE staining image of testicular organoids cultured in vitro for 21 days in Example 3 of this invention.

[0028] Figure 7 This is a PAS staining image of testicular organoids cultured in vitro for 21 days in Example 1 of this invention. Detailed Implementation

[0029] To facilitate understanding of the present invention by those skilled in the art, specific embodiments of the present invention are described below with reference to the accompanying drawings. Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0030] Sources of raw materials and reagents in the examples:

[0031] 1. Penicillin powder, purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0032] 2. P / S, purchased from Cytiva;

[0033] 3. PBS (fetal bovine serum), purchased from Beijing Solarbio Technology Co., Ltd.

[0034] 4. 10cm petri dishes were purchased from Qingdao Jindian Chemical Equipment Co., Ltd.

[0035] 5. 1% P / S DMEM / F12 culture medium was purchased from Thermo Fisher Scientific.

[0036] 6. Collagenase IV, purchased from Sigma;

[0037] 7. TrypLE TM Expression enzymes were purchased from Gibco.

[0038] 8. 40μm filter membrane, purchased from Merck;

[0039] 9. KSR (KSR cell culture medium), purchased from Gibco;

[0040] 10. ITS, purchased from Gibco;

[0041] 11. EGF, purchased from the R&D company (R&D (Research and Development Division)); 12. Paraformaldehyde solution, purchased from Beijing Solarbio Technology Co., Ltd.

[0042] 13. Triton X100, purchased from Beijing Solarbio Technology Co., Ltd.

[0043] 14. BSA (Bovine Serum Albumin), purchased from Beijing Solarbio Technology Co., Ltd.; 15. Goat Serum Albumin, purchased from Boster Biologics Inc.

[0044] 16. Tween 20, purchased from Beijing Solarbio Technology Co., Ltd.

[0045] 17. β-catenin, purchased from Huabio Biotechnology Co., Ltd.

[0046] 18. Hoechst 33342, purchased from Beijing Solarbio Technology Co., Ltd.

[0047] 19. Antiquenching agents were procured from Boster Biologics.

[0048] 20. Hematoxylin, purchased from Beijing Solarbio Technology Co., Ltd.

[0049] 21. Erythril, purchased from Beijing Solarbio Technology Co., Ltd.

[0050] 22. Neutral resin, purchased from Beijing Solarbio Technology Co., Ltd.

[0051] Example 1

[0052] A method for efficiently constructing porcine testicular organoids in vitro, such as Figure 1 As shown, the methods include the following:

[0053] The first step is the enzymatic digestion of testicular tissue:

[0054] Testicular tissue from 4-week-old piglets taken from the pig farm was first rinsed and soaked in physiological saline containing 0.2% penicillin powder. Next, the testicular tissue was disinfected with alcohol, placed in a sterile beaker, and then rinsed three times with physiological saline containing 1% P / S.

[0055] After cleaning, the testicular tissue was transferred to a 10cm culture dish lined with PBS for immersion. The epididymis was removed using sterilized scissors and forceps, removing as much remaining tissue as possible. The separated testicular tissue was transferred to a new 10cm culture dish and rinsed again with PBS containing 1% P / S. The testis was held in place by forceps in the left hand, and longitudinally cut open with scissors in the right hand.

[0056] Then, the testis was detached from the testicular membrane using forceps and transferred to a 10cm culture dish containing 1% P / S DMEM / F12 medium. The testicular tissue was minced using a sterile scalpel, and the white connective tissue was removed using sterile forceps. The minced tissue was then transferred to a 45mL centrifuge tube. 5mL of PBS and 2mg / mL collagenase IV were added to 5mL of the minced tissue, and the mixture was digested in a 37°C water bath for 15-20 minutes. The centrifuge tube was gently inverted every 5 minutes, and the digestion status of the testis was constantly monitored during the process.

[0057] After digestion, collect 20 μL of the supernatant and observe it under a microscope for the presence of numerous tubular structures. Then, transfer the supernatant to a 15 mL centrifuge tube and centrifuge at 90 g for 5 min. After centrifugation, discard the supernatant and add 3-5 mL of TrypLE to the 15 mL centrifuge tube. TM Enzymatic digestion was performed using Expression enzyme. The entire digestion process took approximately 3-5 minutes, and the digestion status needed to be observed every 2 minutes during the process. 20 μL of tissue fluid was placed in a 10 cm culture dish, and the dish was observed under a microscope to check if the testicular tissue had been digested into single cells. If most of the testicular tissue block had been digested into single cells, it was immediately centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, and DMEM / F12 medium containing 10% FBS was added to terminate the digestion.

[0058] Step 2, testicular cell resuspension and counting:

[0059] To remove tissue fragments and undigested tissue fragments, the cell suspension was filtered using a 40 μm filter membrane. The filtered filtrate was centrifuged at 300 x g for 5 min, and the supernatant was discarded after centrifugation. Subsequently, the cells were resuspended in DMEM / F12 medium containing 1% P / S in centrifuge tubes and centrifuged at 300 x g for 3 min. Finally, the cells were counted using a hemocytometer, resuspended in DMEM / F12 medium, and centrifuged again at 300 x g for 5 min. The supernatant was discarded after centrifugation, and the cells were added to the culture system. In this example, the uniformly suspended cells were sequentially transferred to 96-well plates, with approximately 5 × 10⁶ cells per well. 4The culture system in this example (hereinafter referred to as K3 medium) consists of 5% FBS (fetal bovine serum), 10% KSR (KSR cell culture medium), and 1% P / S in DMEM basic medium.

[0060] The third step is the in vitro culture and observation of testicular tissue:

[0061] First, testicular cells were divided into three 200 μL drops of K3 medium in a 10 cm culture dish. Next, the testicular cell clusters were washed three times with the K3 medium droplets, using a new pipette tip each time. Then, the counted cell clusters were aspirated using a pipette and transferred to U-bottom 96-well plates for culture. High cell density promotes cell-cell interactions, but excessive density can lead to overly large testicular organoids, limiting nutrient and oxygen supply. Conversely, low cell density may hinder organoid formation. Therefore, this application selected 5 × 10⁶ cells per organoid. 4 Cells were collected and 200 μL of K3 medium was added to each well. Finally, the U-bottom 96-well plate was placed in an incubator containing 5% carbon dioxide for gas-liquid culture, ensuring simultaneous gas and nutrient passage. The medium in the U-bottom 96-well plate was changed every other day, with fresh K3 medium added. After three weeks of culture using the above method, testicular organoids were harvested every week for morphological and histological observation. Figure 2 As shown.

[0062] Example 2

[0063] Unlike Example 1, in the second step of this example, 200 μL of DMEM / F12 medium containing 1% ITS, 0.1% EGF, and 1% P / S was added to each well to complete the cell resuspension culture. The above medium is referred to as To medium.

[0064] In the third step, firstly, three 200 μL drops of To medium were prepared in a 10 cm culture dish. The testicular cell clusters were then washed three times sequentially in the To medium drops. Afterward, the separated cell clusters were transferred to U-bottom 96-well plates for culture. 5 × 10⁵ cells were added to each well of the U-bottom 96-well plate. 4 Add 200 μL of To medium to each cell. Finally, place the U-bottom 96-well plate in an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through simultaneously. During culture, change the medium every 24 hours by adding fresh To medium.

[0065] Example 3

[0066] Unlike Example 1, in the third step of this example, 200 μL of medium containing 10% KSR and 1% P / SDMEM basic was added to each well to complete the cell resuspension culture. The above medium is referred to as K4 medium.

[0067] In the third step, firstly, three 200 μL drops of K4 medium were prepared in a 10 cm culture dish. The testicular cell clusters were then washed three times sequentially in the K4 medium drops. Afterward, the separated cell clusters were transferred to U-bottom 96-well plates for culture. 5 × 10⁵ cells were added to each well of the U-bottom 96-well plate. 4 Add 200 μL of K4 medium to each cell. Finally, place the U-bottom 96-well plate in an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through simultaneously. During the culture process, change the medium every 24 hours by adding fresh K4 medium.

[0068] Example 4

[0069] Cell type identification of testicular organoids:

[0070] Spermatogenesis in mammals is a highly coordinated process regulated by a variety of somatic cells, including Sertoli cells, testicular interstitial cells, and peritubular myoid cells. Sertoli cells, which are in close contact with testicular germ cells, play a crucial role in the development and maturation of spermatogenic cells into functional sperm. Therefore, this application collected testicular organoids cultured for 21 days and examined specific cell types within them.

[0071] First, the testicular organoids were washed three times in physiological saline and transferred to 3 cm culture dishes containing 4% paraformaldehyde solution, then fixed overnight at 4°C. The next day, the fixed testicular organoids were wrapped in a gauze mask and subjected to gradient alcohol dehydration. Finally, they were immersed in melted paraffin overnight. Then, the testicular organoids were embedded in fresh paraffin. The solidified paraffin blocks were fixed on a microtome, and sections were cut to a thickness of 5 μm. The paraffin sections of the testicular organoids were baked in a 65°C oven for 1 hour, rehydrated with gradient alcohol, washed in PBS for 10 minutes, and then retrieval in antigen retrieval solution at 96°C for 10 minutes. They were then cooled to room temperature.

[0072] Next, wash the slides in 0.2% Triton X100 PBS for 15 min, then wash three times with TBS for 5 min each time. Place the slides flat in a humidified chamber, and add 50 μL of PBS solution containing 3% BSA, 10% goat serum albumin, and 2 μL L-20 to each slide. Cover the sample loading area of ​​the slide with sealing film, close the lid of the humidified chamber, and incubate at 37°C for 45 min. After incubation, remove the sealing film, add 50 μL of primary antibody dilution buffer (β-catenin diluted 1:200) to the slide, and then close the sealing film again. Place the humidified chamber in a sealed bag and incubate overnight at 4°C.

[0073] The next day, the humidified chamber was removed and allowed to warm to room temperature for 30 minutes. Then, the slides were washed three times with TBST for 10 minutes each. Next, the slides were placed back into the humidified chamber, and 50 μL of fluorescent secondary antibody dilution buffer was added to each slide. The slides were then sealed and incubated at 37°C for 45 minutes. After incubation, the slides were placed in a wash cup and washed three times with TBST for 10 minutes each. 50 μL of Hoechst 33342 was added to each slide, and nuclei were stained at room temperature for 6 minutes. After nuclei staining, the slides were washed three times with PBS for 2 minutes each. Finally, the slides were mounted with an anti-quenching agent.

[0074] On the third day, from Figure 3 As can be seen, both the testicular tissue cultured in vivo for 28 days and the testicular organoids cultured in vitro for 21 days clearly expressed β-catenin positive signals, indicating that the testicular organoids cultured in vitro have similar biological functions to the testicular tissue cultured in vivo.

[0075] Example 5

[0076] Development of testicular organoids:

[0077] To observe the development of testicular organoids, testicular organoids cultured for 21 days in Examples 1, 2, and 3 were collected and subjected to HE staining. The staining results are as follows: Figure 4 , Figure 5 and Figure 6 As shown. The specific method is as follows: testicular organoids are cut into 5μm sections, and the slides are placed in an oven to bake for 1 hour, followed by dewaxing and graded alcohol rehydration. Next, hematoxylin staining is performed for 5 minutes, followed by rinsing with tap water for 5 minutes, and then rinsing with distilled water for 2 minutes. Then, the slides are immersed in 1% hydrochloric acid alcohol for differentiation three times, 3 seconds each time.

[0078] Next, the slides were blued with tap water at 45°C for 5 minutes, followed by washing with distilled water for 2 minutes. The slides were then sequentially immersed in 50% ethanol, 70% ethanol, and 80% ethanol for 2 minutes each, stained with eosin for 1 minute, then stained with 95% ethanol I and 95% ethanol II for 3 minutes each, 100% ethanol I and 100% ethanol II for 5 minutes each, and xylene I and xylene II for 5 minutes each. Finally, neutral resin was added to the slides, and a coverslip was placed on top to seal them. After sealing, the slides were air-dried in a fume hood for 2 hours, after which they could be stored at room temperature in a slide holder. Observation of HE staining revealed that the developmental process of testicular organoids cultured in vitro using the K3 culture system of Example 1 was similar to that in vivo, with complete tubular structures appearing in the testicular tissue, such as… Figure 4 As shown in Example 2. Figure 5 As shown in Example 3 Figure 6 As shown, no tubular structures appeared in the testicular organoids under the culture system, and the testicular organoids varied in size.

[0079] The blood-testis barrier participates in the construction of the spermatogenic microenvironment, provides an immune barrier, and maintains normal spermatogenesis. To assess the integrity of the blood-testis barrier in cultured testicular organoids, PAS staining was performed on 21-day cultured testicular organoids. Figure 7 As shown.

[0080] The specific method is as follows: Slides cut to 5 μm are first dried in an oven for 1 hour, then rinsed with xylene I and xylene II for 5 minutes each, followed by washing with 100% ethanol for 5 minutes, 90% ethanol, 80% ethanol, and 70% ethanol for 2 minutes each. Next, periodic acid-Schiff staining is performed according to the Beyotime reagent kit instructions. 100 μL of periodic acid solution is added to each slide, and the slide is incubated in a humidified chamber in the dark for 10 minutes, followed by rinsing with distilled water for 5 minutes. 100 μL of Schiff's reagent is added to each cleaned slide, and the slide is placed flat in a humidified chamber and stained in a 37°C oven in the dark for 45 minutes. After incubation, the slide is rinsed with distilled water for 5 minutes to remove the staining solution. Then, the slide is stained with hematoxylin for 30 seconds. After staining, the slide is rinsed three times with distilled water for 3 seconds each time to remove the staining solution. After rinsing with distilled water, the slides were placed in 1% hydrochloric acid ethanol for 30 seconds for differentiation, then rinsed twice with tap water for 3 seconds each time to remove the differentiation solution. The slides were then allowed to return to their original blue state with tap water for 5 minutes. Next, the slides were immersed in 90% ethanol and 100% ethanol for 2 minutes each, followed by xylene I and xylene II for 5 minutes each. Finally, the slides were mounted with neutral resin and then air-dried in a fume hood for 2 hours. Figure 7 PAS staining revealed that the blood-testis barrier of the in vitro cultured testicular organoids remained intact, exhibiting structural features similar to those of in vivo tissues.

[0081] Therefore, by optimizing the culture system, this invention simplifies the experimental process and successfully utilizes 5×10⁻⁶ cells / year. 4 Testicular organoids were constructed using cells in a U-shaped 96-well plate, successfully culturing testicular organoids with distinct seminiferous tubule structures under completely in vitro conditions. This could provide direction for solving the problem of clinical infertility in humans.

[0082] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for efficiently constructing porcine testicular organoids in vitro, characterized in that, Including the following methods: S1: Enzymatic hydrolysis of piglet testicular tissue: The piglets referred to are newborn piglets to 4-week-old piglets; S2: Testicular cell resuscitation and counting: Testicular cells obtained after S1 enzymatic digestion were filtered through a 40 μm filter membrane; the filtered cell suspension was centrifuged, and the supernatant was discarded; the cells were resuspended in DMEM / F12 containing 1% P / S and centrifuged again; after centrifugation, the supernatant was collected, and the cells were counted using a hemocytometer, and the cells were resuspended in DMEM / F12 medium; after centrifugation again, the supernatant was discarded, and the well-resuspended cells were collected sequentially into U-bottom 96-well plates, with 5 × 10⁶ cells in each well. 4 100 cells; add 200 μL of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S to each well to complete the cell resuspension culture; S3: In vitro culture of testicular organoids: First, prepare three 200 μL droplets of DMEM basic medium containing 5% FBS, 10% KSR, and 1% P / S in a culture dish; wash the testicular cell clusters three times sequentially in droplets of DMEM basic medium containing 5% FBS, 10% KSR, and 1% P / S; then, transfer the separated cell clusters to U-bottom 96-well plates for culture; add 5 × 10⁶ cells / well to each well of the U-bottom 96-well plate. 4 Add 200 μL of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S to each cell. Finally, place the U-bottom 96-well plate into an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through simultaneously. Change the medium every 24 hours during the culture process.

2. The method for efficiently constructing porcine testicular organoids in vitro according to claim 1, characterized in that: S1 involves rinsing the testicular tissue of 4-week-old piglets with physiological saline, then disinfecting it with alcohol, and placing it in a sterile beaker for rinsing with physiological saline containing 1% P / S. After rinsing, the testicular tissue is transferred to a culture dish filled with PBS for soaking. Excess testicular tissue is removed, and the separated testicular tissue is transferred to a new culture dish and rinsed again with PBS containing 1% P / S. After rinsing, the testicular tissue was cut into small pieces using a sterile scalpel, the white connective tissue was removed, and the tissue was transferred to centrifuge tubes. PBS and collagenase IV were added to the minced meat, and it was digested in a 37°C water bath for 15-20 minutes. After digestion, the supernatant was collected and centrifuged. After centrifugation, the supernatant was discarded, and TrypLE was added to the centrifuge tubes. TM The testicular tissue was digested with Expression enzyme; after the tissue blocks were digested into single cells, they were centrifuged; after centrifugation, the supernatant was discarded and DMEM / F12 containing 10% FBS was added to terminate the digestion.

3. The method for efficiently constructing porcine testicular organoids in vitro according to claim 1, characterized in that: The volume of the testicular organoids cultured by this method for 21 days was 649 × 301 μm.

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