Method for efficiently constructing pig testis organs in vitro

Abstract

The invention relates to the technical field of reproductive biology, in particular to a method for efficiently constructing pig testis organs in vitro. Comprising the following steps: S1, enzymolysis of testicular tissues of piglets; and S2, testicular cell resuspension counting: uniformly suspending the testicular cells obtained after enzymolysis in the step S1, sequentially collecting the cells into a 96-well plate with a U-shaped bottom, and adding a DMEM base culture medium containing 5% of FBS, 10% of KSR and 1% of P / S into each well to complete resuspension culture of the cells. S3, testicular organ in-vitro culture: separating testicular cell masses, transferring the separated cell masses into a 96-well plate with a U-shaped bottom, and adding the cell masses into a DMEM base culture medium containing 5% of FBS, 10% of KSR and 1% of P / S for culture; and the quantity of cells in the 96-well plate with the U-shaped bottom is 5 * 104 cells per well. After the culture system is optimized, the experimental process is simplified, the testicular organs can be directly cultured in the 96-well plate with the U-shaped bottom, the replacement frequency is reduced, the pollution risk is reduced, the number of adopted cells is remarkably reduced, and the culture efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of reproductive biology, and in particular to a method for efficiently constructing pig testicular organoids in vitro. Background Art

[0002] In recent years, about 15%-20% of couples worldwide have faced the challenge of infertility, of which male factors account for about half. There are many causes of infertility. For men, testicular development or dysfunction leads to various diseases, including disorders of sexual development (DSD) and infertility. The causes of male infertility are complex and diverse, and clinically manifest as oligospermia, asthenozoospermia, teratozoospermia or azoospermia. Among them, azoospermia is the most serious condition of male infertility. According to the presence or absence of sperm tract obstruction, azoospermia is divided into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). Since NOA patients lack functional sperm, assisted reproductive technology cannot be used as an effective treatment. In addition, male cancer patients can preserve fertility by freezing sperm before receiving reproductive toxic chemotherapy and radiotherapy, but this method is not feasible for prepubertal boys whose spermatogenesis has not yet started. 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 complex organ in the male reproductive system that has the dual functions of producing germ cells and secreting androgens. In mice, the testis develops in the gonad from embryonic day E11.5 (E11.5). This stage is co-regulated by the sex-determining gene Sry and its downstream target gene Sox9. During development, the testis eventually forms a tubular region and an interstitial region. The tubular lumen is composed of immature testicular cords in neonates and mature seminiferous tubules (including the lumen) in adult males. The tubular lumen is mainly composed of two types of somatic cells, peritubular myoid cells (PTMCs) and supporting cells, which contain various developing germ cells. The interstitial lumen is composed of connective tissue, blood vessels, and interstitial cells with the ability to produce androgens. Therefore, testicular organogenesis is considered to be a key process in embryonic development and has long-term effects on its reproductive potential. In summary, it is of great significance to develop an organoid system that can restore in vivo testicular tissue and can be applied to large animal models.

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

[0005] However, most traditional testicular organoid studies use rodent cells as the research object, which has model limitations. In addition, traditional testicular organoid culture methods have the problem of using a large number of cells and the cumbersome culture method, resulting in low culture efficiency. Therefore, there is an urgent need to improve the existing methods of constructing testicular organoids in vitro. Summary of the invention

[0006] In order to solve the shortcomings of the existing technology, pigs have gradually become an important model in biomedical research due to their similarities with humans in anatomical and physiological characteristics. This application uses piglet testicular cells as the starting material, and successfully cultivates testicular organoids with obvious seminiferous tubules structure by optimizing cell cluster culture and culture system. The construction of a complete testicular organoid development and in vitro spermatogenesis process can provide direction for solving the problem of human clinical infertility.

[0007] The technical solution of the present invention is:

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

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

[0010] S2: Testicular cell resuspension counting: The testicular cells obtained after enzymatic hydrolysis in S1 were uniformly suspended and collected in turn into a U-bottom 96-well plate, and DMEM basic culture 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: The testicular cell clusters were separated and transferred to a U-bottom 96-well plate and cultured in DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S. The number of cells in each well of the U-bottom 96-well plate was 5×104 cells; place the U-bottom 96-well plate in an incubator containing 5% carbon dioxide for suspension culture. During the culture process, the culture medium was replaced every 24 hours, and the testicular organoids could be cultured in vitro for 3 weeks.

[0012] Preferably, S1 is to rinse the testicular tissue of a 4-week-old piglet with normal saline, then rinse it with alcohol for disinfection, place it in a sterile beaker, and then rinse it with normal saline containing 1% P / S. After washing, transfer the testicular tissue to a culture dish covered with PBS for soaking; remove excess testicular tissue, and transfer the separated testicular tissue to a new culture dish, and rinse it again with PBS containing 1% P / S. After rinsing, cut the testicular tissue into small pieces, remove the white connective tissue, and transfer it to a centrifuge tube; add PBS and collagenase IV to the minced meat, and digest it in a 37°C water bath for 15-20 minutes. After digestion, take the supernatant and centrifuge it. After centrifugation, discard the supernatant and add TrypLE to the centrifuge tube. TM Expression enzyme for enzymatic digestion; after the testicular tissue blocks were digested into single cells, they were immediately placed in a centrifuge for centrifugation. After the centrifugation, the supernatant was discarded and DMEM / F12 containing 10% FBS was added to terminate the digestion.

[0013] Preferably, S2 is to filter the testicular cells obtained after enzymatic hydrolysis through a 40 μm filter membrane. The filtered cell suspension is centrifuged and the supernatant is discarded. The cells are resuspended in DMEM / F12 containing 1% P / S and centrifuged again; after centrifugation, the supernatant is taken, the cells are counted using a hemocytometer, and the cells are resuspended in DMEM / F12 medium. After centrifugation again, the supernatant is discarded, and the evenly suspended cells are collected in turn into a U-bottom 96-well plate, with 5×10 4 cells; 200 μL of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S was added to each well to complete the resuspension culture of the cells.

[0014] Preferably, S3 is: first, make 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 in the droplets of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S. Then, transfer the separated cell clusters to a U-bottom 96-well plate for culture. Add 5×10 4cells, and then add 200 μL of DMEMbasic medium containing 5% FBS, 10% KSR and 1% P / S. Finally, the U-bottom 96-well plate was placed in an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through at the same time; during the culture process, the medium was replaced every 24 hours.

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

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

[0017] The beneficial effects achieved by the present invention are:

[0018] 1. The present invention simplifies the experimental process by optimizing the culture system. Testicular organoids can be cultured directly in a U-bottom 96-well plate without first culturing cell clusters on an agar block, thereby reducing the number of replacements, reducing the risk of contamination, and improving the efficiency of culture.

[0019] 2. Based on the medium selected by the present invention containing 5% FBS, 10% KSR and 1% P / S, each testicular organoid only needs 5×10 4 cells, compared with the previous 1×10 6 Compared with 100 cells, the number of cells used is significantly reduced, which can increase the utilization rate of cells.

[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 in a larger and similar volume and are more uniform in size, which is convenient for subsequent observation and experiments.

[0021] 4. The present invention can culture testicular organoids with seminiferous tubules, providing a basis for constructing a spermatogenic microenvironment, providing an immune barrier and maintaining normal spermatogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a flow chart of the in vitro culture of testicular organoids of the present invention.

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

[0024] Figure 3It is a β-catenin immunofluorescence staining image of the testicular tissue in vivo for 28 days and the testicular organoid cultured in vitro for 21 days of the present invention.

[0025] Figure 4 This is a HE staining image of the testicular organoid cultured in vitro for 21 days in Example 1 of the present invention.

[0026] Figure 5 This is a HE staining image of the testicular organoid cultured in vitro for 21 days in Example 2 of the present invention.

[0027] Figure 6 This is a HE staining image of the testicular organoid cultured in vitro for 21 days in Example 3 of the present invention.

[0028] Figure 7 This is a PAS staining image of the testicular organoid cultured in vitro for 21 days in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] To facilitate those skilled in the art to understand the present invention, the specific embodiments of the present invention are described below in conjunction with the accompanying drawings. The materials and reagents used in this example are reagents and materials obtained from commercial sources unless otherwise specified.

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

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

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

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

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

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

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

[0037] 7. TrypLE TM Expression enzyme, purchased from Gibco;

[0038] 8. 40 μm 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 R&D (Research and Development Division); 12. Paraformaldehyde solution, purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio);

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

[0043] 14. BSA (bovine serum albumin, purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio); 15. Goat serum albumin, purchased from Boster Biotechnology Co., Ltd. (Boster);

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

[0045] 17. β-catenin, purchased from Huaan Biotechnology Co., Ltd. (Huabio);

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

[0047] 19. Anti-quenching agent, purchased from Boster;

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

[0049] 21. Yihong, purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio);

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

[0051] Example 1

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

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

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

[0055] After washing, transfer the testicular tissue to a 10 cm culture dish covered with PBS for soaking. Use high temperature sterilized scissors and forceps to remove the epididymis and cut the remaining epididymal tissue as clean as possible. Transfer the separated testicular tissue to a new 10 cm culture dish and rinse it again with PBS containing 1% P / S. Hold the testicle with forceps in the left hand and cut the testicle longitudinally with scissors in the right hand.

[0056] Then, use forceps to peel the testis from the testicular membrane and transfer it to a 10 cm culture dish containing 1% P / S DMEM / F12 medium. Use a sterile scalpel to cut the testicular tissue into minced meat, cut it into pieces, use sterile forceps to remove the white connective tissue, and transfer it to a 45 mL centrifuge tube. Add 5 mL of PBS and 2 mg / mL collagenase IV to 5 mL of minced meat and digest it in a 37°C water bath for 15-20 minutes. Gently invert the centrifuge tube every 5 minutes and observe the digestion status of the testis at all times during the digestion process.

[0057] After digestion, take 20 μL of supernatant and observe under a microscope whether there are a large number of tubular structures. Then, take the supernatant to a 15 ml centrifuge tube and centrifuge at 90 g for 5 minutes. After centrifugation, discard the supernatant and add 3-5 mL of TrypLE TM Expression enzyme for enzymatic hydrolysis. The whole enzymatic hydrolysis process takes about 3-5 minutes, and the enzymatic hydrolysis status needs to be observed every 2 minutes during the enzymatic hydrolysis process. Pipette 20μL tissue fluid into a 10cm culture dish, and place the culture dish under a microscope to observe whether the testicular tissue is enzymatically hydrolyzed into single cells. If most of the testicular tissue blocks have been enzymatically hydrolyzed into single cells, immediately place them in a centrifuge and centrifuge at 300g for 5 minutes. After centrifugation, discard the supernatant and add DMEM / F12 medium containing 10% FBS to terminate digestion.

[0058] Step 2: Resuspend and count testicular cells:

[0059] In order to remove tissue chunks and undigested tissue chunks, filter the cell suspension using a 40 μm filter membrane. Centrifuge the filtered filtrate at 300 x g for 5 min, and discard the supernatant after centrifugation. Subsequently, add DMEM / F12 medium containing 1% P / S to the centrifuge tube to resuspend the cells, and centrifuge at 300 x g for 3 min. Finally, count the cells using a hemocytometer, resuspend the cells with DMEM / F12 medium, and continue to centrifuge at 300 x g for 5 min. Discard the supernatant after centrifugation and add the culture system. In this example, the evenly suspended cells are transferred to a 96-well plate in sequence, with approximately 5×10 4The culture system of this embodiment is (hereinafter referred to as K3 culture medium): 5% FBS (fetal bovine serum), 10% KSR (KSR cell culture medium) and 1% P / S DMEM basic culture medium.

[0060] Step 3: In vitro culture and observation of testicular tissue:

[0061] First, place the testicular cells in a 10 cm culture dish and make three 200 μL droplets of K3 medium. Then, wash the testicular cell clusters three times in the droplets of K3 medium, replacing a new pipette tip each time during the washing process. Next, use a pipette to suck up the counted cell clusters and transfer them to a U-bottom 96-well plate for culture. High cell density helps promote cell-to-cell interactions, but too high a density will cause the testicular organoids to be too large, thereby limiting the supply of nutrients and oxygen. A lower cell density may hinder the formation of organoids. Therefore, this application chooses to use 5×10 cells per organoid. 4 cells, and add 200 μL K3 medium to each well. Finally, place the U-bottom 96-well plate in an incubator containing 5% carbon dioxide for gas-liquid culture, ensuring that gas and nutrients pass through at the same time. The medium in the U-bottom 96-well plate was replaced every other day, and fresh K3 medium was added. Cultured according to the above method for three weeks, and testicular organoids were collected every other week for morphological and histological observations, such as Figure 2 shown.

[0062] Example 2

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

[0064] In the third step, first, three 200 μL drops of To medium were made in a 10 cm culture dish. The testicular cell clusters were washed three times in the drops of To medium. After that, the separated cell clusters were transferred to a U-bottom 96-well plate for culture. 5×10 4 cells, and then add 200 μL To medium. Finally, the U-bottom 96-well plate was placed in an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through at the same time. During the culture process, the medium was replaced every 24 hours and fresh To medium was added.

[0065] Example 3

[0066] Different from Example 1, in the third step, 200 μL of medium containing 10% KSR and 1% P / S DMEM basic was added to each well to complete the resuspension culture of cells. The above medium is referred to as K4 medium.

[0067] In the third step, first, three 200 μL droplets of K4 medium were made in a 10 cm culture dish. The testicular cell clusters were washed three times in the droplets of K4 medium. After that, the separated cell clusters were transferred to a U-bottom 96-well plate for culture. 5×10 4 cells, and then add 200 μL K4 medium. Finally, the U-bottom 96-well plate was placed in an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through at the same time. During the culture process, the medium was replaced every 24 hours and fresh K4 medium was added.

[0068] Example 4

[0069] Cell Type Identification of Testicular Organoids:

[0070] Spermatogenesis in mammals is a highly coordinated process regulated by multiple somatic cells, including supporting cells, Leydig cells, and peritubular myoid cells. Supporting cells are in close contact with testicular germ cells and play a key role in the development and maturation of spermatogenic cells into functional sperm. Therefore, this application collected testicular organoids cultured for 21 days and detected specific cell types therein.

[0071] First, the testicular organoids were washed three times in saline, transferred to a 3 cm culture dish containing 4% paraformaldehyde solution, and fixed in a 4°C refrigerator overnight. The next day, the fixed testicular organoids were wrapped with a mask cloth, dehydrated with gradient alcohol, and finally placed in heated and melted paraffin and immersed in wax overnight. Then, the testicular organoids were embedded in new paraffin. The solidified wax block was fixed on a slicer with a slice thickness of 5 μm. The paraffin sections of the testicular organoids were placed in a 65°C oven for 1 hour, rehydrated with gradient alcohol, washed in PBS for 10 minutes, repaired in an antigen repair solution at 96°C for 10 minutes, and cooled to room temperature.

[0072] Next, wash the slides in PBS with 0.2% Triton X100 for 15 minutes, and then wash with TBS three times, 5 minutes each time. Place the slides flat in a wet box, and add 50 μL of PBS solution containing 3% BSA, 10% goat serum albumin, and 2 μL Tween 20 to each slide. Cover the slide with a sealing film, cover the wet box lid, and seal at 37°C for 45 minutes. After the sealing is completed, remove the sealing film, add 50 μL of primary antibody dilution to the slide, and dilute the primary antibody with β-catenin at 1:200. After adding the primary antibody, cover with a sealing film, put the wet box in a sealing bag, and incubate overnight at 4°C.

[0073] On the second day, take out the wet box and rewarm at room temperature for 30 minutes. Then, wash three times with TBST, 10 minutes each time. Next, place the slide in the wet box, add 50μL of fluorescent secondary antibody dilution to each slide, cover with sealing film, and incubate in a 37°C oven for 45 minutes. After incubation, place the slide in a wash cup and wash three times with TBST, 10 minutes each time. Add 50μL of Hoechst33342 to each slide and stain the nucleus at room temperature for 6 minutes. After staining the nucleus, wash three times with PBS, 2 minutes each time. Finally, use an anti-quencher to seal the slide.

[0074] On the third day, from Figure 3 It can be seen that both the testicular tissue 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 we cultured in vitro have similar biological functions as the testicular tissue in vivo.

[0075] Example 5

[0076] Development of Testicular Organoids:

[0077] In order to observe the development of testicular organoids, the testicular organoids cultured for 21 days in Example 1, Example 2 and Example 3 were collected for HE staining. The staining results are shown in FIG. Figure 4 , Figure 5 and Figure 6 As shown. The specific method is to cut the testicular organoids into 5μm slices, put the slides in an oven for 1 hour, and then dewax and rehydrate with gradient alcohol. Then, perform hematoxylin staining for 5 minutes, rinse with tap water for 5 minutes, and then wash with distilled water for 2 minutes. Then, put the slides into 1% hydrochloric acid alcohol for differentiation 3 times, each time for 3 seconds.

[0078] Afterwards, bluing was performed with 45°C tap water for 5 minutes, and then washed with distilled water for 2 minutes. The slides were placed in 50% alcohol, 70% alcohol, and 80% alcohol for 2 minutes each, eosin stained for 1 minute, and then 95% alcohol I and 95% alcohol II for 3 minutes each, 100% alcohol I and 100% alcohol II for 5 minutes each, and xylene I and xylene II for 5 minutes each. Finally, neutral resin was added to the slide and covered with a coverslip. After sealing, the slides need to be air-dried in a fume hood for 2 hours, after which the slides can be placed in a slide box and stored at room temperature. By observing HE staining, we found that the developmental process of testicular organoids cultured in vitro using the K3 culture system of Example 1 was similar to that in vivo, and complete tubular structures appeared in the testicular tissue, such as Figure 4 As shown in Example 2. Figure 5 As shown in Example 3 Figure 6 As shown, the testicular organoids under the culture system did not have tubular structures, and the sizes of the testicular organoids varied.

[0079] The blood-testis barrier is involved in the construction of the spermatogenic microenvironment, provides an immune barrier, and maintains normal spermatogenesis. To evaluate the integrity of the blood-testis barrier in testicular organoids cultured in vitro, PAS staining was performed on testicular organoids cultured for 21 days. Figure 7 shown.

[0080] The specific method is to put the slices cut into 5μm into the oven for 1h, xylene I and xylene II for 5min each, and then wash with 100% alcohol for 5min, 90% alcohol, 80% alcohol and 70% alcohol for 2min each. Then perform periodic acid Schiff staining according to the instructions for use of the kit of Biyuntian Company. Add 100μL of periodic acid solution to each slide, react in a wet box in the dark for 10min, and then wash with distilled water for 5min. Add 100μL of Schiff reagent to each washed slide, place the slide flat in a wet box, and stain in a 37℃ oven in the dark for 45min. After incubation, wash with distilled water for 5min to remove the staining solution. Then, place the slide in a hematoxylin washing cup and stain for 30s. After staining, rinse with distilled water three times, 3s each time, to remove the staining solution. After rinsing with distilled water, place the slide in 1% hydrochloric acid alcohol for differentiation for 30 seconds, rinse twice with tap water for 3 seconds each time, and remove the differentiation solution. Let the tap water turn blue for 5 minutes. Place the slide in 90% alcohol and 100% alcohol for 2 minutes each, and xylene I and xylene II for 5 minutes each. Finally, use a neutral resin to seal the slide, and place the sealed slide in a fume hood to air dry for 2 hours. Figure 7 PAS staining showed that the blood-testis barrier of testicular organoids cultured in vitro was intact, showing structural characteristics similar to those of in vivo tissues.

[0081] Therefore, the present invention simplifies the experimental process by optimizing the culture system and successfully utilizes 5×10 4 Testicular organoids were constructed from cells in a U-bottom 96-well plate, and testicular organoids with obvious seminiferous tubules structure were successfully cultured under completely in vitro conditions, which can provide direction for solving the problem of clinical infertility in humans.

[0082] The above-described embodiments of the present invention do not constitute a limitation on the protection scope of the present invention. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for efficiently constructing pig testicular organoids in vitro, characterized in that: The following methods are included: S1: Enzymatic hydrolysis of piglet testicular tissue: the piglets are newborn to 4-week-old piglets; S2: Testicular cell resuspension counting: The testicular cells obtained after enzymatic hydrolysis in S1 were uniformly suspended and collected in turn into a U-bottom 96-well plate, and DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S was added to each well to complete the cell resuspension culture; S3: In vitro culture of testicular organoids: The testicular cell clusters were separated and transferred to a U-bottom 96-well plate and cultured in DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S; the number of cells in each well of the U-bottom 96-well plate was 5×10 4 cells; place the U-bottom 96-well plate in an incubator containing 5% carbon dioxide for suspension culture; during the culture process, replace the culture medium every 24 hours. Testicular organoids can be cultured in vitro for 3 weeks.

2. The method for efficiently constructing pig testicular organoids in vitro according to claim 1, characterized in that: S1 is to rinse the testicular tissue of 4-week-old piglets with normal saline, then rinse with alcohol for disinfection, and place it in a sterile beaker and rinse with normal saline containing 1% P / S; after washing, transfer the testicular tissue to a culture dish covered with PBS and soak it; remove excess testicular tissue, and transfer the separated testicular tissue to a new culture dish, and rinse it again with PBS containing 1% P / S; after rinsing, cut the testicular tissue into small pieces, remove the white connective tissue, and transfer it to a centrifuge tube; add PBS and collagenase IV to the minced meat, and digest it in a 37°C water bath for 15-20 minutes; after digestion, take the supernatant and centrifuge; after centrifugation, discard the supernatant, and add TrypLE TM Expression enzyme for enzymatic hydrolysis; after the testicular tissue blocks are enzymatically hydrolyzed into single cells, they are placed in a centrifuge for centrifugation; after the centrifugation is completed, the supernatant is discarded, and DMEM / F12 containing 10% FBS is added to terminate the digestion.

3. The method for efficiently constructing pig testicular organoids in vitro according to claim 1, characterized in that: S2 is to filter the testicular cells obtained after enzymatic hydrolysis through a 40 μm filter membrane; centrifuge the filtered cell suspension and discard the supernatant; resuspend the cells in DMEM / F12 containing 1% P / S and centrifuge again; take the supernatant after centrifugation, count the cells using a hemocytometer, and resuspend the cells in DMEM / F12 culture medium; centrifuge again and discard the supernatant, collect the evenly suspended cells in a U-bottom 96-well plate in turn, and each well has 5×10 4 cells; 200 μL of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S was added to each well to complete the resuspension culture of the cells.

4. The method for efficiently constructing pig testicular organoids in vitro according to claim 1, characterized in that: S3: First, make 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 in droplets of DMEM basic medium containing 5% FBS, 10% KSR and 1% P / S; then, transfer the separated cell clusters to a U-bottom 96-well plate for culture; add 5×10 4 cells, and then add 200 μL of DMEM basic culture medium containing 5% FBS, 10% KSR and 1% P / S; finally, put the U-bottom 96-well plate into an incubator containing 5% carbon dioxide for suspension culture to allow gas and nutrients to pass through at the same time; during the culture process, the culture medium was replaced every 24 hours.

5. The method for efficiently constructing pig testicular organoids in vitro according to claim 1, characterized in that: Both the 21-day testicular organoids cultured by this method and the 28-day testicular tissue in vivo clearly expressed β-catenin positive signals.

6. The method for efficiently constructing pig testicular organoids in vitro according to claim 1, characterized in that: The volume of testicular organoids cultured for 21 days using this method was 649*301μm.

Images