Application of pirfenidone in preparation of testicular organs

Abstract

The invention relates to application of pirfenidone in preparation of testicular organs, and the pirfenidone can improve one or more properties and / or functions of the testicular organs, especially improve blood testis barriers and improve spermatogenesis efficiency. The invention also provides a method, a culture medium and a kit for preparing the testicular organoid.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology. More specifically, the present invention relates to the application of pirfenidone in testicular organoid culture and a testicular organoid culture method. Background Art

[0002] One in seven couples in the world suffers from infertility, of which male factors account for about 30-50%. Although there are many reports on the causes of male infertility, 60-75% of male infertility cases are classified as idiopathic spermatogenesis disorders. Establishing an in vitro spermatogenesis research platform is the first stage in studying the pathogenesis of infertility. Testicular organoids can simulate the tissue structure, microenvironment and physiological function of the testicle in vitro, which provides an ideal model for studying testicular development and infertility treatment.

[0003] At present, the method of constructing testicular organoids mainly involves digesting the testicular tissue from newborn mice into single cells and then reaggregating them, forming seminiferous tubule-like structures through spontaneous reaggregation of supporting cells, and encapsulating germ cells in them to continue the arrangement of germ cells similar to that in vivo. However, no testicular organoids that can complete meiosis and obtain functional haploid germ cells have been reported so far, regardless of whether they are made with the help of biological materials such as soft agar or methylcellulose, or even with the help of 3D bioprinting.

[0004] The inventor team successfully established testicular organoids that can produce functional sperm by optimizing the system. This method is recorded in the Chinese invention patent announcement number CN112961822B. However, data analysis shows that the testicular organoids are still different from the testicles in vivo, such as: microenvironment disorder, significant downregulation of the expression of proteins related to the blood-testis barrier, spermatocyte apoptosis, and low sperm quantity and quality, resulting in low live birth rate of offspring.

[0005] The art still needs a method for obtaining testicular organoids with better spermatogenesis function, as well as reagents and culture methods that effectively improve the culture efficiency of testicular organoids, repair the blood-testis barrier, improve the microenvironment in the testis and / or promote microenvironment homeostasis in the testis. Summary of the invention

[0006] The inventors of the present invention have discovered a new use of pirfenidone (PFD). When used for testicular organoid culture, pirfenidone can significantly promote spermatogenesis in testicular organoids. Specifically, the inventors found that pirfenidone can increase the expression level of blood-testis barrier-related constituent proteins in testicular organoids, has the effect of repairing the blood-testis barrier and optimizing the spermatogenic microenvironment, thereby promoting the generation of spermatocytes, and ultimately increasing the number and quality of sperm produced by testicular organoids. The improvement in sperm quantity and quality is reflected in the increased live birth rate of offspring. The present invention applies pirfenidone to the culture of testicular organoids for the first time, and is the first to discover its above-mentioned functions, thereby completing the present invention.

[0007] Therefore, in a first aspect, the present invention provides a method for culturing testicular organoids, the method comprising culturing testicular cells from testicular tissue into testicular organoids in the presence of pirfenidone. Preferably, pirfenidone is used at a concentration of 10-1000 μM.

[0008] In a second aspect, the present invention provides a method for culturing testicular organoids, the method comprising the following steps:

[0009] (1) providing dissociated testicular cells from testicular tissue;

[0010] (2) culturing the dispersed testicular cells of step (1) into aggregate spheres;

[0011] (3) differentiating the polymer spheres of step (2) into testicular organoids in the presence of pirfenidone; and

[0012] (4) Optionally, further culturing the testicular organoids obtained in step (3).

[0013] In a third aspect, the present invention provides a testicular organoid obtained by the method of the first aspect and the second aspect.

[0014] In a fourth aspect, the present invention provides use of pirfenidone in improving the blood-testis barrier in testicular organoids, improving the microenvironment in testicular organoids and / or promoting microenvironment homeostasis in testicular organoids.

[0015] In a fifth aspect, the present invention provides use of pirfenidone in improving spermatogenesis in testicular organoids.

[0016] In a sixth aspect, the present invention provides a culture medium for culturing testicular organoids, comprising pirfenidone.

[0017] In a seventh aspect, the present invention provides a kit for culturing testicular organoids, comprising the culture medium of the seventh aspect.

[0018] In each of the aforementioned aspects, the testicular organoid can be from an animal having testicles, such as a mammal, for example, rat, mouse, pig, dog, cat, monkey and human.

[0019] The present invention has at least the following advantages.

[0020] The testicular organoids obtained after adding pirfenidone in the present invention are similar to the testicles in vivo, specifically, they have a tissue structure, and various types of germ cells (such as spermatogonial stem cells, spermatocytes and sperm cells) and testicular somatic cells are present in the testicular organoids.

[0021] The use of pirfenidone repairs the blood-testis barrier in testicular organoids, improves the microenvironment, and significantly increases the number of spermatocytes and sperm.

[0022] The use of pirfenidone significantly increases the live birth rate of offspring after the haploid sperm cells produced by the cultured testicular organoids are used to form embryos and transplanted into the mother.

[0023] The testicular organoids established by the method of the present invention can produce functional sperm with advantages in quantity and quality, providing a better platform for related research using testicular organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 1 is a schematic diagram showing the process of screening small molecule compounds using testicular organoids in Example 1. Candidate small molecule compounds are added in two culture stages, and the types and quantities of the candidate small molecules are shown in the figure.

[0025] Figure 2 The scatter plot shows the percentage of haploid cells obtained after treating testicular organoids with various candidate small molecules in Example 1, and the abscissa is the data points of small molecule drugs, a total of 142 small molecule drugs. The data points treated with pirfenidone are shown with arrows.

[0026] Figure 3 This is the structural staining result of testicular organoids treated with pirfenidone. Figure 3 a is the bright field photos of testicular organoids and HE staining of sections on days 1, 8, 14, and 25. Figure 3 b is the antibody fluorescence staining result of somatic cell markers in testicular organoids on day 25. Figure 3 c is the result of antibody fluorescent staining of germ cell-specific markers in testicular organoids on day 25.

[0027] Figure 4 Graph showing the expression levels of blood-testis barrier-related proteins TJP1 and GJA1 in the pirfenidone group and the control group. Figure 4 a is the immunofluorescence staining image, Figure 4b is the statistical analysis chart of the results.

[0028] Figure 5 The expression of markers of germ cells at various levels (ZBTB16, STRA8, XY body (a form of H2AFX protein) and CREM) in testicular organoids of the pirfenidone group and the control group. Figure 5 a is the statistical analysis diagram of immunofluorescence results, Figure 5 b is the corresponding coloring image.

[0029] Figure 6 Results of single-cell sequencing of testicular organoids from in vivo control (testes were harvested from wild-type C57 mice 30 days after birth and digested into single cells for analysis), pirfenidone group, and control group. Figure 6 a shows that Figure 6 Figure b shows the results of expression analysis of sperm stage-expressed genes.

[0030] Figure 7 The figure shows the embryonic development and offspring obtained after haploid round sperm injection (ROSI; Round Spermatid Injection) of haploid round sperm obtained by testicular organoid sorting in each experimental group in Example 5, Figure 7 a is a photo of 2-cell embryos in the control group and pirfenidone group, and a white light photo of 2-cell embryos obtained by injection of round spermatozoa from the testicular organoid tubules of the DMSO or SM group. Scale bar = 100 μm. Figure 7 b is the embryonic development table of the in vivo, control group and pirfenidone group, and the embryonic development and offspring statistics of the round spermatozoa obtained from the testicular organoids. **p<0.01, chi-square test. Figure 7 c is the photos of the offspring in the control group and pirfenidone group.

[0031] Figure 8 The physiological characteristics of the offspring in Example 5 are shown. Figure 8 a is the placenta weight, Figure 8 b is the body weight at different time points after birth (0 weeks, 1 week, 2 weeks, 3 weeks), Figure 8 c is a photo of the offspring and its offspring.

[0032] Fig. 9 The results of germ cell staining of human testicular organoids obtained in Example 6 using culture medium supplemented with or without pirfenidone are shown. Fig. 9 a shows the proportion of apoptotic cells that co-express DDX4 and Cleaved-caspase3; Fig. 9 b shows the proportion of apoptotic cells expressing DDX4; Fig. 9 c shows a photograph of fluorescent staining.

[0033] Fig.10 The staining results of the blood-testis barrier-related proteins TJP1 and GJA1 of human testicular organoids obtained using culture medium with or without the addition of pirfenidone in Example 6 are shown. Fig.10 a shows a fluorescent staining photograph; Fig.10 b shows the quantified fluorescence intensity of two blood-testis barrier-related proteins, TJP1 and GJA1. In vivo: data of intact tissues without in vitro culture; control group: no pirfenidone added; pirfenidone group: pirfenidone added. DETAILED DESCRIPTION

[0034] 1. Definition

[0035] The term "pirfenidone" is a small molecule compound with the chemical name 5-methyl-1-phenyl-2-(1H)pyridone. Pirfenidone is a white or light yellow crystalline powder and is easily soluble in water.

[0036] The term "testicular organoid" in the context of the present invention refers to a three-dimensional model cultured in vitro, composed of cells and having a structure and function similar to that of the testis.

[0037] The term "blood-testis barrier (BTB)" refers to an important ultrastructure of the mammalian testicular spermatogenic epithelium, located near the basement membrane and composed of multiple connections between adjacent supporting cells, including tight junctions, basal extracellular specializations (ES, ectoplasmic specializations), desmosomal junctions and gap junctions.

[0038] The term "testicular cells" in the context of the present invention encompasses all cell types in testicular tissue. Therefore, the testicular cells in the context of the method of the present invention are a cell mixture, preferably obtained directly by digesting testicular tissue. There are 17 cell types among the cells dispersed from testicular tissue, 14 of which are testicular germ cells at various stages of differentiation, and the other 3 are testicular microenvironment somatic cells. The 14 testicular germ cells include: 3 spermatogonia, including spermatogonial stem cells (SSC), differentiating spermatogonia (Diff.ing SPG), and differentiated spermatogonia (Diff.ed SPG); 7 spermatocytes, including 3 leptotene spermatocytes (L1-L3), zygotene spermatocytes (Z), pachytene spermatocytes (P), diplotene spermatocytes (D), primary spermatocytes and secondary spermatocytes (SPC7) in the mitotic stage; and 4 spermatids (S1-S4). The somatic cells in the testicular microenvironment are mainly divided into three categories: supporting cells, Leydig cells and myoid cells.

[0039] The term "Sertoli cells" or "Sertoli cells" are also called somatic cells present in the seminiferous tubules. Sertoli cells participate in the formation of the blood-testis barrier and provide protection for spermatocyte meiosis and spermatogenesis.

[0040] The term "spermatogenesis" in the context of the present invention refers to the process of development from spermatogonia to mature sperm, during which the spermatogonia will successively go through the stages of "round spermatozoa" and "elongated spermatozoa" and finally develop into mature sperm. All cells in the process of spermatogenesis are collectively referred to as "spermatogenic cells".

[0041] The terms "haploid differentiation efficiency" or "haploid efficiency" have the same meaning in the context of the present invention, and refer to the percentage of haploid cells (e.g., sperm cells) in the total number of organoid cells cultured in vitro.

[0042] The term "offspring live birth rate" in the context of the present invention refers to the percentage of offspring formed by in vitro fertilization that have one of the four vital signs of heartbeat, breathing, umbilical cord pulsation, and voluntary muscle contraction after delivery, compared to the number of implanted embryos.

[0043] The term "FFPE sample" refers to forminaldehyde-fixed paraffin-embedded samples, which is a form of preserved tissue specimens well known in the art.

[0044] The term "subject" or "animal" in the context of the present invention refers to any animal with testicles. For example, the subject or animal can be a vertebrate, preferably a mammal. The mammal includes, but is not limited to, rodents, monkeys, farm animals, sports animals and pets, such as mice, rats, rabbits, dogs, cats, monkeys, pigs. More preferably, the subject or animal is a human.

[0045] 2. Pirfenidone

[0046] The present invention relates to the application of pirfenidone in promoting spermatogenesis in testicular organoids. This application mode is completely different from the previously known applications of pirfenidone.

[0047] Pirfenidone is known to be an oral anti-fibrotic and anti-inflammatory drug, the main pathway of action of which is to inhibit the production of TGF-β and collagen stimulated by TGF-β, and to reduce the production of TNF-α and IL-1β. Pirfenidone is commonly used to prevent or treat various fibrotic and inflammatory diseases. In the reproductive system, pirfenidone repairs the ovarian function of elderly and obese female mice by reducing inflammation and fibrosis.

[0048] In the present invention, pirfenidone is used in cell culture to obtain testicular organoids, and the testicular organoids obtained thereby have improved spermatogenesis. When used for in vitro testicular organoid culture, pirfenidone is used at a concentration of 10-1000 μM, preferably at a concentration of 50-500 μM, and more preferably at a concentration of 100-200 μM. For example, pirfenidone is added to one or more culture media of the organoid culture process at the above concentrations.

[0049] 3. Culture of Testicular Organoids

[0050] "Organoids" are cell aggregates constructed through three-dimensional culture that can represent some key characteristics of organs and reproduce some of the functions of the corresponding organs.

[0051] "Testicular organoids" refer to aggregates constructed through three-dimensional culture with a tubular structure similar to that of testicles. Sperm is not necessarily present in testicular organoids. Sperm production is required by promoting the differentiation of germ cells.

[0052] The present invention provides a method for culturing a testicular organoid, the method comprising culturing a testicular organoid in the presence of pirfenidone. The culturing method of the present invention is performed in vitro. The culturing of the testicular organoid can be performed using methods known in the art. In a preferred embodiment, dispersed testicular cells, such as dispersed testicular cells obtained by digesting testicular tissue, are used for the culturing of testicular organoids.

[0053] More specifically, the testicular organoid culture method of the present invention comprises the following steps:

[0054] (1) Provide dispersed testicular cells;

[0055] (2) culturing the dispersed testicular cells of step (1) into aggregate spheres;

[0056] (3) culturing the polymerized spheres of step (2) into testicular organoids in the presence of pirfenidone; and

[0057] (4) Optionally, further culturing the testicular organoids obtained in step (3).

[0058] In a more specific embodiment, the testicular organoid culture method of the present invention comprises the following steps:

[0059] (1) Provide dispersed testicular cells;

[0060] (2) resuspending the testicular cells in a polymerase culture medium and culturing at 34-37° C. for 40-72 hours to obtain polymerases;

[0061] (3) culturing the cells on a solid matrix using a medium supplemented with pirfenidone at 34 to 37° C. for at least 5 days; and

[0062] (4) Optionally, the testicular organoids obtained in step (3) are further cultured in a culture medium with or without pirfenidone at 34-37°C.

[0063] 3.1 About step (1)

[0064] The purpose of step (1) is to provide a starting cell mixture for testicular organoid culture. In terms of cell composition, the dispersed testicular cells used in step (1) should include somatic cells contained in testicular tissue, which are used to form the main structural part of the testicular organoid; and spermatogenic cells, which will develop into sperm under appropriate conditions. In some embodiments, the testicular cells at least include supporting cells and spermatogonia, preferably each of interstitial cells, supporting cells, myoid cells and spermatogonia.

[0065] The testicular cells of the present invention can be derived from the testicular tissue of any animal with testicles. The animals are mammals, such as mice, rats, rabbits, dogs, cats, pigs, monkeys and humans, birds, etc. The testicular cells can be from healthy individuals or individuals with diseases. For example, the testicular cells can be from a biopsy sample.

[0066] In some embodiments, the mixture of testicular cells in step (1) is directly obtained from testicular tissue. Partial or complete testicular tissue can be used so that the testicular cells used for culture contain at least supporting cells and spermatogonia. In a preferred embodiment, complete testicular tissue is used to ensure that various necessary somatic cells and spermatogenic cell types are contained. When using partial testicular tissue, the testicular tissue contains one or more cells selected from interstitial cells, supporting cells, myoid cells and spermatogonia, preferably at least supporting cells and spermatogonia, and more preferably each of interstitial cells, supporting cells, myoid cells and spermatogonia. Testicular tissue can be processed into discrete testicular cells by physical or chemical means. The physical means are, for example, broken into small pieces. The chemical means are, for example, digested using enzymes. In a preferred embodiment, the dispersed testicular cells are obtained by enzymatic digestion. Enzymatic digestion can remove the structure that originally exists between cells to connect cells, thereby dispersing cells.

[0067] Preferably, the enzyme used in step (1) is collagenase and / or trypsin. The collagenase is, for example, collagenase I, collagenase II, collagenase III and collagenase IV. More preferably, the collagenase is collagenase IV. More preferably, after treatment with collagenase such as collagenase IV, trypsin is used for treatment. Using two enzyme treatments in succession can better disperse the cells. After using enzyme dispersion, collect all cells in polymeric sphere culture medium and perform step (2).

[0068] In one embodiment, the dispersed testicular cells are obtained by the following two-step enzyme digestion method, specifically comprising: taking the testicles of an animal, removing the testicular capsule, mixing with collagenase IV, digesting, centrifuging and removing the supernatant; then mixing with trypsin, digesting, terminating the digestion, centrifuging and removing the supernatant, to obtain dispersed spermatogonial stem cells. Preferably, the animal is a mouse.

[0069] In some embodiments, the mixture of testicular cells in step (1) can be subjected to expansion culture before being used in subsequent culture steps. In some embodiments, the mixture is subjected to the expansion culture. In some embodiments, different types of cells in the mixture are separately expanded and cultured, and remixed after culture for use in subsequent steps.

[0070] 3.2 About step (2)

[0071] In some embodiments of the present invention, in step (2), the dispersed testicular cells of step (1) are cultured into aggregates using an aggregate medium.

[0072] The polymeric sphere culture medium comprises a basal culture medium and a nutritional additive. The basal culture medium may be a MEM culture medium or a modified form thereof, such as a MEMα culture medium. The nutritional additive may include one or more of serum or serum substitutes, amino acids, pyruvic acid or sodium pyruvate, antioxidants, and neurotrophic factors. The amino acids may be L-glutamine.

[0073] In a preferred embodiment, the polymerase culture medium comprises MEMα medium and KOSR (knockout serum replacement, a serum substitute). Preferably, the polymerase culture medium is based on a mixture of MEMα and KOSR, wherein the volume ratio of MEMα to KOSR is (88-92): (8-12).

[0074] In a specific embodiment, the polymeric sphere culture medium is obtained by adding KOSR, L-glutamine and glial cell line derived neurotrophic factor (GDNF) to MEMα culture medium. Preferably, the volume ratio of MEMα to KOSR is (88-92): (8-12). Preferably, the concentration of L-glutamine is 0.1mM-2.5mM, preferably about 2mM. Preferably, the concentration of GDNF is 10ng / mL-30ng / mL, more preferably about 20ng / mL. For example, the polymeric sphere culture medium has a composition as shown in Table 1.

[0075] In some embodiments of the present invention, the initial seeding density of the culture in step (2) is about 30,000-60,000 cells / well.

[0076] In some embodiments of the present invention, the culture in step (2) is a suspension culture. Preferably, the culture in step (2) is carried out using a U-bottom culture plate, such as a 96-well U-bottom culture plate. Preferably, the culture in step (2) is carried out at 34 to 37° C. and / or lasts for 40 to 72 hours. When the cells are observed to form relatively compact and round aggregates, step (2) can be terminated and step (3) can be started.

[0077] In a specific embodiment of the present invention, step (1) and step (2) can be carried out according to the method described in CN112961822B.

[0078] 3.3 About step (3)

[0079] The key step of the culture method of the present invention is the step of culturing the polymerized spheres into testicular organoids in the presence of pirfenidone, i.e., step (3) in the above method. The culture process in step (3) includes using a culture medium with differentiation inducing factors to promote the formation of organoids.

[0080] In some embodiments, step (3) can be divided into two culturing stages, in which different culture media are used, which are sequentially referred to as steps (3a) and (3b) for the convenience of description.

[0081] In step (3a), the aggregates are cultured in a spermatogonial stem cell culture medium, and in step (3b), the aggregates are cultured in a testicular differentiation medium to promote the formation of testicular organoids.

[0082] In some embodiments of the present invention, the spermatogonial stem cell culture medium used in step (3a) comprises a basal medium and a nutritional additive. The basal medium can be a StemPro34 culture medium, which is a serum-free culture medium. The nutritional additive can include one or more of a supplement, serum or serum substitute, carbohydrate, vitamin, amino acid, cytokine, hormone, antioxidant and neurotrophic factor adapted to the culture medium. The cytokine can be a stem cell factor.

[0083] In one embodiment, the spermatogonial stem cell culture medium comprises the following components: StemPro34 basal medium, StemPro34 supplement, L-glutamine, basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). Preferably, the spermatogonial stem cell culture medium also comprises the following components: glucose, bovine serum albumin (BSA), ascorbic acid, and fetal bovine serum (FBS). More preferably, the spermatogonial stem cell culture medium is a mixture of StemPro34 basal medium and StemPro34 supplement as the basal medium, wherein the volume ratio of StemPro34 basal medium to StemPro34 supplement is (95-98): (2-5). Preferably, the final concentration of the non-essential amino acids is 0.5-2 mM, the final concentration of L-glutamine is 0.1-2.5 mM, the final concentration of bFGF is 5-15 ng / mL, the final concentration of LIF is 100-1500 U / mL, the final concentration of EGF is 5-15 ng / mL, the final concentration of GDNF is 10-30 ng / mL, the final concentration of glucose is 0-8 mg / mL, the final concentration of BSA is 0-10 mg / mL, the final concentration of ascorbic acid is 0-150 mM, and / or the final concentration (volume concentration) of FBS is 0-1.5%. In a specific embodiment, the spermatogonial stem cell culture medium has the composition shown in Table 2.

[0084] In some embodiments of the present invention, the testicular differentiation medium used in step (3b) comprises a basal medium and a nutritional additive. The basal medium may be a MEM medium or a modified form thereof, such as a MEMα medium. The nutritional additive may include one or more of serum or serum substitutes, amino acids, and antioxidants. In a specific embodiment, the polymerase culture medium is obtained by adding KOSR to a MEMα medium. Preferably, the volume ratio of MEMα to KOSR is (88-92): (8-12). In a specific embodiment, the spermatogonial stem cell culture medium has a composition as shown in Table 3.

[0085] As an essential step of the present invention, pirfenidone is added to at least one of the spermatogonial stem cell culture medium and the testicular differentiation culture medium, preferably both. The amount of pirfenidone used is 10 μm-1 mM, preferably 100 μm. Pirfenidone can be added during the preparation of the culture medium, or after the culture medium is prepared.

[0086] In some embodiments of the present invention, the culture of step (3) such as step (3a) and step (3b) includes culturing the polymeric spheres on a solid support such as an agar block to achieve gas-liquid interface culture. The concentration of the agar block can be 1-1.5%, and its size can be placed in the hole of the culture plate, for example, it can have a size of about 1.5cm x 1.5x 1cm. As a method of gas-liquid interface culture, culture medium can be added to the hole of the culture plate where the agar block is placed, so that the liquid level of the culture medium reaches 70% to 98% of the height of the agar block, preferably 80% to 95%, and more preferably 90%. Preferably, the agar block is pre-soaked in the culture medium for 1 to 6 hours before starting the culture step.

[0087] In a specific embodiment of the present invention, the culturing in step (3a) is performed at 34-37° C. and / or lasts for 4-6 days.

[0088] In a specific embodiment of the present invention, the culturing in step (3b) is performed at 34-37° C. and / or lasts for 5-35 days, preferably 7-25 days.

[0089] In a specific embodiment, for example, when the testicular cells are derived from a non-human mammal, such as a mouse, the culture in step (3a) is cultured at 34-37° C. for 4-6 days; and the culture in step (3b) is cultured at 34-37° C. for 18-25 days. Preferably, pirfenidone is added to the culture medium in both step (3a) and step (3b).

[0090] In some embodiments, step (3a) may not be performed, i.e., the cells are directly cultured in a testicular differentiation medium. For example, when the testicular cells are derived from humans, the testicular cells are preferably cultured in the testicular differentiation culture of the present invention at 34 to 37° C. for 5 to 10 days, such as about 7 days.

[0091] At the end of step (3), the formation of testicular organoids with a lumen structure similar to that of the seminiferous tubules of the testis can be observed. The testicular organoids formed by the present invention contain a variety of germ cells, such as spermatogonia, spermatocytes and sperm, especially functional sperm.

[0092] 3.4 About step (4)

[0093] After step (3), step (4) may be optionally included to further culture the obtained testicular organoids, such as maintaining the culture.

[0094] The culture medium for maintaining culture may be the same or different from step (3). When step (3) uses more than one culture medium, the culture medium for maintaining culture may be the same or different from the culture medium used in the last step in step (3). In order to simplify the method, it is preferred to use a culture medium identical to step (3) or the last step in step (3), such as a testicular differentiation culture medium. In step (4), pirfenidone may or may not be included in the culture medium. When pirfenidone is included, pirfenidone may be added with reference to the concentration used in step (3).

[0095] The maintenance culture can be carried out at 34-37° C. for a relatively long period of time, for example, up to 40 days, 50 days, 60 days, 70 days, 80 days, 90 days or 100 days or even longer. When using the method of the present invention, it was found that when the testicular organoids were cultured for more than 100 days, the presence of sperm could still be observed.

[0096] 4. Effects of Pirfenidone

[0097] 4.1 Improvement of blood-testis barrier

[0098] The present invention finds that when pirfenidone is used in the culture of testicular organoids, the blood-testis barrier of the testicular organoids can be improved, the (spermatogenic) microenvironment can be improved, and the recovery of the microenvironmental homeostasis can be promoted. Therefore, the present invention provides the use of pirfenidone for improving the blood-testis barrier of testicular organoids, improving the microenvironment and / or promoting the recovery of the microenvironmental homeostasis.

[0099] In one embodiment, the present invention provides the use of pirfenidone to promote spermatogenesis by optimizing the testicular organoid microenvironment. The optimization of the microenvironment is mainly reflected in promoting the expression of blood-testis barrier junction proteins, such as gap junction proteins, tight junction proteins, etc.

[0100] Blood-testis barrier is known in the art to be of vital importance for normal spermatogenesis, and therefore improving the structural integrity and function of blood-testis barrier is conducive to spermatogenesis. The improvement of the structural integrity of blood-testis barrier can be measured by conventional methods in this area. For example, the state of blood-testis barrier can be measured by detecting the expression of blood-testis barrier-related proteins (specific markers). Such blood-testis barrier-related proteins such as TJP1 (tight junction protein 1, also referred to as ZO-1) or GJA1 (gap junction protein α-1).

[0101] In preferred embodiments, pirfenidone upregulates the expression of one or more blood-testis barrier-related proteins by at least 50%, such as about 60%, about 70%, about 80%, about 90%, about 100% or more, compared to not treated with pirfenidone.

[0102] 4.2 Improve spermatogenesis, sperm quality and quantity

[0103] The inventors also found that when pirfenidone is used in the culture of testicular organoids, spermatogenesis in the testicular organoids can be improved. Therefore, the present invention provides the use of pirfenidone for improving spermatogenesis in testicular organoids, and / or for improving the quality and / or quantity of sperm in testicular organoids.

[0104] In one embodiment, the present invention relates to the use of pirfenidone in increasing the number of germ cells in the middle and late stages of male spermatogenesis, specifically including increasing the number of pachytene and diplotene spermatocytes and the number of haploid spermatids in spermatocytes.

[0105] In one embodiment, the present invention relates to the use of pirfenidone in improving the quality of male germ cells. Pirfenidone can optimize the quantity and quality of sperm in testicular organoids, promote their maturation, and increase the live birth rate of their offspring.

[0106] The spermatogenesis process in animals includes multiple stages. First, spermatogonia undergo mitosis in the testicles, and the cells that proliferate and form the potential to form mature sperm are primary spermatocytes; primary spermatocytes undergo meiosis (meiosis I) to form secondary spermatocytes; each secondary spermatocyte divides into two equal haploid spermatids (spermatids) through meiosis (meiosis II); spermatids further mature and change their formation to form sperm with tails (spermatozoa). The cells in the aforementioned stages can be collectively referred to as "germ cells". Because spermatids are round haploid cells, they are also called "round spermatids" in some cases.

[0107] The improvement of spermatogenesis by pirfenidone can be reflected in the increase of the number and / or ratio of one or more germ cells relative to the case where pirfenidone is not used. The germ cells include but are not limited to: spermatogonia, spermatocytes (primary spermatocytes, secondary spermatocytes), spermatocytes and sperm, especially spermatocytes.

[0108] The spermatogenesis-related protein can be a protein specifically expressed by testicular tissue, or a protein specifically expressed by cells at various stages during spermatogenesis. The number and / or proportion of the germ cells can be determined by detecting the expression of the spermatogenesis-related protein.

[0109] Examples of spermatogenesis-related proteins include, but are not limited to: PLZF (promyelocytic leukemia zinc finger protein, an indicator molecule for differentiated spermatogonia), DDX4 (DEAD [Asp-Glu-Ala-Asp] box polypeptide 4), STRA8 (retinoic acid 8, a marker molecule for the initiation of meiosis in differentiated spermatogonia), SYCP3 (synaptonemal complex protein 3), γH2AX and CREM (cAMP response element regulator), KI67, ZBTB16 (indicator molecule for undifferentiated spermatogonia), γH2AX, SYCP1.

[0110] In a preferred embodiment, pirfenidone increases the number of multiple germ cells in the organoids: spermatogonia, spermatocytes and haploid sperm, compared to the case of no pirfenidone treatment. The ultimate goal of spermatogenesis is to form mature sperm, i.e., functional sperm, so the expression of mature sperm-related genes can also be used to indicate whether the spermatogenesis process is successful or whether it has been improved.

[0111] Mature sperm-related genes include but are not limited to Dnalc2, Spata46, Pdilt, Zdhhc19, Chd5, Ubr7, Akap4, Ica1, Acrv1, Fhl5, Capza3 and Csnka2ip.

[0112] In a preferred embodiment, pirfenidone upregulates the expression of one or more mature sperm-related genes in total germ cells by at least 50%, such as about 60%, about 70%, about 80%, about 90%, about 100% or more, compared to a condition in which pirfenidone is not used, meaning an increase in the number of mature sperm and the proportion of mature sperm in total germ cells.

[0113] Methods for measuring specific markers are known in the art, including but not limited to immunofluorescence staining, flow cytometry, transcriptome analysis, and the like.

[0114] The live birth rate of offspring is another indicator that can reflect the quality of sperm. Specifically, the live birth rate of offspring is the percentage of offspring that use sperm cells or sperm to form fertilized eggs, implant them into the mother for development, and eventually deliver and have one of the four vital signs of heartbeat, breathing, umbilical cord pulsation, and voluntary muscle contraction after delivery, accounting for the number of implanted embryos.

[0115] In a preferred embodiment, pirfenidone increases the live birth rate of offspring to that in vivo compared to not treated with pirfenidone.

[0116] 5. Uses of Testicular Organoids

[0117] The present invention also provides testicular organoids produced by the method of the present invention. Depending on the source of the cells as the starting material, the testicular organoids produced by the present invention can be organoids corresponding to any animal with testicles, such as mammalian testicular organoids, for example, rat, mouse, pig, dog, cat, monkey or human testicular organoids.

[0118] Compared with the case where pirfenidone is not used for treatment but other conditions are equivalent, the testicular organoids obtained by the method of the present invention have improved blood-testis barrier, spermatogenesis and other properties. It should be noted that even if the testicular organoids that are not treated with pirfenidone continue to be cultured according to the original culture conditions, they will only enter the maintenance culture state, and the spermatogenesis ability cannot be further increased, and they cannot be converted into the organoids of the present invention. This means that the testicular organoids of the present invention are organoids that are "qualitatively" different from those with cell composition, tissue structure and gene expression levels that are closer to the body.

[0119] The testicular organoids of the present invention are generally used for non-therapeutic purposes. In other words, the method for preparing testicular organoids of the present invention is a culture method, not a method for the purpose of treating a disease.

[0120] In some embodiments, the testicular organoids of the present invention can be used for scientific research purposes. The testicular organoid system reproduces the tissue structure and physiological environment of the mammalian testis, can observe and track the specific process of spermatogenesis, study sperm production and maturation, and provide an in vitro platform for reflecting the cellular environment of the testis in vivo. It can also be produced from cryopreserved testicular cells and cryopreserved. In addition, germ cell-somatic cell interactions and regulatory mechanisms can be studied.

[0121] In some embodiments, the present invention provides the use of the testicular organoids in disease model construction and drug screening, providing an accurate preclinical pipeline for drug testing and discovery and drug resistance research. The testicular organoids obtained by the present invention can be used to screen small molecule compounds that promote spermatogenesis, for example. Based on this, it can also be used to simulate infertility diseases and screen infertility drugs, as well as to evaluate the reproductive toxicity of drugs in the development of other drugs.

[0122] The present invention includes the following specific embodiments.

[0123] 1. A method for preparing testicular organoids, comprising culturing testicular cells into testicular organoids in the presence of pirfenidone.

[0124] 2. The method of claim 1, wherein the pirfenidone is added to a culture medium.

[0125] 3. The method of item 1 or 2, wherein the pirfenidone is used at a concentration of 10-1000 μM.

[0126] 4. A method for preparing testicular organoids, comprising the following steps:

[0127] (1) Provide dispersed testicular cells;

[0128] (2) culturing the dispersed testicular cells of step (1) into aggregates in an aggregate medium;

[0129] (3) culturing the polymerized spheres of step (2) into testicular organoids in the presence of pirfenidone; and

[0130] (4) Optionally, the testicular organoids obtained in step (3) are further cultured in a culture medium with or without pirfenidone at 34-37°C.

[0131] 5. The method according to any one of items 1 to 4, wherein the dispersed testicular cells in step (1) are obtained by digesting testicular tissue.

[0132] 6. The method of claim 5, wherein the digestion is performed by collagenase, such as collagenase IV.

[0133] 7. The method of any one of items 1-6, wherein the testicular cells comprise at least supporting cells and spermatogonia.

[0134] 8. The method of claim 7, wherein the testicular cells comprise interstitial cells, supporting cells, myoid cells and spermatogonia.

[0135] 9. The method according to any one of items 4 to 8, wherein the aggregate sphere culture medium comprises MEMα culture medium and KOSR.

[0136] 10. The method of claim 9, wherein the polymersphere culture medium further comprises L-glutamine and / or glial cell line-derived neurotrophic factor (GDNF).

[0137] 11. The method according to any one of items 4 to 10, wherein in step (2), the culture is carried out at 34 to 37°C for 40 to 72 hours.

[0138] 12. The method according to any one of items 4 to 11, wherein step (3) comprises culturing on a solid matrix at 34 to 37° C. for 5 to 40 days using a culture medium supplemented with pirfenidone.

[0139] 13. The method of claim 12, wherein step (3) comprises steps (3a) and (3b):

[0140] (3a) culturing on a solid matrix using a spermatogonial stem cell culture medium supplemented with pirfenidone at 34 to 37° C. for 4 to 6 days; and

[0141] (3b) The cells were cultured in a testicular differentiation medium supplemented with pirfenidone at 34 to 37°C for 18 to 25 days.

[0142] 14. The method according to claim 12, wherein in step (3), the cells are cultured in a testicular differentiation medium at 34-37°C for 5-10 days, for example, about 7 days.

[0143] 15. The method according to any one of items 4 to 14, wherein step (4) is performed by further culturing at 34 to 37° C. in a culture medium with or without pirfenidone added.

[0144] 16. The method according to any one of items 4 to 15, wherein step (4) is performed using testicular differentiation medium.

[0145] 17. The method of item 4-16, wherein step (4) can last for about 1 to 100 days.

[0146] 18. The method of any one of items 4-17, wherein pirfenidone is used in an amount of 10-1000 μM.

[0147] 19. The method of any one of items 13-18, wherein the spermatogonial stem cell culture medium comprises StemPro34 basal medium and StemPro34 supplement.

[0148] 20. The method described in item 19, wherein the spermatogonial stem cell culture medium is supplemented with one or more selected from the following group: L-glutamine, basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF).

[0149] 21. The method of item 19 or 10, wherein the spermatogonial stem cell culture medium is further supplemented with one or more selected from the following group: glucose, bovine serum albumin (BSA), ascorbic acid and fetal bovine serum (FBS).

[0150] 22. The method according to any one of items 13 to 21, wherein the testicular differentiation medium comprises MEMα medium and KOSR, or consists of MEMα medium and KOSR.

[0151] 23. The method of any one of items 1-22, wherein the testicular cells are from an animal having testicles.

[0152] 24. The method of claim 23, wherein the animal is a mammal.

[0153] 25. The method of claim 24, wherein the animal is a rat, a mouse, a pig, a dog, a cat, a monkey, or a human.

[0154] 26. A testicular organoid obtained by the method of any one of items 1 to 25, wherein the testicular organoid is capable of producing round spermatids.

[0155] 27. The use of pirfenidone for improving the functions and / or properties of testicular organoids prepared in vitro, wherein the functions and / or properties are selected from one or more of the following groups: blood-testis barrier, spermatogenic microenvironment, spermatogenic microenvironment homeostasis, spermatogenesis, the number of germ cells in the middle and late stages of spermatogenesis, the quality of germ cells, the number of haploid sperm generated and the quality of sperm generated.

[0156] 28. A culture medium for preparing testicular organoids, comprising pirfenidone.

[0157] 29. The culture medium described in item 28, wherein the concentration of pirfenidone is 10-1000 μM.

[0158] 30. The culture medium described in item 28 or 29, which is a culture medium for culturing aggregate spheres.

[0159] 31. The culture medium of item 30, which is a spermatogonial stem cell culture medium, a testicular differentiation culture medium or a combination thereof.

[0160] 32. A kit for preparing testicular organoids, comprising the culture medium of any one of items 28-31.

[0161] 33. A method for improving spermatogenesis in testicular organoids, comprising using pirfenidone in the process of preparing the testicular organoids.

[0162] 34. The method of claim 33, wherein pirfenidone is used at a concentration of 10-1000 μM.

[0163] Example

[0164] In order to more fully understand and apply the present invention, the present invention will be described in detail below with reference to embodiments and drawings, which are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

[0165] Unless otherwise specified, the materials and reagents used in this example were obtained from commercial sources.

[0166] Example 1. Screening of small molecule compounds that promote spermatogenesis

[0167] This example describes a method for screening small molecule compounds that promote spermatogenesis using testicular organoids. In this example, 142 kinds of candidate small molecules were added to the culture medium of testicular organoids, including small molecules with antioxidant, anti-apoptotic, anti-inflammatory or other effects, in order to screen small molecules with the ability to promote spermatogenesis.

[0168] The test steps are as follows Figure 1 The method is described in detail as follows.

[0169] (1) The testicles of newborn mice (3 days old, ICR mice) were removed by laparotomy. The tissue capsule was removed with forceps in the culture medium and the testicles were torn into small pieces. The testicles were washed twice with PBS. 2 mL of collagenase IV (2 mg / mL) was added and digested at 37°C for 3 minutes. The testicles were then centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the testicles were washed once with PBS.

[0170] (2) Add 2 mL of 0.05% trypsin and digest at 37°C for 5 minutes. Add 2 mL of DMEM (12800017, Life Technologies) + 10% FBS (SE200-ES, VISTECH) to terminate the digestion. Centrifuge at 1000 rpm for 3 minutes and discard the supernatant to obtain testicular cells.

[0171] (3) The testicular cells obtained in step (2) were resuspended in a polymerase medium (the composition and content of the polymerase medium are shown in Table 1) to obtain a testicular cell resuspension with a concentration of 700 cells / μL. The testicular cell resuspension (70 μL per well) was pipetted into a U-bottom 96-well plate using a spray gun, and the plate was placed in an incubator and cultured at 37° C. for 36-48 hours to allow the cells to form spherical polymerases with a size of about 300-500 μm.

[0172] Table 1. Composition and content of polymer sphere culture medium

[0173] volume Final concentration Part Number brand MEMα 45mL - 12571063 Thermo Fisher KOSR 5mL - A3181502 Thermo Fisher L-Glutamine 500μL 2mM 25030081 Gibco GDNF 5μL 20 ng / mL 512-GF-050 R&D

[0174] (4) The polymerized spheres obtained in (3) were transferred to an agar block and cultured with a spermatogonial stem cell culture medium (the components and contents are shown in Table 2) (the number of days is recorded as D1). After 6 days (D6), the old culture medium was replaced with a testicular differentiation medium (the components and contents of the testicular differentiation medium are shown in Table 3), and the culture was continued until the 25th day (D25).

[0175] The cell culture was divided into a control group and treatment groups 1-142, each group having 3 wells. During the entire polymer sphere culture process in step (4), a total of 142 candidate small molecules of various types were added to the culture medium of the treatment groups 1-142, one for each group, including small molecules with antioxidant, anti-apoptotic, anti-inflammatory or other effects, and the final concentration after addition was 10 μM. The above small molecules were not added to the culture medium of the control group.

[0176] Table 2. Composition of spermatogonial stem cell culture medium

[0177] Element volume Final concentration Part Number brand StemPro-34SFM 45mL 10639011 Gibco L-Glutamine 500μL 2mM 25030081 Gibco StemPro supplement 1.3mL 10639011 Gibco FGF 5μL 10 ng / mL 3139-FB-025 R&D Mouse LIF 5μL 1000U / mL ESG1107 Merck Millipore Mouse EGF 5μL 10 ng / mL 2028-EG-200 R&D Mouse GDNF 5μL 10 ng / mL 512-GF-050 R&D glucose 0.3g 6mg / ml G7528-KG Sigma BSA 0.25g 5mg / ml A1933-100G Sigma FBS 500μL FBS10270106 Life ascorbic acid 50μL <![CDATA[10 -4 M]]> PHR1008-2G Sigma-Aldrich

[0178] Table 3. Composition of testicular differentiation medium

[0179] Element volume Part Number brand MEMα 45mL 12571063 ThermoFisher KOSR 5mL A3181502 ThermoFisher

[0180] (5) After culturing for 25 days (D25), the cell culture was digested into single cells with 2 mL of collagenase IV (2 mg / mL) and then fixed with 70% ethanol.

[0181] (6) The single cells fixed with 70% ethanol were centrifuged at 1300 rpm for 5 min, the supernatant was discarded, and the cells were washed once with PBS. PI (ethidium bromide) was added for staining at room temperature for 15 min. The haploid cells were then sorted using a flow cytometer, and the proportion of haploid cells in the total number of cells was analyzed to determine the haploid differentiation efficiency.

[0182] The sorted haploid cells were observed under white light, and the sorted haploid cells had the typical morphological characteristics of mouse round sperm, with a diameter of about 8-9 μm, small and round cells, and prominent chromatin regions in the nuclear region. The results showed that round sperm were obtained when the testicular organoids collected for flow sorting were collected at D25 in all control and treatment groups.

[0183] The results of flow cytometry analysis of each group of organoids are shown in Figure 2 Each set of data is the average of 3 wells. Figure 2 In the scatter plot, each dot represents a treatment group or a control group. The middle dotted line is the average value of the DMSO group (control group), which is about 3%, and the upper and lower dotted lines are standard deviations. Figure 2 As shown, the proportion of haploid cells in the pirfenidone group was higher than 5%, which is marked by arrows.

[0184] The results of the screening experiment showed that among the 142 small molecules tested, the percentage of haploid cells in cell cultures treated with pirfenidone was the highest, significantly better than most candidate small molecules ( Figure 2), indicating that pirfenidone can significantly improve the haploid differentiation efficiency of testicular organoids and can be used as an enhancer of organoid differentiation efficiency. Therefore, pirfenidone was selected for further study.

[0185] Example 2. Structure and composition of testicular organoids treated with pirfenidone

[0186] The preparation scheme of the testicular organoids used in Example 2 is the same as that of Example 1, except that a higher concentration of pirfenidone is added, as follows.

[0187] For the treatment group, in step (4), pirfenidone at a final concentration of 100 μM was added to the spermatogonial stem cell culture medium (culture time: D1-D6) and testicular differentiation medium (culture time: D6-D25) of the treatment group, and cultured at 37°C until D25.

[0188] For the control group, pirfenidone was replaced with DMSO (dimethyl sulfoxide), and the rest was the same.

[0189] On the 25th day of the testicular organoid culture prepared above, the testicular organoids obtained by adding pirfenidone to the treatment group were taken to observe their structure and constituent cell types. Specifically, the organoid samples were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned, and the tissue sections were stained with HE and immunofluorescence to detect the tissue structure of the testicular organoids and the presence of germ cells and somatic cells at all levels.

[0190] HE staining was performed using conventional methods, and the results are shown in Figure 3 a.

[0191] Immunofluorescence staining used cell-specific markers to detect somatic cells (Sertoli cells, Leydig cells, and Myoid cells) and germ cells at various levels in testicular organoids. The primary antibodies used for immunofluorescence staining are as follows: anti-SOX9 (ab5535, abcam), anti-3βHSD (sc-515120, Santa), anti-ACTA2 (sc-53142, Santa), anti-KI67 (ab2796, abcam), anti-ZBTB16 (sc-28319, Santa), anti-γH2AX (ab26350, abcam), anti-SYCP3 (ab15093, abcam), anti-SYCP1 (ab27591, abcam), anti-DDX4 (ab5535, abcam), and the following procedures were followed. The above primary antibodies and peanut agglutinin PNA were added. Place in a 4°C refrigerator overnight. Add fluorescently labeled secondary antibodies (Goat Alexa Fluor 488anti-rabbit IgG, Goat Alexa Fluor 594anti-rabbit IgG, Goat Alexa Fluor 488anti-mouse IgG or Goat Alexa Fluor 594anti-mouse IgG) at a dilution ratio of 1:500 and leave at room temperature for 1 hour. Use Hoechst 33342 to stain the nucleus at a dilution ratio of 1:1000 and stain at room temperature for 10 minutes. After staining, observe the sections and take pictures, and the results are shown in Figure 3 bc.

[0192] like Figure 3 a shows that in the testicular organoids obtained by the method of the present invention on days 14 and 25, similar to the seminiferous tubules present in the testis in vivo, there are also lumen-like structures (circled with a dotted circle). In the testicular organoids on day 1, the region where the lumen-like structure begins to differentiate can be identified (circled with a dotted circle).

[0193] like Figure 3 b shows that in the testicular organoids obtained by the present invention, there are Sertoli cells (SOX9 + ), Leydig cells (3βHSD + ) and Myoid cells (ACTA2 + At the same time, the relative positions of these somatic cells form a distinct tubular structure, proving the existence of the lumen (circled with a dotted circle).

[0194] like Figure 3c shows that according to the staining results of specific markers of germ cells at each stage, it can be seen that in the testicular organoids obtained by the method of the present invention on the 25th day, similar to the in vivo testis, there are spermatogonia, spermatocytes (leptotene, zygotene, pachytene, diplotene) and haploid sperm (PNA + DDX4 + ).

[0195] The above results indicate that the organoids cultured in the presence of pirfenidone using the method of the present invention have a structure and cell composition similar to that of the testis in vivo, especially germ cells of various levels including haploid cells.

[0196] Example 3. Blood-testis barrier of testicular organoids treated with pirfenidone

[0197] In this example, the effect of the small molecule pirfenidone on the blood-testis barrier of testicular organoids was tested.

[0198] The testicular organoids obtained in Example 2 were fixed with 4% paraformaldehyde, then embedded in paraffin and sliced, and the tissue sections were subjected to immunofluorescence staining to detect the expression levels of blood-testis barrier-related proteins TJP1 and GJA1. The specific operations were as follows.

[0199] Add primary antibodies anti-TJP1 (ab221547, abcam), anti-GJA1 (ab235585, abcam), and anti-WT1 (sc-7385, Santa) at dilution ratios of 1:600, 1:600, and 1:50, respectively. Place in a 4°C refrigerator overnight. Add fluorescently labeled secondary antibodies (goat Alexa Fluor 488 anti-rabbit IgG and goat Alexa Fluor 594 anti-rabbit IgG) at a dilution ratio of 1:500 and let stand at room temperature for 1 hour. Use Hoechst33342 to stain the nucleus at a dilution ratio of 1:1000 and stain at room temperature for 10 minutes. After staining, observe the sections and take photos for statistics. The stained cell photos are shown in Figure 4 a, and the relative quantitative results of the fluorescence intensity of a single supporting cell are shown in Figure 4 b.

[0200] like Figure 4 a and Figure 4 As shown in b, compared with the control group, pirfenidone treatment significantly increased the expression of blood-testis barrier-related proteins TJP1 and GJA1. This result indicates that pirfenidone has the effect of improving the blood-testis barrier and improving the microenvironment of testicular organoids.

[0201] Example 4. Effect of pirfenidone on spermatogenesis in testicular organoids

[0202] In this example, the effect of the small molecule pirfenidone on spermatogenesis in testicular organoids was tested by immunofluorescence staining and single-cell sequencing analysis.

[0203] The testicular organoids (D25) obtained in Example 2 were fixed with 4% paraformaldehyde and then embedded in paraffin and sliced. The tissue slices were subjected to immunofluorescence staining to detect the levels of spermatogenesis-related proteins, and the specific operation was as follows.

[0204] The following primary antibodies were added: (anti-PLZF (SC-28319, Santacruz) and anti-DDX4 (ab13840, abcam); anti-STRA8 (ABN1656, Millipore) and anti-DDX4 (ab13840, abcam); anti-DDX4 (ab15093, abcam) and anti-γH2AX (ab26350, abcam); anti-DDX4 (ab13840, abcam) and anti-CREM (sc-390426, Santa), the antibody dilution ratio was 1:400, and placed in a 4°C refrigerator overnight. Secondary antibodies Alexa Fluor 488 (115-545-003, Jackson), Alexa Fluor 594 (111-585-003, Jackson), antibody dilution ratio 1:400, stand at room temperature for 1 hour. Use Hoechst33342 (H3570, Thermo Fisher) to stain the nucleus, dilution ratio 1:1000, stain at room temperature for 10 minutes. After staining, observe the sections and take pictures for statistics. The pictures of the stained cells are shown in Figure 5 b, and the quantitative results are shown in Figure 5 a.

[0205] like Figure 5 a and Figure 5 As shown in b, compared with the control group, in the testicular organoids of the pirfenidone-added group, the proportion of undifferentiated spermatogonia (spermatogonia undifferentiation indicator molecule ZBTB16) had no significant difference, the proportion of differentiated spermatogonia (differentiated spermatogonia indicator molecule PLZF, differentiated spermatogonia marker molecule STRA8) in the pirfenidone-added group was significantly reduced, and the proportion of XY body spermatocytes (XY body) and round spermatids (CREM) was significantly increased, indicating that pirfenidone can significantly promote the number of spermatocytes and spermatids.

[0206] In addition, single-cell sequencing analysis was performed on testicular organoids, controls, and in vivo controls, which used testicular tissue from mice.

[0207] The specific steps of single-cell sequencing are as follows.

[0208] 1) Single-cell collection of testicular organoids: Digest the testicular organoids differentiated to 25 days with collagenase and trypsin, and resuspend them in DMEM culture medium. Pipette 10-20μl of the cell suspension into a culture dish, and add 100μL HSA (Human Serum Albumin) to dilute the cells under an optical microscope. At the same time, prepare three HSA droplets for washing capillary glass needles. Prepare a capillary glass needle with a tip of 30-40μm, and pick up single cells into the prepared cell lysis solution under an optical microscope.

[0209] For the in vivo control group, testicular tissue from wild-type C57 mice at 30 days after birth was obtained and digested using the same procedure as the testicular cells used to prepare organoids, and the single cells obtained by digestion were subjected to subsequent library construction and sequencing steps.

[0210] 2) Single-cell library construction: The single-cell RNA-seq library was prepared using the STRT-seq method, as follows. Testicular organoids were digested with collagenase to form a single-cell suspension. Single cells were picked under a stereoscope and placed in a PCR tube containing oligo (dT) primers and lysis buffer. Reverse transcription was performed in a PCR instrument to synthesize the first strand of cDNA, and then the second strand was synthesized and the cDNA was amplified. All cDNAs in the 96-well plate were combined together, and the biotinylated 5' end was enriched with magnetic beads with streptavidin, fragmented by an ultrasonic crusher, and the 3' end sequencing adapter was introduced by adding A to the end, and then the other end sequencing adapter was introduced by PCR, and finally 150bp paired-end sequencing was performed on the Illumina HiSeq XTEN platform (sequencing by Novogene).

[0211] Through single-cell sequencing-based transcriptome analysis, it was found that compared with the in vitro organoid control group, the expression of key genes for mature sperm (Dnalc2, Spata46, Pdilt, Zdhhc19, Chd5, Ubr7, Akap4, Ica1, Acrv1, Fhl5, Capza3, Csnka2ip) in the testicular organoids treated with pirfenidone was significantly upregulated, indicating that pirfenidone promoted the maturation of sperm in testicular organoids.

[0212] Example 5. Effects of pirfenidone on the function of sperm produced by testicular organoids and offspring mice

[0213] This example studies the sperm function of haploid cells generated from testicular organoids obtained by the method of the present invention.

[0214] The pirfenidone-treated testicular organoids (D25) obtained in Example 2, the DMSO-treated testicular organoids (organoid control group), and the testicular samples of wild-type C57 mice on the 30th day after birth (in vivo group control group) were digested according to the methods of steps (5) and (6) in Example 1, and haploid cells were sorted by flow cytometry.

[0215] The haploid cells obtained by sorting were used for round sperm injection (ROSI, Round Spermatid Injection) in the oocyte cytoplasm. Specifically, the oocytes (derived from female mice with C57 and DBA backgrounds, purchased from Guangdong Medical Experimental Animal Center) were treated with activation solution (M2 (M7167, Sigma), cytochalasin (C6762, Sigma)) for 10 minutes to activate the oocytes. The round spermatids obtained by the above sorting were then injected into the activated oocytes and cultured in the activation solution at 37°C for 6 hours. The culture medium M16 (M7292, Sigma) without activation solution was then replaced and culture was continued. At 24 hours, it developed into a 2-cell embryo ( Figure 7 a) The resulting 2-cell embryos were transplanted into the oviducts of pseudopregnant female mice.

[0216] At 19.5 days after transplantation, the mother mice with transplanted embryos were killed by cervical dislocation and the fetuses (offspring mice) were quickly removed. The fetuses in the pirfenidone treatment group, organoid control group, and in vivo control group were statistically analyzed and the results are shown in Figure 7 b and Figure 7 c.

[0217] like Figure 7 b and Figure 7 As shown in c, the efficiency of offspring generation (live birth rate: the percentage of live-born offspring to the number of transferred embryos) in the pirfenidone-treated group was more than 4 times that of the organoid control group.

[0218] In the testicular organoid patent of the inventor team CN112961822B, it has been proved that the offspring produced by the organs and organoids in the body are fertile, and the hair color trait separation occurs in the second generation, indicating that the haploid cells obtained by the organoid culture method in the patent have the potential to develop into individuals. In the present invention, pirfenidone is further used on the basis of the organoid culture method of the patent, and the offspring of the organoid control group without pirfenidone and the pirfenidone group are compared. Specifically, the placenta weight of these offspring mice was measured, and the results are shown in Figure 8 a; The weight of mice was measured during growth and the results are shown in Figure 8 b. In addition, after 5 weeks, the male and female offspring mice were mated together and successfully produced fertile offspring (second-generation mice), confirming their fertility ( Figure 8 c).

[0219] like Figure 8 a- Figure 8 As shown in c, there were no significant differences in placental weight, body weight, and fertility in the offspring mice of the pirfenidone group compared with the offspring mice of the organoid control group. These results demonstrated the safety of using pirfenidone in organoid culture.

[0220] Example 6. Effect of pirfenidone on increasing the number of germ cells in human testicular organoids and improving the blood-testis barrier

[0221] In this example, the effect of pirfenidone on human testicular organoids in maintaining the number of germ cells and improving the blood-testis barrier was studied by staining germ cells, apoptotic cells and blood-testis barrier-related proteins.

[0222] The culture of human testicular organoids was carried out as follows. Aggregate spheres were obtained by reaggregating interstitial cells, supporting cells, peritubular myoid cells and spermatogonia from the subjects in a U-shaped culture dish and culturing for 48 hours. Then, the aggregate spheres were transferred to an agar block (D0) and cultured in a testicular differentiation medium (components as shown in Table 3) with the addition of pirfenidone at a final concentration of 100 μM, and cultured at 34°C until D7 to obtain human testicular organoids. A control group was also included, in which testicular differentiation medium without the addition of pirfenidone was used.

[0223] The obtained human testicular organoid samples (pirfenidone group and control group) were fixed with 4% paraformaldehyde and then embedded in paraffin and sectioned. The tissue sections were subjected to immunofluorescence staining to detect the number and apoptosis of germ cells, as well as the expression levels of blood-testis barrier-related proteins TJP1 and GJA1. The specific operations are as follows.

[0224] The following three groups of primary antibodies were added respectively: anti-DDX4 (ab13840, abcam) and anti-Cleaved-caspase3 (9661, cell signaling technology); anti-TJP1 (ab221547, abcam) and anti-Vimentin (10366-1-AP, proteintech); and anti-GJA1 (ab235585, abcam) and anti-Vimentin (10366-1-AP, proteintech), and placed in a 4°C refrigerator overnight. Fluorescently labeled secondary antibodies (goat Alexa Fluor488 anti-rabbit IgG and goat Alexa Fluor 594 anti-rabbit IgG) were added at an antibody dilution ratio of 1:500 and allowed to stand at room temperature for 1 hour. Nuclei were stained with Hoechst33342 at a dilution ratio of 1:1000 and stained at room temperature for 10 minutes. After staining, the sections were observed and photographed for statistics. The staining and statistical results of germ cells and apoptosis are shown in Fig. 9 The staining and fluorescence intensity quantification results of blood-testis barrier-related proteins are shown in Fig.10 .

[0225] like Fig. 9 a and Fig. 9 As shown in b, compared with the control group, the proportion of germ cells (expressing the germ cell indicator molecule DDX4) in human testicular organoids in the pirfenidone group increased significantly ( Fig. 9 b), while the proportion of apoptotic cells (expressing both the germ cell indicator molecule DDX4 and the apoptotic cell indicator molecule Cleaved-caspase3) in germ cells was significantly reduced ( Fig. 9 a). Fig. 9 c is a fluorescent staining photograph of germ cells and apoptotic cells in the pirfenidone group and the control group, which shows that pirfenidone can significantly reduce the apoptosis of germ cells in human testicular organoids and increase the number of surviving germ cells, thereby helping to maintain the survival rate of germ cells.

[0226] in addition Fig.10 a and Fig.10 As shown in b, compared with the control group, pirfenidone treatment significantly increased the expression of blood-testis barrier-related proteins TJP1 and GJA1.

[0227] The above results indicate that pirfenidone also has the effect of improving the blood-testis barrier and improving the microenvironment of testicular organoids in human testicular organoids.

Claims

1. A method for preparing testicular organoids, comprising the following steps: (1) Provide dispersed testicular cells; (2) culturing the dispersed testicular cells of step (1) into aggregates in an aggregate medium; (3) culturing the polymerized spheres of step (2) into testicular organoids in the presence of pirfenidone; and (4) Optionally, further culturing the testicular organoids obtained in step (3).

2. The method of claim 1, wherein the dispersed testicular cells of step (1) are obtained by digesting testicular tissue and / or at least comprise supporting cells and spermatogonia.

3. The method according to claim 1 or 2, wherein the step (2) is carried out by culturing at 34-37°C for 40-72 hours.

4. The method of any one of claims 1 to 3, wherein The step (3) is performed by culturing on a solid matrix using a culture medium supplemented with pirfenidone at 34-37° C. for at least 5 days; and / or The step (4) is carried out at 34-37° C. in a culture medium with or without pirfenidone added.

5. The method of any one of claims 1 to 4, wherein pirfenidone is used in an amount of 10-1000 μM.

6. A testicular organoid obtained by the method of any one of claims 1 to 5, wherein the testicular cells are from an animal having testicles, such as a mammal, for example, a rat, a mouse, a pig, a dog, a cat, a monkey or a human.

7. The use of pirfenidone for improving the functions and / or properties of testicular organoids prepared in vitro, wherein the functions and / or properties are selected from one or more of the following groups: blood-testis barrier, spermatogenic microenvironment, spermatogenic microenvironment homeostasis, spermatogenesis, the number of germ cells in the middle and late stages of spermatogenesis, the quality of germ cells, the number of haploid sperm generated and the quality of sperm generated.

8. A culture medium for preparing testicular organoids, comprising pirfenidone.

9. A kit for preparing testicular organoids, comprising the culture medium of claim 8.

10. A method for improving the spermatogenesis function of testicular organoids, comprising using pirfenidone in the process of preparing the testicular organoids.

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