Testis organ as well as preparation method and application thereof

Abstract

The invention discloses a testis organ as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing printing ink and carrying out photocuring 3D printing, wherein the printing ink comprises photocuring hydrogel and testicular cells. The testicular organ has similar cell type composition, morphology and expression of a cell specific marker as the testicular organ. The method for preparing the testicular organ is easy to operate, rapid and convenient, and the 3D cultured testicular organ is formed through the 3D photocuring printing technology. The testicular organ can be used for evaluating the reproductive toxicity of a compound such as a medicine to male, and has reliability and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of reproductive toxicity evaluation of chemicals, and in particular to a testicular organoid and a preparation method and application thereof. Background Art

[0002] Infertility is a global disease. The WHO predicts that infertility will become the third largest disease after tumors and cardiovascular and cerebrovascular diseases in this century. About 15% of couples of childbearing age suffer from it, and men account for more than 40% of diagnosable causes. The causes of male infertility vary greatly, but may be related to congenital, acquired or idiopathic factors that damage spermatogenesis. The fact that bisphenol A, phthalates and their metabolites are widely present in human urine, blood, and even male semen has attracted the attention of reproductive toxicity researchers. Male reproductive toxicity refers to the damage of foreign substances to the normal physiological functions of the male reproductive system, which is an important part of the non-clinical safety evaluation of drugs.

[0003] At present, a variety of in vitro toxicity test methods for testicular cells have been established at home and abroad. The main problems include that the test of a single cell can only study the specific toxicity and mechanism of the cell, which is difficult to be used as an evaluation method for reproductive toxicity, and the effect on spermatogenic cell differentiation, i.e. sperm production, cannot be observed. Traditional two-dimensional culture models generally have their own limitations, such as the inability to simulate the testicular microenvironment, blood-testis barrier and sperm formation. In fact, all cells in the body are in a three-dimensional environment, which is very critical for cell growth and metabolism.

[0004] In the prior art, there are few testicular organoid models with three-dimensional morphology that can be used for in vitro toxicity testing, and there is a lack of a simple and convenient method for preparing a testicular organoid model with three-dimensional morphology. Summary of the invention

[0005] In order to solve the shortcomings of the prior art that there are few testicular organoid models with three-dimensional morphology that can be used for in vitro toxicity testing, and there is a lack of a method for simply and conveniently preparing a testicular organoid model with three-dimensional morphology, the present invention provides a testicular organoid and its preparation method and application. The testicular organoid has a cell type composition, morphology and expression of cell-specific markers similar to those of the testicular organ. The method for preparing testicular organoids provided by the present invention is simple, fast and convenient to operate, and 3D cultured testicular organoids are formed by 3D light-curing printing technology. The testicular organoid can be used to evaluate the reproductive toxicity of compounds such as drugs to males, and is reliable.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A first aspect of the present invention provides a method for preparing testicular organoids, the method comprising the steps of preparing printing ink for photocurable 3D printing; the printing ink comprises a photocurable hydrogel and testicular cells.

[0008] In some embodiments of the present invention, the method comprises the following steps:

[0009] (1) preparing the printing ink;

[0010] (2) dropping the printing ink into the cell suspension chamber;

[0011] (3) Perform 3D printing.

[0012] In some embodiments of the present invention, the printing ink further comprises a photoinitiator.

[0013] In some preferred embodiments of the present invention, the photocurable hydrogel is methacrylic anhydride gelatin; and / or the photoinitiator is a blue light initiator.

[0014] In some specific embodiments of the present invention, the blue light initiator is LAP.

[0015] In some embodiments of the present invention, the printing ink satisfies one or more of the following conditions:

[0016] The concentration of the methacrylic anhydride gelatin is 5.5-6.5%;

[0017] The concentration of LAP is 0.15%-0.25%;

[0018] The concentration of the testicular cells was 4.2×10 7 - 4.4×10 7 Pieces / mL.

[0019] In some specific embodiments of the present invention, the printing ink satisfies one or more of the following conditions:

[0020] The concentration of the methacrylic anhydride gelatin is 6%;

[0021] The concentration of LAP is 0.2%;

[0022] The concentration of the testicular cells was 4.4×10 7 Pieces / mL.

[0023] In some embodiments of the present invention, the drop amount of the printing ink is 15-25 μL.

[0024] In some specific embodiments of the present invention, the drop amount of the printing ink is 20 μL.

[0025] In some embodiments of the present invention, the printing parameters of the 3D printing include: an exposure time of 15-16 s; and / or an exposure intensity of 19%-21%.

[0026] In some specific embodiments of the present invention, the exposure time is 15 s, and / or the exposure intensity is 20%.

[0027] In some embodiments of the present invention, in the printing ink: the concentration of the methacrylic anhydride gelatin is 5.5-6.5%; the concentration of the LAP is 0.15%-0.25%; and the content of the testicular cells is 4.2×10 7 - 4.4×10 7 The printing parameters of the 3D printing include: an exposure time of 15-16 s, and / or an exposure intensity of 19%-21%.

[0028] In some embodiments of the present invention, the testicular cells are derived from rodents, humans or non-human primates; and / or the testicular cells are a cell composition obtained after enzyme digestion.

[0029] In some preferred embodiments of the present invention, the testicular cells are derived from mice or rats.

[0030] In some preferred embodiments of the present invention, the testicular cells are derived from 3-14 day old rodents, humans or non-human primates.

[0031] In some specific embodiments of the present invention, the testicular cells are derived from 5-7 day old mice.

[0032] In some embodiments of the present invention, the testicular cells are obtained by enzymatic digestion.

[0033] In some preferred embodiments of the present invention, the enzymatic digestion method is a three-step enzymatic digestion method; the first step of enzymatic digestion uses type I collagenase, the second step of enzymatic digestion uses one or more of type I collagenase, hyaluronidase and DNase I enzymes; and / or, the third step of enzymatic digestion uses TrypLE digestion enzyme; and / or, the concentration of the enzyme is 0.5-2 mg / mL; and / or, the time for each step of enzymatic digestion is 6-20 minutes.

[0034] In some embodiments of the present invention, the enzymatic digestion method comprises the following steps:

[0035] The first step of enzyme digestion is to use type I collagenase, the concentration of type I collagenase is 0.5-2 mg / mL, and the digestion time is 8-20 minutes;

[0036] The second step of enzymatic digestion uses type I collagenase, hyaluronidase and DNase I enzymes; the concentration of type I collagenase is 1 mg / mL, the concentration of hyaluronidase is 1 mg / mL and the concentration of DNase I enzyme is 0.5 mg / mL; the digestion time is 12-20 minutes;

[0037] The third step of enzyme digestion uses TrypLE digestion enzyme; the concentration of the TrypLE digestion enzyme is 1×; the digestion time is 4-10 minutes.

[0038] In some specific embodiments of the present invention, the enzymatic digestion method comprises the following steps:

[0039] After killing the mice, the testicles were removed and transferred to a 7 mL EP tube. The testicles were washed three times with washing solution and the washing solution was discarded.

[0040] First, add 0.5 mL of 1 mg / mL type I collagenase and cut the testicles into 1 mm pieces with scissors. 3 Transfer the testicular fragments within 100 ml into a 15 mL centrifuge tube, add 5.5 mL type I collagenase, seal the tube and place it on a shaker at 37°C 120 rpm for the first digestion. After digestion for 10 minutes, place the centrifuge tube vertically for 10 minutes to allow the sperm tubule fragments to precipitate.

[0041] Separate the precipitate and supernatant, add stop solution (α-MEM + 5% FBS + 1% pen / strep) to the supernatant to stop digestion, add 1 mg / mL type I collagenase and hyaluronidase and 0.5 mg / mL DNase Ⅰ enzyme to the precipitate for a second digestion, seal and place on a shaker at 37°C and 120 rpm for a second digestion for 15 minutes, add stop solution to stop digestion, and finally pass the two tubes of cell suspension together through a 70 μm sieve and centrifuge at 400×g at 4°C for 5 minutes.

[0042] After centrifugation, the supernatant was discarded, 2 mL of TrypLE digestion enzyme was added for digestion for 6 minutes, and 4 mL of stop solution was added to terminate the digestion.

[0043] The stiffness of the testis is about 3 kPa. Since the main component of the testicular extracellular matrix is ​​GelMA, different concentrations of GelMA are added and mixed with photosensitive materials and PBS and then printed by a 3D printer. Through mechanical testing, when the testicular organoids are prepared with the printing ink provided by the present invention, the hardness of the testicular organoids obtained is very close to that of the testicular tissue.

[0044] In some embodiments of the present invention, the method further comprises the step of culturing the prepared testis-like model;

[0045] In some preferred embodiments of the present invention, the culture is a two-step culture:

[0046] (i) 7-10 days in maintenance medium; (ii) 1-7 days in differentiation medium;

[0047] The maintenance medium and the differentiation medium are serum-free medium.

[0048] In some preferred embodiments of the present invention, the maintenance medium comprises: Ad-DMEM-F12 basal medium, Gibco's B-27 supplement, Gibco's GlutaMAX, N-acetyl-L-cysteine, EGF, FSH, IGF-1, FGF-9, activin A and T3.

[0049] In some embodiments of the present invention, the differentiation medium comprises: Ad-DMEM-F12 basal medium, B-27 supplement, Glutamax, N-acetyl-L-cysteine, EGF, FSH, testosterone and RA.

[0050] In some preferred embodiments of the present invention, the maintenance medium and the differentiation medium further contain antibiotics, and the antibiotics are selected from one or more of penicillin, streptomycin and gentamicin.

[0051] In some more preferred embodiments of the present invention, the maintenance medium is based on Ad-DMEM-F12 as the basic medium, comprising 1×B-27 supplement, 1×GlutaMAX, 1-2 mM N-acetyl-L-cysteine, 40-60 ng / mL mouse EGF, 20-25ng / mL FSH, 90-120ng / mL IGF-1, 40-60μg / mL FGF-9, 40-60 ng / mL activin A, 90-110nM T3, 90-110 U / mL penicillin, 0.1-0.2 mg / mL streptomycin and 40-60 μg / mL gentamicin.

[0052] In some more preferred embodiments of the present invention, the differentiation medium is based on Ad-DMEM-F12, comprising 1×B-27 additive, 1×GlutaMAX, 1-2 mM acetyl-L-cysteine, 40-60 ng / mL mouse EGF, 20-25 ng / mL FSH, (1-1.5)×10 -6 M testosterone, (1-1.5) 10 -6 M RA, 90-110 U / mL penicillin, 0.1-0.2 mg / mL streptomycin, and 40-60 μg / mL gentamicin.

[0053] In some specific embodiments of the present invention, the maintenance medium is based on Ad-DMEM-F12 as the basal medium, comprising 1×B-27 supplement, 1×GlutaMAX, 1.25 mM N-acetyl-L-cysteine, 50 ng / mL mouse EGF, 22 ng / mL FSH, 100 ng / mL IGF-1, 50 μg / mL FGF-9, 50 ng / mL activin A, 100 nM T3, 100 U / mL penicillin, 0.1 mg / mL streptomycin and 50 μg / mL gentamicin.

[0054] In some specific embodiments of the present invention, the differentiation medium is based on Ad-DMEM-F12 as a basic medium, comprising 1×B-27 additive, 1×GlutaMAX, 1.25 mM acetyl-L-cysteine, 50 ng / mL mouse EGF, 22 ng / mL FSH, 10 -6 M testosterone, 10 -6 M RA, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 50 μg / mL gentamicin.

[0055] A second aspect of the present invention provides a testicular organoid, which is prepared by the method described in the first aspect.

[0056] The third aspect of the present invention provides a use of the testicular organoid as described in the second aspect in drug screening or in vitro reproductive toxicity evaluation.

[0057] In some embodiments of the present invention, the application is to apply the drug to be screened after the testicular organoids mature, and screen and evaluate the reproductive risks of the drug to males by analyzing the developmental indicators of the testicular organoids.

[0058] In some preferred embodiments of the present invention, the developmental indicators include changes in the morphology of testicular organoids and the developmental levels of each cell in the testicular organoids.

[0059] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0060] The reagents and raw materials used in the present invention are commercially available.

[0061] Advantages and benefits of the present invention:

[0062] The organoid model provided by the present invention can be used to predict and evaluate the potential toxicity of drugs to male reproduction, and has the following advantages:

[0063] 1) By optimizing the culture method and conditions, a mouse testicular organoid model was established that can observe the functional changes of Sertoli cells and Leydig cells, as well as the differentiation of spermatogonia;

[0064] 2) Due to the use of in vitro culture system, the use of experimental animals is reduced, which complies with ethical requirements and reduces experimental costs and time;

[0065] 3) The use of 3D light-curing printing equipment provides a relatively fast and accurate screening platform suitable for high-throughput drug screening;

[0066] 4) It can quantitatively analyze the changes in cell morphology and function caused by drugs, providing valuable data for early safety assessment of drugs;

[0067] 5) The repeatability and comparability of experimental results are ensured through standardized testing procedures.

[0068] In the present invention, testicular cells of 5-7 day old C57BL / 6J mice are used to prepare testicular organoids, which are significantly stronger than mature testicular cells in self-organization in in vitro culture, have pluripotency and high activity. The method for preparing testicular organoids provided by the present invention is simple, quick and convenient to operate, and 3D cultured testicular organoids are formed by 3D light-curing printing technology. The prepared testicular organoids can observe the functional changes of Sertoli cells and Leydig cells, and can observe spermatogonia differentiation, have high-throughput characteristics, and can be used for large-scale drug raw material detection and screening work, and have high accuracy. Cell morphology, biochemical markers and certain molecular events, such as gene expression, intracellular signal transduction, etc., can be detected by microscopic observation, qPCR, immunofluorescence and other methods, such as evaluating the reproductive toxicity of compounds such as drugs to males, which is reliable and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The growth status of the testicular organoids of the present invention in the bright field at different days. The scale bar represents 500 μm.

[0070] Figure 2 Immunofluorescence staining was used to detect the four main cell staining results of testicular tissue sections, including peritubular myoid cells (α-SMA), supporting cells (GATA4), germ cells (MHV), and interstitial cells (β-HSD), as well as the results of H&E staining and DAPI staining.

[0071] Figure 3 The co-staining results are for peritubular myoid cells (red) and germ cells (green).

[0072] Figure 4The results of co-staining of supporting cells (red) and germ cells (green).

[0073] Figure 5 The results are shown for co-staining of interstitial cells (red) and germ cells (green).

[0074] Figure 6 These are the CTG results under different culture media.

[0075] Figure 7 CTG results for different printing materials. Updated printing materials ( Figure 6 Compared with the old printing material (GelMA:HAMA:LAP=8:1:1), the cells proliferated faster after the same number of days of culture.

[0076] Figure 8 The results of curing degree under different printing parameters.

[0077] Fig. 9 Results of the identification of marker genes for the developmental status of supporting cells (Fshr, Sox9, Amh), germ cells (Oct4, Scyp3, Acrv1), interstitial cells (β-HSD, Star) and peritubular myoid cells (α-SMA) in organoids at the 0th, 3rd, 7th, 14th and 21st time points by RT-qPCR method. Data are expressed as mean ± standard deviation. There is statistical significance compared with the control condition (*p < 0.05, **p < 0.01, ***p < 0.001).

[0078] Fig.10 The results of the identification of the marker genes for the developmental status of supporting cells (Fshr, Sox9, Amh), germ cells (Scyp3), interstitial cells (3β-HSD, Star) and peritubular myoid cells (α-SMA) in organoids after triptolide were detected by RT-qPCR. The data are expressed as mean ± standard deviation. Compared with the control group (DMSO), there is statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001). Among them, GAPDH was used as the internal reference in the experiment, and the relative expression level of the control group was taken as 1 for normalized data processing. DETAILED DESCRIPTION

[0079] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0080] The reagents used in the examples of the present invention are shown in Table 1:

[0081] Table 1 Reagents used

[0082]

[0083] Example 1 Construction of testicular organoid model

[0084] The testicular organoid model construction of the present invention comprises the following steps:

[0085] 1. Obtaining primary testicular cells from young mice

[0086] 1) 5-7 day old male C57BL / 6J mice Source: The animals in this study were obtained from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.

[0087] 2) After killing the mouse, remove the testicles, transfer them to a 7 mL EP tube, wash them three times with washing solution, and discard the washing solution.

[0088] 3) First add 0.5 mL of 1 mg / mL type I collagenase, and cut the testicles into 1 mm pieces with scissors. 3 Transfer the testicular fragments within 100 ml into a 15 mL centrifuge tube, add 5.5 mL type I collagenase, seal the tube and place it on a shaker at 37°C 120 rpm for the first digestion. After digestion for 10 minutes, place the centrifuge tube vertically for 10 minutes to allow the sperm tubule fragments to precipitate.

[0089] 4) Separate the precipitate and supernatant, add stop solution (α-MEM + 5% FBS + 1% pen / strep) to the supernatant to stop digestion, add 1 mg / mL type I collagenase and hyaluronidase and 0.5 mg / mL DNase I enzyme to the precipitate for a second digestion, seal the tube and place it on a shaker at 37°C and 120 rpm for a second digestion for 15 minutes, then add stop solution to stop digestion. Finally, the two tubes of cell suspension were passed through a 70 μm sieve together and centrifuged at 400×g at 4°C for 5 minutes.

[0090] 5) After centrifugation, discard the supernatant, add 2 mL of 1×TrypLE digestion enzyme and digest for 6 minutes, and add 4 mL of stop solution to stop the digestion.

[0091] 6) After centrifugation at 400×g for 5 minutes, remove the supernatant, add culture medium and trypan blue for counting.

[0092] 2. Light-curing 3D printing model establishment

[0093] 1) After counting, dilute the cell suspension to a concentration of 1.46 × 10 8The printing material and cell suspension were mixed according to the printing ink composition ratio: 10% GelMA: 2% LAP: cell suspension = 6: 1: 3 (V / V), and the final cell concentration was 4.4E7 / mL (the printing ink contained 6% GelMA, 0.2% LAP and 4.4×10 7 The composition and ratio (and concentration) of the above printing ink components are determined by experimental adjustment based on the hardness of testicular tissue being approximately 3K Pa. GelMA is methacrylic anhydride gelatin, and LAP is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0094] 2) Set the parameters of the light-curing 3D printer;

[0095] 3) Drop 20 μL of printing ink in the middle of each cell suspension chamber of the 12-well plate and then print. Finally, a round cake-shaped gel model with a height of about 0.5 mm is formed in each cell suspension chamber. After washing once with PBS, culture is performed.

[0096] The printer parameters were adjusted, and the results are shown in Table 1 and Figure 8 As shown. When the exposure intensity is 19%-21% and the exposure time is 15-16s, it meets the best state after the bioprinting ink is cured. When the exposure time is greater than 16s and the exposure intensity is higher than 21%, the printed adhesive surface will be too sticky and stick to the lens or crack. When the exposure time is less than 15s and the exposure intensity is less than 19%, some liquid ink will remain on the printed adhesive surface, and cells will escape after a period of culture. Printing mode: single image static printing, printed image: all white.

[0097] Table 2 Printing parameters

[0098]

[0099] 3. Testicular organoid culture

[0100] 1) The printed model is cultured in a testicular organoid maintenance medium that can maintain and enhance the immature cell state for ten days, and the medium is changed every other day. The testicular organoid maintenance medium used in the present invention is the testicular organoid maintenance medium CM1 or the testicular organoid maintenance medium CM2 before improvement.

[0101] Improved testicular organoid maintenance medium CM1: Ad-DMEM-F12 as basal medium, supplemented with 1% penicillin-streptomycin-gentamicin solution (working concentrations of 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 50 μg / mL gentamicin), 1× B-27 supplement (diluted B-27 TMSupplement), 1× GlutaMAX, 1.25 mM N-acetyl-L-cysteine, 50 ng / mL mouse EGF (epidermal growth factor), 22 ng / mL FSH (follicle-stimulating hormone), 100 ng / mL IGF-1 (insulin-like growth factor-1), 50 μg / mL FGF-9 (fibroblast growth factor 9), 50 ng / mL activin A, and 100 nMT3 (3,3',5'-triiodo-L-thyronine).

[0102] Testicular organoid maintenance medium CM2: α-MEM + 10 KnockOut serum replacement + 1% pen / strep + 10 -7 M melatonin.

[0103] The CTG method was used to measure the cell metabolic activity as an indicator of cell viability. On the 0th, 1st, 5th and 9th day of culture, the 12-well plate containing the testicular organoids was removed from the incubator, the culture medium was removed and 100 μL of detection reagent (CTG (Abcam, Cat. No.: ab211091) : PBS = 1:1 volume ratio) was added to each well in the dark, and the shaker was shaken at 350 rpm for 30 min in the dark. After 30 minutes, the reagent was aspirated into each well of the white opaque 96-well plate, and the absorbance at OD590 nm was measured with an enzyme reader.

[0104] The results are as follows Figure 6 As shown, the printed organoid models had higher cell activity when cultured in CM1, and the cells proliferated faster in the same number of days.

[0105] 2) After culturing in CM1 for ten days, the culture medium was changed to a medium that promotes the state of Sertoli cells. The medium is based on Ad-DMEM-F12 supplemented with 1% triple antibody, 1×B-27, 1×Glutamax, 1.25 mM acetyl-L-cysteine, 50 ng / mL mouse EGF, 22 ng / mL FSH, 10 -6 M testosterone and 10 -6 M RA (retinoic acid), also change the solution every other day.

[0106] The organoid model was observed under a microscope on the 0th, 3rd, 7th and 14th day of culture. Figure 1 As shown, on the 0th day, it showed a uniform single-cell morphology, began to gradually aggregate into clusters on the third day, began to appear hollow tubular structures on the 7th day, and formed obvious tubular structures on the 10th day.

[0107] Try to prepare testicular organoids using other schemes. Prepare a mixed solution of 10% GelMA: 10% HAMA: 2% LAP = 8:1:1 (V / V), and mix the mixed solution: cell suspension = 1:1 (V / V) as printing ink (4% GelMA, 0.5% HAMA, 0.1% LAP, final cell concentration of 4.4E7 / mL). Follow the above steps for printing and organoid model culture (using CM2 culture medium), and detect cell activity on day 0, day 1, day 5, and day 9 of culture using the above CTG method. The results are as follows: Figure 7 As shown in the figure, as the number of culture days increases, the cell activity decreases, which shows that it is not suitable to prepare testicular organoids using this scheme. The cell activity of the testicular organoids prepared using this printing ink scheme is significantly lower than that of the testicular organoids prepared using the scheme provided in Example 2.

[0108] 4. Immunofluorescence staining and RT-qPCR analysis

[0109] (1) H&E staining and immunofluorescence staining of testicular tissue sections

[0110] H&E staining:

[0111] 1. Dewaxing of paraffin sections to water

[0112] Sequentially put the slices into xylene Ⅰ for 10 min-xylene Ⅱ for 10 min-anhydrous ethanol Ⅰ for 5 min-anhydrous ethanol Ⅱ for 5 min-95% alcohol for 5 min-90% alcohol for 5 min-80% alcohol for 5 min-70% alcohol for 5 min-distilled water for washing. ("Ⅰ" and "Ⅱ" only represent different steps)

[0113] 2. Hematoxylin staining of cell nuclei

[0114] The sections were stained with Harris hematoxylin for 3-8 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0115] 3. Eosin staining of cytoplasm

[0116] Stain the sections in eosin solution for 1-3 min.

[0117] 4. Dehydration and sealing

[0118] Put the slices into 95% alcohol I for 5 min-95% alcohol II for 5 min-absolute ethanol I for 5 min-absolute ethanol II for 5 min-xylene I for 5 min-xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene and let them dry slightly, then seal them with neutral gum. (“Ⅰ” and “Ⅱ” only indicate different steps)

[0119] 5. Microscope examination, image acquisition and analysis.

[0120] Immunofluorescence staining

[0121] Reagent preparation:

[0122] Blocking solution: 3% BSA reagent (diluted with DPBS);

[0123] Primary antibody dilution (double staining; 1:100): Add 1 μL of GATA4 and 1 μL of DDX4 to every 100 μL of PBS; Add 1 μL of 3β-HSD and 1 μL of DDX4 to every 100 μL of PBS; Add 1 μL of α-SMA and 1 μL of DDX4 to every 100 μL of PBS;

[0124] Secondary antibody dilution (double staining; 1:200): add 1 μL of each secondary antibody to every 500 μL of PBS;

[0125] 0.1%TritonX-100: 1 μL TritonX-100 + 1000 μL PBS.

[0126] dyeing:

[0127] 1) Cell preparation and washing: When the cells reach the sampling time, remove the culture medium and wash once with PBS.

[0128] 2) Fixation: Add 4% PFA paraformaldehyde to the well plate (just cover it), incubate at 4°C overnight, remove the paraformaldehyde, treat it as waste liquid, rinse the well plate 3 times / 5 min with 1 mL PBS, and then dry it with PBS.

[0129] 3) Permeabilization: Add the prepared 0.1% TritonX-100 to the well plate and place it at room temperature for 15 min. Discard the TritonX-100 and rinse the well plate 3 times / 10 min with 1 mL PBS.

[0130] 4) Blocking: Block with blocking solution (just cover) at room temperature, aspirate after about 1 hour, and do not wash;

[0131] 5) Primary antibody incubation: Add primary antibody dilution, 200 μL / well, cover the plate, and incubate at 4°C overnight.

[0132] 6) Recovery of primary antibody: The collected primary antibody is stored at -20℃.

[0133] 7) Fluorescent secondary antibody incubation: rinse the plate with PBS 3 times / 10 min, add 200 μL / well of fluorescent secondary antibody diluent. This step should be performed in a dark environment (wrapped in tin foil). Take out the plate at room temperature for 2 h and rinse it with PBS 3 times / 10 min.

[0134] 8) Counterstaining: Add 500 μL DAPI to each well to counterstain the cell nuclei (just cover the nuclei) and incubate at room temperature for 10 min.

[0135] 9) Rinse the plate with PBS 3 times / 10 min and observe.

[0136] The results are as follows Figure 2 As shown in the figure, four main cells can be seen in the testicular tissue section: peritubular myoid cells (α-SMA), supporting cells (GATA4), germ cells (MHV), and interstitial cells (β-HSD). Among them, peritubular myoid cells form the main structure of the tubules, germ cells and supporting cells are mixed and distributed inside the tubules, and interstitial cells are distributed between the tubules.

[0137] (2) Immunofluorescence staining of testicular organoid model

[0138] After the culture in the chamber, the membrane of the model-bearing type at the bottom of the culture chamber was cut off with surgical scissors. The immunofluorescence staining steps in 4. (1) of this example were followed.

[0139] The results are as follows Figure 3-Figure 5 As shown, Figure 3 The co-staining results of peritubular myoid cells (red) and germ cells (green) show that the peritubular myoid cells self-organize into tubular structures, and the germ cells are distributed on the inner side of the tubules. Figure 4 The co-staining results for supporting cells (red) and germ cells (green) show that the two are mixed together. Figure 5 The results of co-staining of interstitial cells (red) and germ cells (green) show that interstitial cells are distributed around the tubules. The above results are consistent with the staining results of testicular tissue ( Figure 2 ) are consistent.

[0140] (3) qPCR detection of markers

[0141] First, samples were collected on days 0, 3, 7, 14, and 21 during the culture process, and total RNA was extracted and then reverse transcribed into cDNA. qPCR was performed using a 2×Color SYBR Green gPCR Master Mix (ROX2 plus) kit, and relative gene expression was quantified using GAPDH as an internal reference.

[0142] The results are as follows Fig. 9As shown in the figure, during the organoid culture process, the expression of different related genes showed specific time dynamic changes. In the initial stage, Fshr, Sox9, and Amh were markers of testicular support. Among them, Fshr and Amh were initially expressed at a high level, but as the culture progressed, their expression showed a downward trend, while Sox9 showed an increasing trend in the early stage of culture and then gradually decreased. Star and 3β-HSD are markers of testicular interstitial cells. The expression of Star increased significantly in the early stage, then reached a peak and gradually decreased; the overall trend of 3β-HSD was also an increase first and then a decrease, but the difference was not large. α-SMA is a marker of peritubular myoid cells, and its expression showed an overall downward trend, which is consistent with the gene expression in vivo. The expression genes of germ cells are Acrv1, Oct4, and Scyp3. The expression of Oct4 showed an increasing trend with the culture time. The two genes Acrv1 and Scyp3 decreased significantly in the early stage of culture and had no obvious changes afterwards. The expression changes of these genes reflect the different stages and processes of testicular organoid culture.

[0143] Example 2 Administration of drugs to be screened and evaluation of their effects

[0144] After obtaining a stable testicular organoid model (after two weeks of culture), the drug to be screened is administered to the testicular organoid model.

[0145] Administration and effect evaluation of triptolide, a drug to be screened:

[0146] 1) Preparation before drug administration: First, prepare a stock solution of triptolide using DMSO as a solvent with a drug concentration of 10 mM / mL.

[0147] 2) Triptolide concentration setting: Considering the clinical application dose of triptolide, as well as its effective concentration and cytotoxicity threshold in in vitro experiments, the concentration range of the drug was initially set. Based on the pre-conducted dose-response curve analysis, a non-cytotoxic concentration that can simulate clinical treatment conditions was selected and triptolide was added to the culture medium.

[0148] 3) Continuous administration throughout the culture process: Starting from the 14th day of culture, a given concentration of triptolide was added to the culture medium and continued to exist throughout the culture process. Considering that the cell culture medium needs to be replaced every other day, the same concentration of triptolide needs to be added again every time the culture medium is replaced to keep the drug concentration constant.

[0149] 4) Evaluation of cell survival rate: During drug treatment, the growth rate and survival rate of testicular Sertoli cells, Leydig cells, peritubular myoid cells, and germ cells were regularly evaluated by the expression levels of specific markers of the three cells, such as immunofluorescence staining to detect changes in cell self-organization morphology. At the same time, CTG or flow cytometry and other methods were used to quantitatively analyze the survival rate and proliferation of cells.

[0150] 5) Functional evaluation: Record images of testicular cell self-organization morphological changes and perform quantitative analysis of cell migration using image analysis software.

[0151] 6) Observation of morphological changes: Observe and photograph the morphological changes of cells under a microscope, and pay attention to whether the cells have irregular morphology.

[0152] 7) Data recording and analysis: All experimental data should be recorded and organized, including cell morphology images before and after drug treatment, immunofluorescence staining images, and real-time quantitative PCR results. Use statistical software for data analysis and compare the differences between the control group and the drug-treated group to evaluate the effects of triptolide on the growth and function of testicular organoids.

[0153] The administration and effect evaluation process of the drug to be screened, triptolide, in testicular organoids are described in detail as follows:

[0154] 1) Drug preparation: First, prepare a stock solution of triptolide using DMSO as solvent with a final concentration of 10 mM / mL.

[0155] 2) Concentration setting: Taking into account the clinical application dose of triptolide, as well as its effective concentration and cytotoxicity threshold in in vitro experiments, the concentration range of the drug was preliminarily set, and the final drug concentrations of 0.01 μM, 0.1 μM and 1 μM were determined through CTG cytotoxicity experiments.

[0156] 3) Drug administration: The cells are cultured in a gas-liquid culture in a cell suspension chamber (cultured in the culture medium promoting the Sertoli cell state in Example 1), a predetermined concentration of triptolide is added, and the cells are continuously exposed to the drug environment.

[0157] 4) Culture monitoring: During the entire drug-addition culture process, observe and record the morphological development and size changes of the cell clusters daily, and collect samples after 72 hours of drug-addition culture.

[0158] 5) Cell apoptosis and differentiation index test: To evaluate the effect of triptolide on the development of testicular organoids, the expression of cell-related genes was evaluated by immunofluorescence staining to identify cell apoptosis. At the same time, RT-qPCR was used to analyze Sertoli cells, Leydig cells, peritubular myoid cells and germ cell-specific markers, Sertoli cells (Fshr, Amh, Sox9), Leydig cells (3β-HSD, Star), peritubular myoid cells (α-SMA) and germ cells (Scyp3).

[0159] 6) Data collection and analysis: Use statistical software such as SPSS or GraphPad Prism to analyze the data and evaluate the differences between the treatment and control groups.

[0160] 5. Data Analysis

[0161] 1) Collect experimental data, including experimental pictures, video records, cell counts, and fluorescence intensity;

[0162] 2) Use statistical software to analyze the data and determine the significant differences between the drug-treated group and the control group;

[0163] 3) Uncover the reproductive toxicity mechanism of drugs through data analysis, which can be inferred by observing how drugs affect specific signaling pathways or gene expression;

[0164] 4) Finally, the data at the morphological and molecular levels are integrated to draw conclusions on the potential harm of the screened drugs to male reproductive toxicity.

[0165] RT-qPCR analysis results Fig.10 As shown in the figure, during the drug-added culture process, when the drug concentration was 0.01 μM, most genes did not show a significant decrease in expression, only Amh decreased significantly, and the expression of Sox9 and Fshr increased. When the drug concentration was 0.1 μM, the expression of Star and Sox9 genes did not decrease significantly, and the remaining genes all decreased to below 50%, indicating obvious toxicity. When the drug concentration was 1 μM, the expression of all genes except Star was significantly affected, indicating that the reproductive toxicity of triptolide was obvious at a drug concentration of 1 μM.

[0166] In previous in vivo experimental data, after male animals were treated with different concentrations of reproductively toxic cadmium, the gene expression levels of Fshr and 3β-HSD decreased in a dose-dependent manner, indicating that cadmium damaged the function of testicular interstitial cells and supporting cells. In addition, according to literature reports, after male animals were given triptolide, the 3β-HSD protein level was significantly reduced. The above gene expression changes disclosed in the prior art are similar to Fig.10This shows that the testicular organoid model can truly reflect the reproductive toxicity risk of drugs and is reliable when used for drug screening or in vitro reproductive toxicity evaluation.

[0167] The present invention uses photocuring 3D printing technology to prepare a 3D testicular organoid model, which has simple operation steps and stable finished product quality. The organoid model provided by the present invention has high throughput, high sensitivity and specificity, which helps to exclude compounds with potential reproductive toxicity in the early stages of drug development and reduce the risk of failure of later clinical trials.

Claims

1. A method for preparing testicular organoids, characterized in that: The method comprises the steps of preparing printing ink for photocurable 3D printing; the printing ink comprises photocurable hydrogel and testicular cells.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) preparing the printing ink; (2) dropping the printing ink into the cell suspension chamber; (3) Perform 3D printing.

3. The method according to claim 1 or 2, characterized in that The printing ink also includes a photoinitiator; Preferably, the photocurable hydrogel is methacrylic anhydride gelatin; and / or, the photoinitiator is a blue light initiator; More preferably, the blue light initiator is LAP.

4. The method according to claim 3, characterized in that The printing ink meets one or more of the following conditions: The concentration of the methacrylic anhydride gelatin is 5.5-6.5%; The concentration of LAP is 0.15%-0.25%; The concentration of the testicular cells was 4.2×10 7 - 4.4×10 7 Pieces / mL.

5. The method according to any one of claims 1 to 4, characterized in that: The drop amount of the printing ink is 15-25 μL, for example, 20 μL; The printing parameters of the 3D printing include: an exposure time of 15-16 s, for example, 15 s; and / or an exposure intensity of 19%-21%, for example, 20%.

6. The method according to any one of claims 1 to 5, characterized in that: The testicular cells are derived from rodents, humans or non-human primates, such as mice or rats; and / or, the testicular cells are a cell composition obtained after enzyme digestion; and / or, the testicular cells are obtained by enzyme digestion; Preferably, the testicular cells are derived from 3-14 day old rodents, humans or non-human primates, for example, from 5-7 day old mice; and / or, The enzymatic digestion method is a three-step enzymatic digestion method; the first step of enzymatic digestion uses type I collagenase, the second step of enzymatic digestion uses one or more of type I collagenase, hyaluronidase and DNase I enzymes; and / or, the third step of enzymatic digestion uses TrypLE digestion enzyme; and / or, the concentration of the enzyme is 0.5-2 mg / mL; and / or, the time for each step of enzymatic digestion is 6-20 minutes.

7. The method according to any one of claims 1 to 6, characterized in that: The method further comprises the step of culturing the prepared testis-like model; Preferably, the culture is a two-step culture: (i) culturing in maintenance medium for 7-10 days; (ii) culturing in differentiation medium for 1-7 days; The maintenance medium and the differentiation medium are serum-free medium.

8. The method according to claim 7, characterized in that The maintenance medium comprises: Ad-DMEM-F12 basal medium, Gibco's B-27 supplement, Gibco's GlutaMAX, N-acetyl-L-cysteine, EGF, FSH, IGF-1, FGF-9, activin A and T3; and / or, the differentiation medium comprises: Ad-DMEM-F12 basal medium, the B-27 supplement, the Glutamax, N-acetyl-L-cysteine, EGF, FSH, testosterone and RA; Preferably, the maintenance medium and the differentiation medium further contain antibiotics; the antibiotics are selected from one or more of penicillin, streptomycin and gentamicin; More preferably, the maintenance medium is based on Ad-DMEM-F12, comprising 1×B-27 supplement, 1×GlutaMAX, 1-2 mM N-acetyl-L-cysteine, 40-60 ng / mL mouse EGF, 20-25 ng / mL FSH, 90-120 ng / mL IGF-1, 40-60 μg / mL FGF-9, 40-60 ng / mL activin A, 90-110 nM T3, 90-110 U / mL penicillin, 0.1-0.2 mg / mL streptomycin and 40-60 μg / mL gentamicin; The differentiation medium is based on Ad-DMEM-F12, and contains 1×B-27 additive, 1×GlutaMAX, 1-2 mM acetyl-L-cysteine, 40-60 ng / mL mouse EGF, 20-25 ng / mL FSH, (1-1.5)×10 -6 M testosterone, (1-1.5) 10 -6 M RA, 90-110 U / mL penicillin, 0.1-0.2 mg / mL streptomycin, and 40-60 μg / mL gentamicin.

9. A testicular organoid, characterized in that: The testicular organoid is prepared by the method according to any one of claims 1 to 8.

10. Use of the testicular organoid as described in claim 9 in drug screening or in vitro reproductive toxicity evaluation.

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