Testis tissue culture medium, 3D culture method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a testis tissue culture medium, a 3D culture method and application of the testis tissue culture medium. According to the testis tissue culture medium, a double-antibody-containing DMEM low-sugar culture medium serves as a basic culture medium, lycium barbarum polysaccharide, a testis extracting solution and other components are added, a 3D culture system is constructed through biological hydrogel, the in-vivo microenvironment of testis can be simulated, and the testis tissue culture medium can replace animals to conduct drug screening and toxicity evaluation within a certain range; the use time of animals is greatly reduced, the method accords with the '3R 'principle of'reduction, replacement and optimization' of animal protection, and the method has great application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a testicular tissue culture medium and a 3D culture method and applications thereof in pharmacology and toxicology. Background Art

[0002] In drug development, it is crucial to evaluate the adverse effects of toxicity on the male reproductive system, which usually relies on in vivo animal experiments, in vitro cell experiments and sperm experiments. In vitro experiments have the advantages of being fast, simple and economical, and can be used to preliminarily screen the reproductive toxicity and pharmacology of drugs.

[0003] Testicular tissue culture in vitro is usually used to study the regulation mechanism of spermatogenesis. Testicular tissue of embryonic or newly born mice is selected for culture until spermatogenesis, which provides hope for the restoration of fertility in patients with spermatogenesis disorders due to abnormal testicular microenvironment, and provides an important reference for the development of clinical technology for fertility preservation in prepubertal cancer patients. At present, few studies have applied testicular tissue culture technology in vitro in the study of male reproductive toxicity. The testicular in vitro culture system can not only simulate the microenvironment of the testis in vivo, but also replace animals for drug screening and toxicity evaluation within a certain range. This method greatly reduces the use and time of animals and complies with the "3R" principle of animal protection (reduction, replacement, optimization). Therefore, the testicular tissue in vitro culture system has great application potential and is the main development direction of in vitro reproductive toxicity research. Traditional in vitro experiments are basically 2D culture systems, which cannot well simulate the microenvironment of the testis in vivo. It is necessary to develop a 3D culture method system that can better simulate the microenvironment of the testis in vivo, maintain the activity and function of the testicular tissue, and provide strong support for research and treatment. Summary of the invention The present invention provides a testicular tissue culture medium: The basal medium was DMEM low-glucose medium containing double antibodies, and the following components were added: fetal bovine serum (FBS), insulin transferrin selenium supplement, Lycium barbarum polysaccharide, testicular extract, N-acetylcysteine ​​(NAC), vitamin C, testosterone, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1); The concentrations of the added components after being fixed to volume using DMEM low-glucose medium are: 5% FBS, 1x insulin transferrin selenium supplement, 100ug / mL wolfberry polysaccharide, 10uL / mL testis extract, 10ug / mL NAC, 10 -3M vitamin C, 20uM testosterone, 50ng / mL EGF, 50ng / mL bFGF and 20ng / mL TGF-β1.

[0004] The preparation method of the testis extract comprises the following steps: taking fresh adult mouse testis tissue, cutting it into pieces, putting it into a precooled centrifuge tube containing physiological saline, ultrasonicating it in an ice box until the testis tissue is completely broken; centrifuging the suspension obtained by ultrasonication, taking out the supernatant and filtering it to obtain the testis extract.

[0005] Furthermore, the preparation method of the testicular extract is: (1) Sampling: Take 50 mg of fresh adult mouse testicular tissue, cut it into pieces, and place it in a pre-cooled centrifuge tube containing 200 uL of physiological saline.

[0006] (2) Ultrasound: Place the centrifuge tube containing the testicular tissue in an ice box for ultrasonic treatment at 70 Hz, run for 5 seconds, stop for 6 seconds, and cycle 15 times. If the testicular tissue is not completely broken, increase the number of cycles.

[0007] (3) Centrifugation: Centrifuge the suspension obtained by ultrasound at 14,000 rpm for 10 min at 4°C, and transfer the supernatant to a new pre-cooled centrifuge tube.

[0008] (4) Filtration: In a sterile environment, use a 5 mL syringe to draw out the clear liquid, install a sterile syringe filter to filter and obtain the testicular extract, which can be prepared for immediate use.

[0009] A testicular tissue 3D culture method comprises the following steps: S1. Testicular specimen collection and processing: testicular tissue is separated and cut into small tissue blocks suitable for tissue culture; S2. Testicular tissue block inoculation: The cut tissue blocks of S1 were inoculated onto the culture plate containing the biohydrogel, and an appropriate amount of biohydrogel was added to completely encapsulate the testicular tissue. After the biohydrogel solidified, an appropriate amount of the testicular tissue culture medium described above was added to each well for culture; S3. Drug treatment: Replace the testicular tissue culture medium described above containing the test drug for culture; S4. Result analysis: The pharmacology and toxicity of the test drug on testicular tissue were determined through activity detection and tissue morphology observation.

[0010] Furthermore, the activity detection is a CCK-8 test, which converts the absorbance value of each well of the culture plate to calculate the effect of the test drug on the activity of the testicular tissue block.

[0011] The tissue morphology observation is to observe the testicular tissue morphology of each group after culture according to HE staining sections, observe the apoptosis of testicular tissue according to Tunel staining results, and analyze the effect of the test drug on the morphology of testicular tissue blocks.

[0012] Preferably, the biohydrogel is a xeno-free synthetic polysaccharide VitroGel hydrogel, which is a xeno-free synthetic polysaccharide hydrogel system that can simulate the natural extracellular matrix (ECM) environment and can also be replaced by other hydrogels with good biocompatibility, such as type I rat tail collagen, matrigel, nanocellulose hydrogel, sodium alginate hydrogel, etc.

[0013] The testicular tissue is mouse testicular tissue.

[0014] The use of the testicular tissue culture medium or the testicular tissue 3D culture method described above in male reproductive toxicology and pharmacology evaluation.

[0015] Technical effects of the present invention: 1. The testicular tissue culture medium provided by the present invention uses wolfberry polysaccharides to promote the normal development of testicular germ cells and has a repair and protection effect on damaged spermatogenic cell chromosomes. The testicular extract contains abundant molecular peptides with a molecular weight of less than 10KDa and no antigenicity, which synergize with other components to play an important role in maintaining the normal function of the testicles. Compared with the existing culture medium, the testicular tissue cultured in the culture medium of the present invention has almost no dead cells, has a very superior effect in in vitro testicular 3D culture, and maintains the high activity and complete morphology of the testicular tissue.

[0016] 2. The present invention, by screening the hydrogel, preferably uses VitroGel hydrogel to embed testicular tissue to form a three-dimensional structure. When placed in a culture medium, the surrounding tissue culture fluid will penetrate into this structure, providing the testicular tissue with the nutrients needed for survival. And the biohydrogel applies a certain pressure around the testicular tissue, better simulating the testicular microenvironment in the human body. Animals can be replaced to carry out drug screening and toxicity evaluation within a certain range. This method greatly reduces the use and experimental time of animals, meets the "3R" principle (reduction, substitution, optimization) of animal protection, has great application potential, and is the main development direction of in vitro reproductive toxicity research.

[0017] 3. This invention is the first to use adult mouse testicles to apply the testicular tissue 3D culture system in male reproductive toxicology and pharmacology evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison diagram of the effect of culturing testicular tissue using the testicular tissue culture medium of the present invention; Figure 2 This is a diagram showing the results of exploring the effects of shikonin on male reproduction through the 3D culture method of testicular tissue. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. It should be noted that if no specific conditions are specified in the examples, they are carried out under normal conditions. The drawings described below are only some embodiments of the present application and do not limit the present invention in any form. For those skilled in the art, several adjustments and improvements can be made without departing from the concept of the present invention. All technologies implemented based on the above content of the present invention belong to the protection scope of the present invention.

[0020] Example 1. Testicular tissue culture medium of the present invention The present invention provides a testis tissue culture medium, which is based on DMEM low-glucose culture medium (containing double antibodies) (Solarbio, 31600), supplemented with 5% FBS (Excellbio, FSP5000), 1x insulin transferrin selenium supplement (ITS) (Yeasen, 60708ES10), 100 ug / mL wolfberry polysaccharide (MedChemExpress, HY-159062), 10 uL / mL testis extract, 10 ug / mL N-acetylcysteine ​​(MedChemExpress, HY-B0215), 10 -3 M vitamin C, 20 uM testosterone (MedChemExpress), 50 ng / mL EGF (MedChemExpress, HY-P7067), 50 ng / mL bFGF (Peprotech) and 20 ng / mL TGF-β1 (Peprotech).

[0021] The specific components are shown in Table 1. Table 1. Compositions of testicular tissue culture medium of the present invention Preparation method: According to the final concentrations in Table 1, FBS, insulin-transferrin-selenium supplement (ITS), Lycium barbarum polysaccharides, testicular extract, NAC, vitamin C, testosterone, EGF, bFGF and TGF-β1 were calculated and mixed, and then fixed to volume using DMEM low-glucose culture medium.

[0022] Some abbreviations: fetal bovine serum (FBS), N-acetylcysteine ​​(NAC), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1).

[0023] Experimental Example 2. 3D culture method of testicular tissue of the present invention 1. Testis specimen collection: Adult male mice were euthanized by carbon dioxide inhalation, and the testicles were removed from the abdominal area and stored in culture medium at low temperature. The testicles must be used for subsequent experiments within 30 minutes of ex vivo separation to ensure activity and structural integrity.

[0024] 2. Testicular sample processing: Wash the testicles three times with normal saline, gently remove the white membrane, separate the testicular tissue inside, and then cut the testicular tissue inside the white membrane into tiny tissue blocks of the same size. 3. Testicular tissue block inoculation: Add an appropriate amount of VitroGel hydrogel (a xeno-free synthetic polysaccharide hydrogel system) to each well until the bottom of the well plate is submerged, and place in an incubator until the hydrogel solidifies (this step is completed before the second step), and inoculate the cut tissue blocks in step 2 onto a 96-well cell culture plate, with one testicular tissue block inoculated per well (other specifications of culture plates can be inoculated with multiple blocks as needed). Add an appropriate amount of hydrogel to the well again until the testicular tissue is completely wrapped, and place the culture plate in a cell culture incubator at 37°C and 5% CO2 for about half an hour. After the hydrogel is completely solidified, remove the culture plate, add an appropriate amount of the testicular tissue culture medium described in Example 1 to each well, place in an incubator for culture, and change the culture medium every 2-3 days.

[0025] Experimental Example 3. Testicular tissue 3D culture-pharmacology / toxicology detection model of the present invention 1. Testis specimen collection: Adult male mice were euthanized by carbon dioxide inhalation, and the testicles were removed by opening the abdominal area and stored in culture medium at low temperature. The testicles must be used for subsequent experiments within 30 minutes of ex vivo separation to ensure activity and structural integrity.

[0026] 2. Testicular sample processing: Wash the testicles three times with saline, gently remove the tunica albuginea, separate the testicular tissue inside, and then cut the testicular tissue inside the tunica albuginea into tiny tissue blocks of the same size.

[0027] 3. Testicular tissue block inoculation: Add an appropriate amount of VitroGel hydrogel to each well until the bottom of the well plate is submerged, and place in an incubator until the hydrogel solidifies (this step is completed before the second step), and inoculate the cut tissue blocks in step 2 onto a 96-well cell culture plate, with one testicular tissue block inoculated in each well (other specifications of culture plates can be inoculated with multiple blocks as needed). Add an appropriate amount of hydrogel to the well again until the testicular tissue is completely wrapped, and place the culture plate in a cell culture incubator at 37°C and 5% CO2 for about half an hour. After the hydrogel is completely solidified, remove the culture plate, add an appropriate amount of the testicular tissue culture medium described in Example 1 to each well, and place it in an incubator for culture.

[0028] 4. Drug treatment: The testis tissue culture medium was cultured for 24 hours to ensure stability, the original culture medium was discarded, and the testis tissue culture medium described in Example 1 containing the test drug was added and replaced every 24 hours for 3 days (can be shortened or extended as needed).

[0029] 5. Activity detection and tissue morphology observation: Add CCK-8 solution to the wells containing tissues to make the content 10%, place incubator and continue incubation for 4-6 hours, use microplate reader to test the absorbance value of each well. Take out part of the tissue, fix and embed, and do HE staining and Tunel staining.

[0030] 6. Result analysis: According to the absorbance conversion formula, the absorbance value of each well of the well plate was converted, and then the effect of the test drug on the activity of the testicular tissue block was calculated; the testicular tissue morphology of each group after culture was observed according to the HE staining sections, and the apoptosis of the testicular tissue was observed according to the Tunel staining results, thereby reflecting the effect of the test drug on the morphology of the testicular tissue block.

[0031] Comparative Example 1. Comparison of the effects of the testicular tissue culture medium of the present invention and the reported testicular tissue culture medium The testis tissue culture medium of the present invention is shown in Example 1.

[0032] The testicular tissue culture medium reported in the literature is: α-MEM medium, 40 mg / mL bovine serum albumin (Sigma), 600 μM palmitic acid (Sigma), 600 μM oleic acid (Sigma), 100 μM linoleic acid (Sigma), 50 μM linolenic acid (Sigma), 600 μM stearic acid (Sigma), 3.2 μg / mL cholesterol (Sigma), 20 μg / mL phosphatidylcholine (Sigma), 3.5 μg / mL sphingomyelin (Sigma), 1 μM retinoic acid (Sigma), 1 μM retinol (Sigma), 1 μM testosterone (Wako), 2.0 ng / mL triiodothyronine (Sigma), 1 μg / mL luteinizing hormone (Sigma), 1 μg / mL yolk (Sigma), 100 IU / mL penicillin, 100 μg / mL streptomycin, 250 ng / mL amphotericin (ThermoFisher), and 1.82 g / L NaHCO3. (The mentioned literature is: SANJO H, KOMEYA M, SATO T, et al. In vitro mouse spermatogenesis with an organ culture method in chemically defined medium [J]. PLOS ONE, 2018, 13(2): e0192884.) 1. Experimental Methods The testicular tissue culture medium reported in the above literature and the testicular tissue culture medium of the present invention were used respectively, and the testicular tissue 3D culture method of the testicular tissue described in Example 2 was used to culture the testicular tissue of the same adult male mouse for 7 days. Three samples were made in each group as replicates. The activity of the testicular tissues of the two groups after 7 days of culture was compared by trypan blue staining and CCK-8 experiment.

[0033] 2. Experimental Results The experimental results are as follows Figure 1 As shown, after culturing testicular tissue for 7 days in the testicular tissue culture medium of the present invention (new culture medium) and the testicular tissue culture medium reported in the above literature (existing culture medium), Figure 1 The results of A in Figure 1 show that there are almost no dead cells in the testicular tissue cultured with the new culture medium, while the existing culture medium shows some dead cells. Figure 1 It can be seen from B in that the activity of the testicular tissue cultured in the new culture medium is significantly higher than that of the testicular tissue cultured in the existing culture medium. The above results can prove that the testicular tissue culture medium of the present invention can be used for 3D culture of testicular tissue, and can better maintain testicular activity than the existing culture medium, and has a better culture effect.

[0034] Compared with the existing culture medium, the unique beneficial effects of the culture medium of the present invention are mainly due to the following reasons: Lycium barbarum polysaccharides can promote the normal development of testicular germ cells and have a repairing and protective effect on damaged spermatogenic cell chromosomes; the testicular extract contains abundant molecular peptides with a molecular weight of less than 10KDa and no antigenicity, which plays an important role in maintaining the normal function of the testicles; and the culture medium is also beneficial due to the synergistic effect of the composition and optimal ratio of the entire culture medium.

[0035] Example 4. Exploring the effect of shikonin on male reproduction through 3D culture of testicular tissue 1. Experimental Methods 1. Testis specimen collection: Adult male mice were euthanized by carbon dioxide inhalation, and the testicles were removed by opening the abdominal area and stored in culture medium at low temperature. The testicles must be used for subsequent experiments within 30 minutes of ex vivo separation to ensure activity and structural integrity.

[0036] 2. Testicular sample processing: Wash the testicles three times with saline, gently remove the tunica albuginea, separate the testicular tissue inside, and then cut the testicular tissue inside the tunica albuginea into tiny tissue blocks of the same size. 3. Testicular tissue block inoculation: Add an appropriate amount of hydrogel to each well until the bottom of the well plate is submerged, and place in an incubator until the hydrogel solidifies (this step is completed before the second step), and inoculate the cut tissue blocks in step 2 onto a 96-well cell culture plate, with one testicular tissue block inoculated in each well (other specifications of culture plates can be inoculated with multiple blocks as needed). Add an appropriate amount of hydrogel to the well again until the testicular tissue is completely wrapped, and place the culture plate in a cell culture incubator at 37°C and 5% CO2 for about half an hour. After the hydrogel is completely solidified, remove the culture plate, add an appropriate amount of the testicular tissue culture medium described in Example 1 to each well, and place it in an incubator for culture.

[0037] 4. Drug treatment: The testis tissue culture medium was cultured for 24 hours to ensure the state was stable, the original culture medium was discarded, and an appropriate amount of the testis tissue culture medium described in Example 1 containing 20 μM shikonin was added. A control group of the testis tissue culture medium described in Example 1 without drug addition was set up, and the culture medium was replaced every 24 hours. The culture was carried out for 3 days (can be shortened or extended as needed).

[0038] 5. Tissue morphology observation: After the testicular tissue was cultured in vitro for 3 days, some tissue blocks were removed, fixed with 4% paraformaldehyde overnight, and embedded in paraffin. The obtained paraffin blocks were then cut into 5 μM continuous sections and mounted on slides. After dewaxing, the sections were stained with hematoxylin and eosin in turn. Images were captured using an OLYMPUSVS200 digital slice scanner.

[0039] 6. Activity detection: After the tissue block was cultured in a 96-well plate for 3 days, the culture medium was replaced with the fresh culture medium described in Example 1 without shikonin. CCK-8 solution was added to make its content 10%, and the cells were placed in an incubator for further incubation for 4 hours. The absorbance value of each well was tested using an enzyme marker. According to the absorbance value conversion formula, the absorbance value of each well of the well plate was converted to calculate the effect of shikonin on the activity of the testicular tissue block.

[0040] 7. Use YF ® TUNEL apoptosis detection kit (UElandy, Suzhou, China) was used to stain tissue sections, and DAPI solution (Solarbio, Beijing, China) was used to counterstain tissue sections. Images were obtained using a laser confocal microscope (Olympus, Japan).

[0041] 2. Experimental Results The experimental results are as follows Figure 2 As shown by Figure 2 From the results of A cell activity, it can be seen that the testicular tissue activity of the shikonin group was significantly reduced. Figure 2B (the scale bars on the left and right are 100 μm and 20 μm, respectively) showed that the testicular tissue morphology of each group after culture was observed. Compared with the control group, the testicular tissue in the shikonin group was severely damaged, the boundaries of the seminiferous tubules were broken, the blank lumen was enlarged, the germ cells were loosely arranged, and the sperm count in the lumen was reduced. Figure 2 The results in C (scale bar is 100 μm) showed that compared with the control group, the number of apoptotic cells in the testicular tissue treated with 20 μM shikonin was greater, and shikonin could induce apoptosis in testicular tissue.

[0042] 3. Conclusion: Exposure of shikonin to mouse testicles can lead to a significant decrease in testicular tissue activity, an increase in the number of apoptotic cells, and damage to testicular tissue morphology. It can be seen that shikonin has certain reproductive toxicity. Therefore, when using shikonin to treat cancer and other diseases, the potential harm of shikonin to the reproductive system should be considered. It also shows that the testicular tissue culture medium and its 3D culture method of the present invention can be applied to male reproductive toxicology and pharmacology evaluation.

[0043] Summarize: 1. The present invention provides a testicular tissue culture medium, which has a very superior effect in in vitro testicular 3D culture and maintains the high activity and complete morphology of testicular tissue.

[0044] 2. For the first time, the testicular in vitro culture method was systematically applied to male reproductive toxicology and pharmacology evaluation using adult mouse testicles.

[0045] 3. The advantage of this technology is that VitroGel hydrogel is preferably used to embed testicular tissue to form a three-dimensional spatial structure. When placed in a culture medium, the surrounding tissue culture fluid will penetrate into this structure, providing the testicular tissue with the nutrients it needs to survive. In addition, the hydrogel exerts a certain amount of pressure around the testicular tissue, better simulating the testicular microenvironment in the human body.

Claims

1. A testicular tissue culture medium, characterized in that: The basic medium is DMEM low-glucose medium containing double antibodies, and the following components are added: fetal bovine serum, insulin transferrin selenium supplement, Lycium barbarum polysaccharide, testicular extract, N-acetylcysteine, vitamin C, testosterone, epidermal growth factor, basic fibroblast growth factor and transforming growth factor-β1; The concentrations of the added components after being fixed to volume using DMEM low-glucose medium are: 5% fetal bovine serum, 1x insulin transferrin selenium supplement, 100ug / mL wolfberry polysaccharide, 10uL / mL testis extract, 10ug / mL N-acetylcysteine, 10 -3 M vitamin C, 20uM testosterone, 50ng / mL epidermal growth factor, 50ng / mL basic fibroblast growth factor, and 20ng / mL transforming growth factor-β1.

2. The testicular tissue culture medium according to claim 1, characterized in that: The preparation method of the testis extract comprises the following steps: taking fresh adult mouse testis tissue, cutting it into pieces, putting it into a precooled centrifuge tube containing physiological saline, ultrasonicating it in an ice box until the testis tissue is completely broken; centrifuging the suspension obtained by ultrasonication, taking out the supernatant and filtering it to obtain the testis extract.

3. A 3D culture method for testicular tissue, characterized in that: The steps include: S1. Testicular specimen collection and processing: testicular tissue is separated and cut into small tissue blocks suitable for tissue culture; S2. Testicular tissue block inoculation: The cut tissue block of S1 is inoculated onto a culture plate containing a biohydrogel, and an appropriate amount of biohydrogel is added to completely encapsulate the testicular tissue. After the biohydrogel solidifies, an appropriate amount of the testicular tissue culture medium as described in claim 1 is added to each well for culture; S3. Drug treatment: replacing the testicular tissue culture medium as described in claim 1 containing the test drug for culture; S4. Result analysis: The pharmacology and toxicity of the test drug on testicular tissue were determined through activity detection and tissue morphology observation.

4. The testicular tissue 3D culture method according to claim 3, characterized in that: The activity detection is a CCK-8 test, which converts the absorbance value of each well of the culture plate to calculate the effect of the test drug on the activity of the testicular tissue block.

5. The testicular tissue 3D culture method according to claim 3, characterized in that: The tissue morphology observation is to observe the testicular tissue morphology of each group after culture according to HE staining sections, observe the apoptosis of testicular tissue according to Tunel staining results, and analyze the effect of the test drug on the morphology of testicular tissue blocks.

6. The testicular tissue 3D culture method according to claim 3, characterized in that: The biological hydrogel is a xeno-free synthetic polysaccharide VitroGel hydrogel or type I rat tail collagen or matrix gel or nanocellulose hydrogel or sodium alginate hydrogel.

7. The testicular tissue 3D culture method according to claim 6, characterized in that: The biohydrogel is a xeno-free synthetic polysaccharide VitroGel hydrogel.

8. The testicular tissue 3D culture method according to claim 3, characterized in that: The testicular tissue is mouse testicular tissue.

9. Use of the culture medium according to claim 1 or 2 or the culture method according to any one of claims 3 to 8 in male reproductive toxicology and pharmacology evaluation.

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