CN114908032A - Preparation, culture, cryopreservation and resuscitation method and application of testicular-like organ - Google Patents

Preparation, culture, cryopreservation and resuscitation method and application of testicular-like organ Download PDF

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CN114908032A
CN114908032A CN202210271380.7A CN202210271380A CN114908032A CN 114908032 A CN114908032 A CN 114908032A CN 202210271380 A CN202210271380 A CN 202210271380A CN 114908032 A CN114908032 A CN 114908032A
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culture
testicular
organoid
testis
cells
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CN114908032B (en
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杨艳
黄亚东
曹振
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Jinan University
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Abstract

The invention discloses a cultured testicular organoid, and preparation, culture, cryopreservation resuscitation methods and applications thereof. The testis organoid prepared by the invention has the same genetic background height with the testis tissue from which the testis organoid is derived, and meanwhile, the cultured organoid can keep the specific function of the testis for a long time and can be frozen and restored. The preparation and culture processes are simple and convenient to operate, the repeatability is high, the drug effect testing time is short, the culture cost is more economic than that of an animal model, the established testicular organoids can realize high-flux drug screening, and the application prospect is good.

Description

Preparation, culture, cryopreservation and resuscitation method and application of testicular-like organ
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for preparing, culturing, cryopreserving and resuscitating testis organoids and application thereof.
Background
The testis is a reproductive organ of a male animal, is respectively positioned on the left side and the right side of a scrotum and is oval, a fibrous membrane is arranged on the surface of the testis pill and is called as a tunica albuginea, the tunica albuginea thickens along the rear edge of the testis and protrudes into the testis to form a mediastinum, a plurality of connective tissue compartments are emitted from the mediastinum to substantially divide the testis into a plurality of testis lobules, the testis lobules contain convoluted seminal tubules, the epithelium of the seminal tubules can generate sperms, interstitial cells are arranged in the connective tissue between the tubules, and the interstitial cells generate androgen and are closely related to secondary androgenesis, physiological functions and the like.
Organoids (Organoids) belong to the group of (3D) cell cultures, are organ-specific collections of cells derived from stem or precursor cells, contain some of the key features of their representative organs, are able to mimic the organizational structure, cell type composition of real organs, and possess specific functional characteristics, while maintaining the advantages of a simplified and readily available cell culture model. Unlike two-dimensional (2D) cell culture models, organoid culture cultures multiple cell populations that a particular tissue or organ contains in a 3D environment, which cultures a microenvironment system closer to the in vivo system. Therefore, the organoid can be used as an in vitro platform to research the interaction between cells, tissue development and toxicology, effectively supplement the existing animal and two-dimensional (2D) cell culture models, and has wide application prospect in the aspects of basic research of various organ physiopathology, accurate medical treatment, drug screening and development, regenerative medicine and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for preparing, culturing, freezing and recovering testicular organoids and application thereof, so as to solve the problems in the prior art.
Specifically, the invention provides a method for preparing, culturing, freezing and resuscitating mouse testicular organoids and application thereof, wherein the method comprises the following steps: taking a young mouse testicular tissue, and sequentially soaking, washing, removing a capsule, shearing and digesting cells to obtain a mouse testicular cell suspension; carrying out hanging drop suspension culture on the suckling mouse testis cells to obtain a testis organoid; collecting testis organoid by centrifugation, and culturing in suspension with specific culture medium.
Accordingly, in one aspect, the present invention provides a testicular organoid comprising: mesenchymal cells, supporting cells, spermatogonial stem cells, or a combination thereof.
According to a preferred embodiment of the invention, the testicular organoids are obtained by in vitro cell culture and are capable of producing testosterone, preferably the cells are of rodent or non-human primate origin, mouse or rat.
According to a preferred embodiment of the invention, the testicular organoid expresses one or any combination of the following protein markers: fibronectin (Fibronectin), Alpha-smooth muscle actin (Alpha-SMA), Fibronectin 1 (light junction protein, ZO-1), Wilms tumor protein 1 (WT 1), steroid hormone synthetic acute regulatory protein (Star), and Proliferating Cell Nuclear Antigen (PCNA).
According to a preferred embodiment of the invention, the testicular organoids are capable of expressing one of the following genes/proteins or any combination thereof: 3 β -hydroxysteroid dehydrogenase (3-Beta-hydroxysteroid dehydrogenase1, Hsd3b1), 17 β -hydroxysteroid dehydrogenase 3(17 β -hydroxysteroid dehydrogenase 3, Hsd17b3), steroid hormone synthesis acute regulatory protein (Steroidogenic acid regulator, Star), sex-determining region Y box protein 9(Sry related HMG box-9, Sox9), Wilms tumor protein 1(Wilms tumor 1 WT1), Androgen Receptor (Androgen Receptor, Ar), follicle stimulating hormone Receptor (fsher), protooncogene (Ret proto-gene, C-Ret), and cyclic adenosine monophosphate response component binding protein (cyclic AMP-modulator).
According to a preferred embodiment of the invention, the testicular organoids are capable of secreting one of the following factors or any combination thereof: anti-Mullerian hormone (Amh), Desert hedgehog (Desert hedgehog), GLIAL cell line-derived neurotrophic factor (GLIAL cell-line derived neurotrophic factor, GDNF), inhibin b (inhibinb), C-X-C chemokine ligand 12(C-X-C chemokine ligand 12, Cxcl12) and Fibroblast growth factor 2(Fibroblast growth factor 2, Fgf 2).
Another aspect of the present invention provides a method for preparing a testicular organoid, comprising the steps of:
1) obtaining a juvenile animal testicular cell;
2) carrying out hanging drop culture on the obtained testicular cells; and optionally
3) Performing suspension culture on the hanging drop culture product obtained in the step 2), thereby obtaining the testicular organoid.
It should be noted that in the method of the present invention, the hanging drop culture of step 2) has been able to form testicular organoids, and the purpose of the suspension culture of step 3) is to provide nutrients to maintain the long-term culture of testicular organoids.
According to a preferred embodiment of the invention, the animal is a rodent or a non-human primate, a mouse or a rat.
When the animal is a mouse, the young mouse is preferably a mouse 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
According to a preferred embodiment of the present invention, in the step 1), the testis cell of the young animal is obtained by taking the testis tissue of the young animal, sequentially soaking, washing, decapsulating, shearing and cell digestion, wherein preferably, the soaking is performed by using PBS; and/or the washing solution used in the washing is PBS containing penicillin and streptomycin (for example, PBS and 100IU/ml penicillin + 100ug/ml streptomycin are mixed); preferably, the digestion is with pancreatin.
According to a preferred embodiment of the invention, adult cells are used, preferably a collection of cells obtained after digestion of testicular tissue of animals from 3 days to 2 weeks, particularly preferably from 7 to 10 days old.
According to a preferred embodiment of the invention, the PBS used for soaking is ice-precooled.
According to a preferred embodiment of the invention, the hanging-drop cultivation is carried out as follows: testis cells obtained after digestion are dropped on the inside of a dish lid (forming hemispherical droplets), and the dish lid is turned upside down on a dish to which PBS such as PBS is added, followed by culturing in an incubator, thereby obtaining a 3D testis organoid.
Preferably, in the above step 2), the culture medium used for the hanging-drop culture is a serum-free medium containing Epidermal Growth Factor (EGF), such as a basal medium containing EGF and KSR (serum replacement)Culture medium (preferably DMEM, MEM, DMEM/F12 medium) at 34-37 deg.C and 3% -5% CO 2 The suspension drop culture is carried out for 5-7 days at a concentration, preferably, the concentration of EGF in the culture medium is 50-100 ng/ml. Preferably, the culture medium used for the hanging-drop culture is 10% KSR medium by volume containing 50ng/ml EGF and 5% CO at 34 ℃ 2 Hanging drop culture for 5 days under the concentration; and/or
In the above step 3), the culture medium used in the suspension culture is a basal medium (preferably DMEM, MEM, DMEM/F12 medium) containing KSR, LH (luteinizing hormone) and FSH (follicle stimulating hormone), and preferably contains the following components by volume of the culture medium (e.g., DMEM): KSR, 5-10 volume%; LH, 1-10 ng/ml; FSH, 1-10 ng/ml. Preferably, the content of each component is as follows: KSR, 10 vol%; LH, 1 ng/ml; FSH, 1 ng/ml; and/or
Preferably, the hanging drop culture product obtained in the step 2) is centrifuged to enrich cells, added into the culture medium and cultured at 34-37 ℃ and 3-5% CO 2 Culturing at 120-; the medium is preferably changed every 5 days and can be continuously cultured for a long period.
According to a particularly preferred embodiment of the invention, the above EGF, LH and FSH are each of mammalian origin, preferably human or mouse EGF, LH and FSH.
The serum replacement medium (e.g., KSR) used in the present invention has advantages of a definite composition and a high consistency as compared with the serum medium. However, the difference between the batches of the serum culture medium is large, and the possibility of contamination of animal-derived proteins or viruses exists. The medium is therefore preferably a serum-free medium.
According to a particularly preferred embodiment of the present invention, there is provided a method for producing testicular organs, comprising the steps of:
(1) taking male Kunming suckling mouse testis tissue, and soaking in precooled PBS (with double antibodies (penicillin and streptavidin)) added;
(2) taking 6 sterile circular bacteria dishes of 9x9 cm, sequentially adding PBS (phosphate buffer solution) containing double antibodies, numbering 1-6, transferring testis tissues to the bacteria dish of numbering 1 for rinsing for 1-2min, sequentially transferring to the bacteria dishes of No. 2 and No. 3 for rinsing, slightly puncturing a testis capsule by using sterile surgical forceps, taking out the testis tissues, transferring to the bacteria dish of No. 4 for washing for 1-2min, and sequentially transferring to the bacteria dishes of No. 5 and No. 6 for washing;
(3) taking out the tissue from No. 6 bacterial dish, transferring to penicillin bottle, and shearing testis tissue for 5 min;
(4) adding pancreatin, placing in a constant temperature incubator at 37 deg.C, digesting for 15min, and shaking for 2-3 times;
(5) after digestion, adding 10% FBS to neutralize pancreatin, and blowing the suspension for 5min by using a glass pipette;
(6) transferring the mixture into a 15ml centrifuge tube to centrifuge at 1500rpm for 5 min;
(7) discarding the supernatant, resuspending the precipitate with DMEM containing 10 vol% of KSR, standing for about 5min, transferring the supernatant, discarding the bottom precipitate, taking out 90ul of suspension from the supernatant, mixing with 10ul of trypan blue, and counting;
(8) the resuspended testis cells were added to DMEM containing 10 vol% KSR +50ng/ml EGF at 1X10 5 Diluting cells at a cell/ml ratio;
(9) taking 20 mu l of cell dilution liquid to drop in a square bacterial dish cover of 10x10 cm, and inversely buckling the bacterial dish cover on a bacterial dish added with PBS;
(10) the bacterial dish was transferred to a 34 ℃ incubator and cultured by hanging drop for 5 days to obtain 3D testicular organoids with an initial cell count of about 2000/pellet.
Also provided is a method for culturing testicular organoids, comprising the steps of:
(1) taking the primary testicular organoid prepared above, transferring to a 15ml centrifuge tube for enrichment, and centrifuging at 1500rpm for 5 min;
(2) discarding the supernatant and resuspending testicular organoids in DMEM media containing 10 vol% KSR +1ng/ml LH +1ng/ml FSH;
(3) transferring to a 50ml triangular flask, adding 20-30ml DMEM medium containing 10 volume percent KSR +1ng/ml LH +1ng/ml FSH, and performing suspension culture;
(4) the flask was transferred to a constant temperature culture shaker at 34 ℃ for 120-160rpm culture, and the medium was changed every 5 days.
According to a preferred embodiment of the present invention, there is also provided a testicular organoid producible by the method for producing a testicular organoid according to the present invention.
In another aspect, the invention also provides the use of the testicular organoids of the invention in drug screening or determining drug toxicity.
According to a preferred embodiment of the invention, the drug candidate is contacted with a testicular organoid according to the invention.
According to a preferred embodiment of the present invention, there is also provided a use of a testicular organoid for a drug screening test comprising the steps of:
(1) organoids were inoculated into 96-well plates at 5/80 ul, with CdCl of varying concentrations 2 (1.56. mu.M, 3.12. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, 200. mu.M), cultured on a shaker at 160rpm, organoid status was photographed after 3 days and survival was tested by the cck8(cell counting kit-8) method.
In another aspect, the present invention also provides a method for cryopreservation and resuscitation of testicular organoids, comprising the steps of:
(1) freezing and storing: after organoid suspension culture for 5 days, collecting from a triangular flask, centrifuging for 5min in a 15ml centrifuge tube at 1500 rpm;
(2) discarding the supernatant, adding organoid cryopreservation liquid, transferring to a cryopreservation tube, transferring the cryopreservation tube to a cryopreservation box at-80 ℃ overnight, and transferring to liquid nitrogen the next day;
(3) and (3) unfreezing: taking out the frozen organoids from liquid nitrogen, rapidly thawing in 37 deg.C water bath, transferring into a prepared 15ml centrifuge tube containing 10% FBS, centrifuging at 1500rpm for 5min to obtain testis organoids, and culturing according to the above culture method.
The innovation points of the invention at least comprise:
(1) the testis organoid can be obtained by the hanging drop suspension culture method, the operation is simple, and special equipment is not needed.
(2) Can preserve the specific functions of supporting cells and interstitial cells in the testis for a long time and simulate the function of the testis in vitro.
(3) The testicular organoids can be tested for drugs and evaluated for toxicology.
Advantageous effects
The testis organoid prepared by the invention has the same genetic background height with the testis tissue from which the testis organoid is derived, and meanwhile, the cultured organoid can keep the specific function of the testis for a long time and can be frozen and restored. The preparation and culture processes are simple and convenient to operate, the repeatability is high, the drug effect testing time is short, the culture cost is more economic than that of an animal model, the established testicular organoids can realize high-flux drug screening, and the application prospect is good.
Drawings
FIG. 1 is a flow chart of the preparation, suspension culture and formation of testicular organoid hanging drops of the present invention.
FIG. 2 is the area change curve of testis organoid at 5, 10, 15, 20, 25, 30 days in the suspension culture process of testis organoid of the present invention.
FIG. 3 shows HE staining of testis organoids at days 5, 10, 15, 20, 25, and 30 during the testis organoid suspension culture process of the present invention.
FIG. 4 shows immunofluorescence staining of testicular organoids at day 15 during testicular organoid suspension culture according to the present invention.
FIG. 5 shows the gene expression level changes of testis organoid mesenchymal cells, supporting cells and spermatogonial stem cells at days 5, 10 and 15 during the suspension culture of testis organoids according to the present invention.
FIG. 6 shows the change of testosterone in testis organoids at days 5, 10, 15, and 20 during the suspension culture of testis organoids of the present invention.
FIG. 7 shows the variation of the gene expression level of the testis organoid secretion factors in days 5, 10, 15, 20, 25 and 30 during the suspension culture of testis organoids according to the present invention.
FIG. 8 shows the results of TUNEL apoptosis staining experiments performed after cryo-sectioning after 3 days of culture and cryo-preservation and resuscitation of testicular organoids according to the present invention.
FIG. 9 shows that in the process of testis organoid suspension culture according to the present invention, the testis organoids are treated with CdCl of different concentrations at day 15 2 After treatment, cck8 was measured.
Detailed Description
Unless otherwise indicated, the terms used herein have the ordinary technical meaning as understood by those skilled in the art.
Examples
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. The specific techniques or conditions are not specified in the examples, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The reagents or apparatus used are conventional products which are commercially available, not indicated by the manufacturer.
Reagent
Penicillin (MDBio),100 IU/ml;
streptomycin (MDBio),100 ug/ml;
pancreatin (Gibco);
fetal bovine serum fbs (excell bio);
DMEM(Gibco)；
serum replacement KSR (life),10 vol% KSR;
trypan blue (Sciencell);
luteinizing hormone LH (human, Merk Millipore),1 ng/ml;
follicle stimulating hormone FSH (human, Prospec), 1 ng/ml;
organoid cryopreservation was purchased from invasive core international biotechnology (guangzhou) ltd;
cadmium chloride (Alorich), 1.56-200. mu.M
Example 1: preparation method of testis organoid
Taking testis tissue of 7-10 days male Kunming suckling mouse (purchased from Experimental animal center of Guangdong province), placing on ice box, and soaking in PBS containing double antibody (penicillin (MDBio),100IU/ml, and streptomycin (MDBio),100 ug/ml);
taking 6 sterile 9x9 cm circular bacteria dishes, sequentially adding the PBS containing the double antibodies, sequentially numbering 1-6, transferring the testis tissues to the bacteria dish numbered 1 for rinsing for 1-2min, sequentially transferring to the bacteria dishes numbered 2 and 3 for rinsing, slightly puncturing the testis capsule by using sterile surgical forceps, taking out the testis tissues, transferring to the bacteria dish numbered 4 for washing for 1-2min, and sequentially transferring to the bacteria dishes numbered 5 and 6 for washing;
taking out testis tissue from No. 6 bacterial dish with forceps, transferring into penicillin bottle, shearing testis tissue with surgical curved scissors for about 5min until the tissue is homogenized;
adding 10mL of pancreatin (Gibco) into a penicillin bottle, placing the penicillin bottle in a constant-temperature incubator at 37 ℃ for digestion for 15min, taking out the penicillin bottle at intervals of 2-3 times, observing the digestion state and appropriately shaking the penicillin bottle;
after digestion, adding 10% FBS with double volume to neutralize pancreatin, and blowing the suspension for 5min by using a glass pipette until no floccule appears;
transferring the mixture into a 15ml centrifuge tube and centrifuging the mixture for 5min at 1500 rpm;
the supernatant was discarded, resuspended in DMEM containing 10% by volume of KSR (Life), 90. mu.l of the suspension was taken out and mixed with 10. mu.l of Trypan blue (Sciencell), and counted within 3 min;
the resuspended testis cells were added to DMEM medium containing 10 vol% KSR and 50ng/ml EGF (human EGF, Pepro Tech Co., USA, Cat: AF-100-15) at 1X10 5 Diluting the resuspension fluid at an individual cell/ml ratio;
taking 20ul of cell suspension by using a discharging gun, transferring the cell suspension to the inner side of a square bacterium dish cover of 10x10 cm, enabling the bacterium dish cover to be red and hemispherical, and inversely buckling the bacterium dish cover on a bacterium dish added with PBS;
the petri dish was transferred to 34 ℃ with 5% CO 2 And (5) standing the suspension drop in the constant temperature incubator for 5 days to obtain the 3D testis organoid with the initial cell number of about 2000/grain.
Example 2: method for culturing testicular organoids
Taking the primary testis organoid prepared according to the above example 1, transferring the primary testis organoid into a 15ml centrifuge tube, and centrifuging the primary testis organoid at 1500rpm for 5 min;
discarding the supernatant and resuspending testicular organoids in DMEM media containing 10 vol% KSR +1ng/ml LH +1ng/ml FSH;
transferring to 50ml triangular flask, adding 20-30ml DMEM medium containing 10 vol% KSR +1ng/ml LH +1ng/ml FSH according to organoid number, and performing suspension culture;
the flask was transferred to a constant temperature of 34 ℃ with 5% CO 2 The medium was incubated at 120-.
FIG. 2 is the area change curve of testis organoid at 5, 10, 15, 20, 25, 30 days in the suspension culture process of testis organoid of the present invention. The results of the area change curves show that the volume of the testis organoids increases slowly with the increase of the culture time, indicating that the organoids can grow and maintain morphology for a long time.
FIG. 3 shows HE staining of testis organoids at days 5, 10, 15, 20, 25, and 30 during the testis organoid suspension culture process of the present invention. HE staining indicated that cells within the testicular organoids formed a structure resembling testicular tissue.
FIG. 4 shows the fluorescent immunostaining of testis organoids at day 15 during the suspension culture of testis organoids according to the present invention. Immunofluorescent staining revealed that the supporting cells (labeled protein: Fibronectin (Fibronectin), ZO-1, WT1), pericoromyoid cells (labeled protein: α SMA), mesenchymal cells (labeled protein: Star), and spermatogonium (labeled protein: PCNA) in the organoid grew normally and expressed proteins required for maintaining cell growth.
Figure BDA0003553412660000091
FIG. 5 shows the gene expression level changes of testis organoid interstitial cells, supporting cells and spermatogonial stem cells at days 5, 10 and 15 during the suspension culture of testis organoid of the present invention. The PCR result shows that the gene expression of the obtained testis organoid is close to that of adult mouse testis tissue, and the prepared organoid is similar to testis in gene expression level.
Figure BDA0003553412660000101
The primers were synthesized by Huada Gene Co.
FIG. 6 shows the testosterone changes of testis organoids at days 5, 10, 15, and 20 during the suspension culture of testis organoids according to the present invention (testosterone radioimmunoassay kit, northern organisms). The results show that the testis organoid culture system can effectively maintain testosterone secretion within 20 days of culture, and the state is optimal on day 15, which shows that the organoid can normally secrete testosterone and has the function similar to that of testis.
FIG. 7 shows the expression level changes of testis organoid secretion factor genes at days 5, 10, 15, 20, 25, and 30 during the suspension culture of testis organoids according to the present invention. The PCR result shows that the relevant secretion factor of the testis organoid is close to the testis tissue of an adult mouse, which indicates that the prepared organoid is similar to the testis in the expression level of the relevant secretion factor.
Figure BDA0003553412660000111
The primers were synthesized by Huada Gene Co.
Example 3: cryopreservation and resuscitation of testicular organoids
Freezing and storing: after the organoid suspension culture is carried out for 5 days, collecting suspension from a triangular flask, and centrifuging the suspension in a 15ml centrifuge tube at 1500rpm for 5 min;
discarding the supernatant, adding organoid cryopreservation solution (purchased from Chuangzhou International Biotechnology, Limited, Inc.) and transferring to a cryopreservation tube, transferring the cryopreservation tube to a cryopreservation box at-80 deg.C overnight, and transferring to liquid nitrogen the next day;
unfreezing: taking out organoid from liquid nitrogen, rapidly thawing in 37 deg.C water bath, transferring into 15ml centrifuge tube containing 10% FBS, centrifuging at 1500rpm for 5min to obtain revived testis organoid, and culturing according to the above culture method.
FIG. 8 shows the TUNEL apoptosis staining experiment after frozen section after 3 days of culture after cryopreservation and resuscitation of testis organoids according to the present invention. The results show that: the unfrozen group had essentially no apoptosis (green indicates apoptosis), the commercial frozen stock group had a small amount of apoptosis, and the positive control group had all cells withered (FIG. 8, A). Staining statistics shows that the survival rate of the frozen stock solution with DMEM and FBS and DMSO being 6:3:1 after recovery is 83.5%, the survival rate of the commercial frozen stock solution after recovery culture is 89.5%, the survival rate of the cells which are not frozen is about 97.0% (fig. 8, B), and the commercial frozen stock solution is superior to the frozen stock solution with DMEM and FBS and DMSO being 6:3: 1. Indicating that the organoid can maintain the original state after the resuscitation.
Example 4: drug screening test for testicular organoids
Organoids were inoculated into 96-well plates (DMEM medium) at 5/80 ul, with CdCl at different concentrations 2 (1.56. mu.M, 3.12. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, 200. mu.M) were cultured on a shaker (34 ℃ C., 5% CO) 2 Incubator), 3 days later, the state of organs and cck8 method (MCE company, cat #: HY-K0301) for survival rate.
The results are shown in FIG. 9. The result shows that the testis organoid of the invention has good dose-effect curve relation when being used for drug toxicity evaluation, and the testis organoid can be used as a drug toxicity evaluation model.
It will be appreciated by persons skilled in the art that although the invention has been described with reference to specific embodiments thereof, the invention is not limited to these specific embodiments. Based on the teachings of the present invention and the methods and technical solutions thereof, those skilled in the art can make appropriate changes or modifications without departing from the spirit of the present invention, and thus equivalent embodiments are within the scope of the present invention.
Figure IDA0003553412720000011
Figure IDA0003553412720000021
Figure IDA0003553412720000031
Figure IDA0003553412720000041
Figure IDA0003553412720000051

Claims (10)

1. A testicular organoid, comprising: mesenchymal cells, supporting cells, spermatogonial stem cells, or a combination thereof.
2. Testicular organoids according to claim 1, which are obtained by in vitro cell culture and are capable of producing testosterone, preferably the cells are of rodent or non-human primate origin, mouse or rat, most preferably the cells are adult cells, preferably a collection of cells obtained after trypsinization of testicular tissue of an animal 3 to 2 weeks, particularly preferably 7 to 10 days, large.
3. The testicular organoid of claim 1 or 2, expressing one or a combination of the following protein markers: fibronectin, α -SMA, ZO-1, WT1, Star, and PCNA.
4. The testicular organoid of any of claims 1-3, capable of expressing one or a combination of the following genes: hsd3b1, Hsd17b3, Star, Sox9, Wt1, Ar, Fshr, C-ret, and Crem.
5. The testicular organoid of any of claims 1-4, capable of secreting one or a combination of the following factors: amh, Dhh, Gdnf, inhibin B, Cxcl12 and Fgf 2.
6. A method for preparing a testicular organoid, comprising the steps of:
1) obtaining a cell of a testis of a young animal, preferably the cell is an adult cell, more preferably a cell set obtained after digestion of a testis tissue of an animal 3 days to 2 weeks, particularly preferably 7 to 10 days old;
2) carrying out hanging drop culture on the obtained testicular cells; and optionally
3) Performing suspension culture on the hanging drop culture product obtained in the step 2), thereby obtaining the testicular organoid.
7. The method of claim 6, wherein:
a) the animal is a rodent or non-human primate, mouse or rat;
b) in the step 1), taking the testis tissue of the young animal, and sequentially performing soaking, washing, membrane removal, shearing and cell digestion to obtain testis cells of the young animal, wherein preferably, the soaking is performed by using PBS (phosphate buffer solution); and/or the washing solution used in the washing is PBS containing penicillin and streptomycin; preferably, the digestion is with pancreatin;
c) in step 2), the culture medium used in the hanging-drop culture is a serum-free medium containing EGF (preferably DMEM, MEM, DMEM/F12 medium), such as basal medium containing EGF and KSR, and 3% -5% CO at 34 deg.C-37 deg.C 2 Concentration culture conditions under hanging drop culture for 5-7 days, preferably based on the volume of the culture medium, the concentration of EGF in the culture medium is 50-100ng/ml, and the concentration of KSR is 5-10 volume%; and/or
d) In step 3), the culture medium used in the suspension culture is a basal medium (preferably DMEM, MEM, DMEM/F12 medium) containing KSR, LH and FSH, and preferably contains the following components by volume: KSR, 5% to 10% by volume; LH, 1-10 ng/ml; FSH, 1-10 ng/ml; and/or preferably, the hanging drop culture product obtained in the step 2) is centrifugally enriched in cells, and is added into the culture medium, and the temperature is 34-37 ℃, and the CO content is 3-5 percent 2 Culturing at 120 ℃ and 160 rpm.
8. The testicular organoid of any of claims 1-5, which is preparable by the method of claim 6 or 7.
9. Use of a testicular organoid according to any of claims 1-5 and 8 in drug screening or determining drug toxicity.
10. The use according to claim 9, wherein the drug candidate is contacted with a testicular organoid according to any one of claims 1-5 and 8.
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