WO2023173763A1 - Testis organoid preparation, culture and cryopreservation resuscitation methods and application - Google Patents

Testis organoid preparation, culture and cryopreservation resuscitation methods and application Download PDF

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WO2023173763A1
WO2023173763A1 PCT/CN2022/129770 CN2022129770W WO2023173763A1 WO 2023173763 A1 WO2023173763 A1 WO 2023173763A1 CN 2022129770 W CN2022129770 W CN 2022129770W WO 2023173763 A1 WO2023173763 A1 WO 2023173763A1
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testicular
culture
cells
organoids
medium
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Chinese (zh)
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杨艳
黄亚东
曹振
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暨南大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
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    • C12N2513/00—3D culture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention belongs to the field of biomedicine technology, and specifically relates to a method and application for the preparation, culture, cryopreservation and recovery of testicular organoids.
  • the testis is the reproductive organ of male animals. It is located on the left and right sides of the scrotum. It is oval in shape. There is a layer of fibrous membrane on the surface of the testicle, called the tunica albuginea.
  • the tunica albuginea thickens along the trailing edge of the testicle and protrudes into the testicle to form the mediastinum. From the mediastinum It emits many connective tissue septa and divides the testicular parenchyma into many testicular lobules.
  • the testicular lobules contain coiled seminiferous tubules.
  • the epithelium of the seminiferous tubules can produce sperm.
  • Organoids are (3D) cell cultures, which are organ-specific cell collections derived from stem cells or precursor cells. They contain some key characteristics of representative organs and can simulate the tissue architecture and cell type composition of real organs. and possess specific functional characteristics while maintaining the advantages of a simplified and accessible cell culture model. Different from the two-dimensional (2D) cell culture model, organoid culture is to cultivate a variety of cell types contained in specific tissues and organs in a 3D environment, and the microenvironmental system of its culture is closer to that in the body. Therefore, organoids can serve as an in vitro platform to study cell-cell interactions, tissue development and toxicology, effectively complementing existing animal and two-dimensional (2D) cell culture models, and conducting basic research on the physiology and pathology of various organs. , precision medicine, drug screening and development, regenerative medicine, etc., it has broad application prospects.
  • the technical problem to be solved by the present invention is to provide a method and application for the preparation, culture, cryopreservation and recovery of testicular organoids, so as to solve the problems existing in the existing technology.
  • the inventor provides a method and application for the preparation, culture, cryopreservation and recovery of mouse testicular organoids.
  • the method includes the following steps: taking young mouse testicular tissue, soaking, washing, removing the membrane, shearing and Digest the cells to obtain mouse testicular cell suspension; culture the testicular cells of suckling mice through hanging drop suspension to obtain testicular organoids; collect the testicular organoids by centrifugation and culture them in suspension in a specific culture medium.
  • the invention provides a testicular organoid comprising: Leydig cells, Sertoli cells, spermatogonial stem cells, or a combination thereof.
  • the testicular organoid is obtained by in vitro cell culture and is capable of producing testosterone.
  • the cells are derived from rodents or non-human primates, mice or rats.
  • the testicular organoid expresses one of the following protein markers or any combination thereof: fibronectin, ⁇ -smooth muscle actin, ⁇ -SMA ), Tight junction protein (ZO-1), Wilms tumor 1 (WT1), steroidogenic acute regulatory protein (Steroidogenic acute regulatory protein, Star), and proliferating cell nuclear antigen (Proliferating cell nuclear antigen, PCNA).
  • the testicular organoid is capable of expressing one of the following genes/proteins or any combination thereof: 3 ⁇ -hydroxysteroid dehydrogenase1 (Hsd3b1), 17 ⁇ -hydroxysteroid Dehydrogenase 3 (17 ⁇ -hydroxysteroid dehydrogenase 3, Hsd17b3), steroidogenic acute regulatory protein (Steroidogenic acute regulatory protein, 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 (Fshr), proto-oncogene (Ret proto-oncogene, C-ret), and cyclic AMP-responsive element modulator (Crem).
  • Hsd3b1 3 ⁇ -hydroxysteroid dehydrogenase1
  • 17 ⁇ -hydroxysteroid Dehydrogenase 3 17 ⁇ -hydroxysteroid dehydrogenase 3, Hsd
  • the testicular organoid is capable of secreting one of the following factors or any combination thereof: anti-Mullerian hormone (anti-Mullerian hormone, Amh), desert hedgehog factor (Desert hedgehog, Dhh), GLIAL cell-line derived neurotrophic factor (GDNF), inhibinb, C-X-C chemokine ligand 12 (Cxcl12) and fibroblast growth factor 2 (Fibroblast growth factor 2, Fgf2).
  • Another aspect of the invention provides a method for preparing testicular organoids, comprising the following steps:
  • step 3 Perform suspension culture on the hanging drop culture product obtained in step 2), thereby obtaining testicular organoids.
  • the hanging drop culture in step 2) can already form testicular organoids, while the purpose of the suspension culture in step 3) is to provide nutrients to maintain long-term culture of testicular organoids.
  • the animal is a rodent or a non-human primate, a mouse or a rat.
  • the young mouse is preferably 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
  • the testicular tissue of young animals is taken, soaked, washed, decoated, sheared and cell digested in order to obtain testicular cells of young animals, wherein preferably, the soaking Use PBS for soaking; and/or the washing solution used in the washing is PBS containing penicillin and streptomycin (for example, PBS mixed with 100 IU/ml penicillin + 100 ⁇ g/ml streptomycin); preferably, the digestion It is digested using trypsin.
  • the soaking Use PBS for soaking and/or the washing solution used in the washing is PBS containing penicillin and streptomycin (for example, PBS mixed with 100 IU/ml penicillin + 100 ⁇ g/ml streptomycin); preferably, the digestion It is digested using trypsin.
  • the present invention uses adult cells, preferably a cell collection obtained by digesting the testicular tissue of an animal that is 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
  • the PBS used for soaking is pre-cooled with ice.
  • the hanging drop culture is carried out as follows: the testicular cells obtained after digestion are dropped on the inside of the culture dish cover (to form hemispherical droplets), and then the culture dish cover is turned upside down on the inside of the culture dish with the addition of On a petri dish such as PBS, and then cultured in an incubator, 3D testicular organoids were obtained.
  • the culture medium used in the hanging drop culture is a serum-free medium containing epidermal growth factor (EGF), such as a basal culture containing EGF and KSR (serum replacement, Knockot serum replacement) Base (preferably DMEM, MEM, DMEM/F12 medium), and culture in hanging drop at 34°C-37°C, 3%-5% CO 2 concentration for 5-7 days, the concentration of EGF in the medium is preferably 50-100ng /ml.
  • the culture medium used in the hanging drop culture is 10 volume% KSR medium containing 50ng/ml EGF, and the hanging drop culture is carried out at 34°C and 5% CO 2 for 5 days; and/or
  • the medium used for the suspension culture is a basic medium (preferably DMEM, MEM, DMEM/F12 medium) containing KSR, LH (luteinizing hormone) and FSH (follicle stimulating hormone), preferably Based on the volume of the culture medium (such as DMEM), the content of each component is: KSR, 5-10% by volume; LH, 1-10ng/ml; FSH, 1-10ng/ml. Preferably, the content of each component is: KSR, 10% by volume; LH, 1ng/ml; FSH, 1ng/ml; and/or
  • the hanging drop culture product obtained in the above step 2) is centrifuged to enrich the cells, the above medium is added, and cultured at 34°C-37°C, 3%-5% CO 2 , 120-160 rpm; preferably every 5 days
  • the culture medium needs to be changed once and the culture can be continued for a long time.
  • each of the above-mentioned EGF, LH and FSH is derived from mammals, preferably human or mouse EGF, LH and FSH.
  • the serum substitute culture medium used in the present invention has the advantage of clear composition and strong consistency.
  • the serum culture medium has large batch-to-batch differences, and there is a possibility of animal-derived protein or virus contamination. Therefore, the culture medium is preferably a serum-free medium.
  • the present invention provides a method for preparing testicular organoids, comprising the following steps:
  • a method for culturing testicular organoids including the following steps:
  • the present invention also provides testicular organoids that can be prepared by the method for preparing testicular organoids according to the present invention.
  • the present invention also provides use of the testicular organoids of the present invention in drug screening or determination of drug toxicity.
  • the candidate drug is contacted with the testicular organoids according to the invention.
  • the present invention also provides an application of testicular organoids for drug screening testing, which includes the following steps:
  • Organoids are inoculated into a 96-well plate at 5 particles/80ul, and different concentrations of CdCl 2 (1.56 ⁇ M, 3.12 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M) are added at the same time, and placed on a shaker at 120-160rpm After 3 days of culture, the organoid status was photographed and the survival rate was tested using the cck8 (cell counting kit-8) method.
  • the present invention also provides a method for cryopreservation and recovery of testicular organoids, including the following steps:
  • the innovative points of the present invention at least include:
  • Testicular organoids can be obtained through hanging drop suspension culture method, which is simple to operate and does not require special equipment.
  • testicular organoids can be subjected to drug testing and toxicology evaluation.
  • testicular organoids prepared by the present invention are highly consistent with the genetic background of the testicular tissue from which they are derived.
  • the cultured organoids can maintain specific testicular functions for a long time, and can also be cryopreserved and revived.
  • the preparation and culture process is simple and reproducible, the drug efficacy test time is short, and the culture cost is more economical than animal models.
  • the established testicular organoids can achieve high-throughput drug screening and have good application prospects.
  • Figure 1 is a flow chart for the preparation, suspension culture and formation of hanging droplets of testicular organoids of the present invention.
  • Figure 2 shows the area change curve of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention.
  • Figure 3 shows HE staining of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention.
  • Figure 4 shows the immunofluorescence staining of testicular organoids on day 15 during the testicular organoid suspension culture process of the present invention.
  • Figure 5 shows changes in gene expression of testicular organoid interstitial cells, Sertoli cells, and spermatogonial stem cells on days 5, 10, and 15 during the testicular organoid suspension culture process of the present invention.
  • Figure 6 shows the testosterone changes in testicular organoids on days 5, 10, 15 and 20 during the suspension culture process of testicular organoids of the present invention.
  • Figure 7 shows changes in gene expression of testicular organoid secretion factors on days 5, 10, 15, 20, 25 and 30 during the testicular organoid suspension culture process of the present invention.
  • Figure 8 shows the results of the TUNEL apoptosis staining experiment after the testicular organoids of the present invention were cryopreserved and resuscitated, cultured for 3 days, and frozen sectioned.
  • Figure 9 shows the measurement of cck8 after the testicular organoids were treated with different concentrations of CdCl 2 on the 15th day during the suspension culture process of the testicular organoids of the present invention.
  • Fetal bovine serum FBS (ExCell Bio).
  • Luteinizing hormone LH human source, Merk Millipore, 1ng/ml
  • FSH human source, Prospect
  • Organoid cryopreservation solution was purchased from Chuangxin International Biotechnology (Guangzhou) Co., Ltd.;
  • the resuspended testicular cells were added to DMEM medium containing 10 volume% KSR and 50ng/ml EGF (human EGF, American Pepro Tech Company, product number: AF-100-15), and the resuspended cells were diluted according to a ratio of 1x10 5 cells/ml. suspension;
  • a volley gun to take 20ul of the cell suspension and transfer it to the inside of a 10x10cm square bacterial dish cover.
  • the bacterial dish cover will appear in the shape of a red hemisphere. Place the bacterial dish cover upside down on the bacterial dish with PBS;
  • Example 2 Method for culturing testicular organoids
  • the flask was transferred to a 34°C constant-temperature 5% CO 2 culture shaker at 120-160 rpm for culture, and the medium was changed every 5 days to obtain testicular organoids.
  • Figure 2 shows the area change curve of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention.
  • the area change curve results showed that the volume of testicular organoids slowly increased as the culture time increased, indicating that the organoids could grow and maintain their shape for a long time.
  • Figure 3 shows HE staining of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention.
  • HE staining showed that the cells inside the testicular organoids formed a structure similar to testicular tissue.
  • Figure 4 shows the immunofluorescence staining of testicular organoids on day 15 during the testicular organoid suspension culture process of the present invention.
  • Immunofluorescence staining showed that supporting cells (marked proteins: Fibronectin, ZO-1, WT1), peritubular myoid cells (marked proteins: ⁇ SMA), and interstitial cells (marked proteins: ⁇ SMA) in organoids : Star) and spermatogonia (labeled protein: PCNA) are growing normally and expressing proteins required to maintain cell growth.
  • Figure 5 shows changes in gene expression of testicular organoid interstitial cells, Sertoli cells, and spermatogonial stem cells on days 5, 10, and 15 during the testicular organoid suspension culture process of the present invention.
  • the PCR results showed that the gene expression level of the obtained testicular organoids was close to the gene expression level of adult mouse testicular tissue, indicating that the prepared organoids were similar to testises in terms of gene expression levels.
  • Figure 6 shows the testosterone changes in testicular organoids on days 5, 10, 15, and 20 during the testicular organoid suspension culture process of the present invention (testosterone radioimmunoassay kit, Northern Biotech). The results showed that within 20 days of culture, the testicular organoid culture system could effectively maintain testosterone secretion, with the best condition on the 15th day, indicating that the organoids could secrete testosterone normally and have similar functions to testicles.
  • Figure 7 shows changes in gene expression of testicular organoid secretion factors on days 5, 10, 15, 20, 25 and 30 during the testicular organoid suspension culture process of the present invention.
  • the PCR results showed that the secreted factors related to testicular organoids were close to those of adult mouse testis tissue, indicating that the prepared organoids were similar to testis in the expression levels of related secreted factors.
  • Cryopreservation After 5 days of organoid suspension culture, collect the suspension from the Erlenmeyer flask and centrifuge it at 1500 rpm for 5 minutes in a 15 ml centrifuge tube;
  • organoid cryopreservation solution purchased from Chuangxin International Biotechnology (Guangzhou) Co., Ltd.
  • organoid cryopreservation solution purchased from Chuangxin International Biotechnology (Guangzhou) Co., Ltd.
  • transfer the cryopreservation tube to a cryopreservation box at -80°C overnight and transfer it the next day. to liquid nitrogen;
  • Thaw Take the organoids out of liquid nitrogen and quickly thaw them in a 37°C water bath. Move them into a prepared 15ml centrifuge tube containing 10% FBS. Centrifuge at 1500rpm for 5 minutes to obtain the recovered testicular organoids. Culture them according to the above culture method.
  • Figure 8 shows the testicular organoids of the present invention that were cryopreserved and resuscitated, cultured for 3 days, and then frozen sectioned for TUNEL apoptosis staining experiments.
  • the results showed that there was basically no apoptosis in the non-cryopreserved group (green indicates cell apoptosis), a small number of cells in the commercial cryopreservation solution group were apoptotic, and all cells in the positive control group were apoptotic ( Figure 8, A).
  • Organoids were inoculated into a 96-well plate (the culture medium was DMEM) at 5 particles/80ul, and different concentrations of CdCl 2 (1.56 ⁇ M, 3.12 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M) were added and placed on a shaker. Culture (34°C, 5% CO 2 incubator), and after 3 days, the organoid status was photographed and the survival rate was tested using the cck8 method (MCE Company, Cat. No.: HY-K0301).
  • testicular organoids of the present invention have a good dose-effect curve relationship when used for drug toxicity evaluation, indicating that the testicular organoids can be used as a drug toxicology evaluation model.

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Abstract

Provided are a cultured testis organoid, and preparation, culture and cryopreservation resuscitation methods therefor and an application thereof. The testis organoid can maintain specific functions of testes and can realize high-throughput drug screening.

Description

一种类睾丸器官的制备、培养、冻存复苏方法及应用Methods and applications for the preparation, culture, cryopreservation and recovery of testicle-like organs 技术领域Technical field
本发明属于生物医药技术领域，具体涉及一种睾丸类器官的制备、培养、冻存复苏方法及应用。The invention belongs to the field of biomedicine technology, and specifically relates to a method and application for the preparation, culture, cryopreservation and recovery of testicular organoids.
背景技术Background technique
睾丸是雄性动物的生殖器官，分别位于阴囊左右两侧，呈卵圆形，睾丸表面有一层纤维膜，称为白膜，沿睾丸后缘白膜增厚，凸入睾丸内形成纵隔，从纵隔发出许多结缔组织小隔，将睾丸实质分成许多睾丸小叶，睾丸小叶内含有盘曲的精曲小管，精曲小管的上皮能产生精子，小管之间的结缔组织内有间质细胞，间质细胞产生雄激素，与雄性第二性征、生理功能等密切相关。The testis is the reproductive organ of male animals. It is located on the left and right sides of the scrotum. It is oval in shape. There is a layer of fibrous membrane on the surface of the testicle, called the tunica albuginea. The tunica albuginea thickens along the trailing edge of the testicle and protrudes into the testicle to form the mediastinum. From the mediastinum It emits many connective tissue septa and divides the testicular parenchyma into many testicular lobules. The testicular lobules contain coiled seminiferous tubules. The epithelium of the seminiferous tubules can produce sperm. There are interstitial cells in the connective tissue between the tubules. The interstitial cells produce sperm. Androgens are closely related to male secondary sexual characteristics and physiological functions.
类器官(Organoids)属于(3D)细胞培养物，是衍生于干细胞或前体细胞的器官特异性细胞集合，包含了其代表器官的一些关键特征，能够模拟真实器官的组织构架、细胞类型组成，并具备特定的功能学特征，同时保持简化和易获得的细胞培养模型的优势。与二维(2D)细胞培养模型不同，类器官培养是在3D环境中培养出特定组织器官包含的多种细胞类群，其培养的微环境体系更接近于体内。因此，类器官可以作为体外平台来研究细胞与细胞之间的相互作用，组织发育和毒理学，有效的补充现有动物和二维(2D)细胞培养模型，在各种器官生理病理的基础研究、精准医疗、药物筛选和开发、再生医学等方面，有广泛的应用前景。Organoids are (3D) cell cultures, which are organ-specific cell collections derived from stem cells or precursor cells. They contain some key characteristics of representative organs and can simulate the tissue architecture and cell type composition of real organs. and possess specific functional characteristics while maintaining the advantages of a simplified and accessible cell culture model. Different from the two-dimensional (2D) cell culture model, organoid culture is to cultivate a variety of cell types contained in specific tissues and organs in a 3D environment, and the microenvironmental system of its culture is closer to that in the body. Therefore, organoids can serve as an in vitro platform to study cell-cell interactions, tissue development and toxicology, effectively complementing existing animal and two-dimensional (2D) cell culture models, and conducting basic research on the physiology and pathology of various organs. , precision medicine, drug screening and development, regenerative medicine, etc., it has broad application prospects.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种睾丸类器官的制备、培养、冻存复苏方法及应用，以解决现有技术存在的问题。The technical problem to be solved by the present invention is to provide a method and application for the preparation, culture, cryopreservation and recovery of testicular organoids, so as to solve the problems existing in the existing technology.
具体地，本发明人提供一种小鼠睾丸类器官制备、培养、冻存复苏方法及应用，该方法包括以下步骤：取幼年小鼠睾丸组织，依次进行浸泡、洗涤、去被膜、剪切和消化细胞，获得小鼠睾丸细胞悬液；将乳鼠睾丸细胞通过悬滴悬浮培养，获得睾丸类器官；将睾丸类器官离心收集，用特定的培养基悬浮培养。Specifically, the inventor provides a method and application for the preparation, culture, cryopreservation and recovery of mouse testicular organoids. The method includes the following steps: taking young mouse testicular tissue, soaking, washing, removing the membrane, shearing and Digest the cells to obtain mouse testicular cell suspension; culture the testicular cells of suckling mice through hanging drop suspension to obtain testicular organoids; collect the testicular organoids by centrifugation and culture them in suspension in a specific culture medium.
因此，在一个方面，本发明提供了一种睾丸类器官，其包含：间质细胞、支持细胞、精原干细胞或它们的组合。Accordingly, in one aspect, the invention provides a testicular organoid comprising: Leydig cells, Sertoli cells, spermatogonial stem cells, or a combination thereof.
根据本发明的一个优选实施方案，所述睾丸类器官为通过体外细胞培养获得的，并且能够生成睾酮，优选所述细胞是来源于啮齿动物或非人灵长类动物，小鼠或大鼠。According to a preferred embodiment of the present invention, the testicular organoid is obtained by in vitro cell culture and is capable of producing testosterone. Preferably, the cells are derived from rodents or non-human primates, mice or rats.
根据本发明的一个优选实施方案，所述睾丸类器官表达以下蛋白标志物之一或它们的任意组合：纤连蛋白(Fibronectin)，α-平滑肌肌动蛋白(Alpha-smooth muscle actin,α-SMA)，闭锁连接蛋白1(Tight junction protein，ZO-1)，肾母细胞瘤蛋白1(Wilms tumor 1，WT1)，类固醇激素合成急性调节蛋白(Steroidogenic acute regulatory，Star)，和增殖细胞核抗原(Proliferating cell nuclear antigen，PCNA)。According to a preferred embodiment of the present invention, the testicular organoid expresses one of the following protein markers or any combination thereof: fibronectin, α-smooth muscle actin, α-SMA ), Tight junction protein (ZO-1), Wilms tumor 1 (WT1), steroidogenic acute regulatory protein (Steroidogenic acute regulatory protein, Star), and proliferating cell nuclear antigen (Proliferating cell nuclear antigen, PCNA).
根据本发明的一个优选实施方案，所述睾丸类器官能够表达以下基因/蛋白之一 或它们的任意组合：3β-羟类固醇脱氢酶(3-Beta-hydroxysteroid dehydrogenase1，Hsd3b1)，17β-羟类固醇脱氢酶3(17β-hydroxysteroid dehydrogenase 3，Hsd17b3)，类固醇激素合成急性调节蛋白(Steroidogenic acute regulatory，Star)，性别决定区Y框蛋白9(Sry related HMG box-9，Sox9)，肾母细胞瘤蛋白1(Wilms tumor 1，WT1)，雄激素受体(Androgen Receptor，Ar)，促卵泡生成素受体(follicle stimulating hormone receptor，Fshr)，原癌基因(Ret proto-oncogene，C-ret)，和环腺苷酸反应成分结合蛋白(cyclic AMP-responsive element modulator，Crem)。According to a preferred embodiment of the present invention, the testicular organoid is capable of expressing one of the following genes/proteins or any combination thereof: 3β-hydroxysteroid dehydrogenase1 (Hsd3b1), 17β-hydroxysteroid Dehydrogenase 3 (17β-hydroxysteroid dehydrogenase 3, Hsd17b3), steroidogenic acute regulatory protein (Steroidogenic acute regulatory protein, 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 (Fshr), proto-oncogene (Ret proto-oncogene, C-ret), and cyclic AMP-responsive element modulator (Crem).
根据本发明的一个优选实施方案，所述睾丸类器官能够分泌以下因子之一或它们的任意组合：抗缪勒管激素(anti-Mullerian hormone，Amh)，沙漠刺猬因子(Desert hedgehog,Dhh)，胶质细胞系衍生的神经营养因子(GLIAL cell-line derived neurotrophic factor，GDNF)，抑制素B(Inhibinb)，C-X-C趋化因子配体12(C-X-C chemokine ligand 12，Cxcl12)和成纤维细胞生长因子2(Fibroblast growth factor 2，Fgf2)。According to a preferred embodiment of the present invention, the testicular organoid is capable of secreting one of the following factors or any combination thereof: anti-Mullerian hormone (anti-Mullerian hormone, Amh), desert hedgehog factor (Desert hedgehog, Dhh), GLIAL cell-line derived neurotrophic factor (GDNF), inhibinb, C-X-C chemokine ligand 12 (Cxcl12) and fibroblast growth factor 2 (Fibroblast growth factor 2, Fgf2).
本发明的另一方面提供了一种用于制备睾丸类器官的方法，其包括以下步骤：Another aspect of the invention provides a method for preparing testicular organoids, comprising the following steps:
1)获得幼年动物睾丸细胞；1) Obtain testicular cells from young animals;
2)将获得的睾丸细胞进行悬滴培养；和任选地2) subjecting the obtained testicular cells to hanging drop culture; and optionally
3)将步骤2)中获得的悬滴培养产物进行悬浮培养，由此获得睾丸类器官。3) Perform suspension culture on the hanging drop culture product obtained in step 2), thereby obtaining testicular organoids.
应当指出，在本发明的方法中，步骤2)的悬滴培养已经能够形成睾丸类器官，而步骤3)的悬浮培养的目的是提供营养从而维持睾丸类器官的长时间培养。It should be noted that in the method of the present invention, the hanging drop culture in step 2) can already form testicular organoids, while the purpose of the suspension culture in step 3) is to provide nutrients to maintain long-term culture of testicular organoids.
根据本发明的一个优选实施方案，所述动物为啮齿动物或非人灵长类动物，小鼠或大鼠。According to a preferred embodiment of the present invention, the animal is a rodent or a non-human primate, a mouse or a rat.
当所述动物为小鼠时，幼年小鼠优选为3天至2周大、特别优选7～10天大的小鼠。When the animal is a mouse, the young mouse is preferably 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
根据本发明的一个优选实施方案，在上述步骤1)中，取幼年动物睾丸组织，依次进行浸泡、洗涤、去被膜、剪切和细胞消化，获得幼年动物睾丸细胞，其中优选地，所述浸泡使用PBS浸泡；和/或所述洗涤中使用的洗涤液为含有青霉素和链霉素的PBS(例如，PBS和青霉素100IU/ml+链霉素100μg/ml混合而成)；优选地，所述消化是使用胰酶消化。According to a preferred embodiment of the present invention, in the above step 1), the testicular tissue of young animals is taken, soaked, washed, decoated, sheared and cell digested in order to obtain testicular cells of young animals, wherein preferably, the soaking Use PBS for soaking; and/or the washing solution used in the washing is PBS containing penicillin and streptomycin (for example, PBS mixed with 100 IU/ml penicillin + 100 μg/ml streptomycin); preferably, the digestion It is digested using trypsin.
根据本发明的一个优选实施方案，本发明使用的是成体细胞，优选是3天至2周大、特别优选7～10天大的动物的睾丸组织消化后得到的细胞集合。According to a preferred embodiment of the present invention, the present invention uses adult cells, preferably a cell collection obtained by digesting the testicular tissue of an animal that is 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
根据本发明的一个优选实施方案，浸泡使用的PBS是冰预冷过的。According to a preferred embodiment of the present invention, the PBS used for soaking is pre-cooled with ice.
根据本发明的一个优选实施方案，所述悬滴培养是如下进行：将消化后获得的睾丸细胞滴在培养皿盖内侧(形成半球状液滴)，再将该培养皿盖倒扣在加有诸如PBS的培养皿上，之后在培养箱中培养，由此获得3D睾丸类器官。According to a preferred embodiment of the present invention, the hanging drop culture is carried out as follows: the testicular cells obtained after digestion are dropped on the inside of the culture dish cover (to form hemispherical droplets), and then the culture dish cover is turned upside down on the inside of the culture dish with the addition of On a petri dish such as PBS, and then cultured in an incubator, 3D testicular organoids were obtained.
优选地，在上述步骤2)中，所述悬滴培养使用的培养基是含有表皮生长因子(EGF)的无血清培养基，诸如含有EGF和KSR(血清替代物，Knockot serum replacement)的基础培养基(优选DMEM，MEM、DMEM/F12培养基)，并且于34℃-37℃、3％-5％CO 2浓度下悬滴培养5-7天，优选培养基中EGF的浓度为50-100ng/ml。优选地，所述悬滴培养使用的培养基是含有50ng/ml EGF的10体积％KSR培养基，并且于34℃、5％CO 2浓度下悬滴培养5天；和/或 Preferably, in the above step 2), the culture medium used in the hanging drop culture is a serum-free medium containing epidermal growth factor (EGF), such as a basal culture containing EGF and KSR (serum replacement, Knockot serum replacement) Base (preferably DMEM, MEM, DMEM/F12 medium), and culture in hanging drop at 34℃-37℃, 3%-5% CO 2 concentration for 5-7 days, the concentration of EGF in the medium is preferably 50-100ng /ml. Preferably, the culture medium used in the hanging drop culture is 10 volume% KSR medium containing 50ng/ml EGF, and the hanging drop culture is carried out at 34°C and 5% CO 2 for 5 days; and/or
在上述步骤3)中，所述悬浮培养使用的培养基是含有KSR、LH(黄体生成素)和FSH (卵泡刺激素)的基础培养基(优选DMEM，MEM、DMEM/F12培养基)，优选按培养基(如DMEM)的体积计，各成分的含量为：KSR，5-10体积％；LH，1-10ng/ml；FSH，1-10ng/ml。优选地，各成分的含量为：KSR，10体积％；LH，1ng/ml；FSH，1ng/ml；和/或In the above step 3), the medium used for the suspension culture is a basic medium (preferably DMEM, MEM, DMEM/F12 medium) containing KSR, LH (luteinizing hormone) and FSH (follicle stimulating hormone), preferably Based on the volume of the culture medium (such as DMEM), the content of each component is: KSR, 5-10% by volume; LH, 1-10ng/ml; FSH, 1-10ng/ml. Preferably, the content of each component is: KSR, 10% by volume; LH, 1ng/ml; FSH, 1ng/ml; and/or
优选地，将上述步骤2)中获得的悬滴培养产物离心富集细胞，加入上述培养基，于34℃-37℃、3％-5％CO 2、120-160rpm下培养；优选每5天换培养基一次，并且可长期持续培养。 Preferably, the hanging drop culture product obtained in the above step 2) is centrifuged to enrich the cells, the above medium is added, and cultured at 34°C-37°C, 3%-5% CO 2 , 120-160 rpm; preferably every 5 days The culture medium needs to be changed once and the culture can be continued for a long time.
根据本发明的一个特别优选的实施方案，上述EGF、LH和FSH各自是来源于哺乳动物，优选是人或小鼠EGF、LH和FSH。According to a particularly preferred embodiment of the present invention, each of the above-mentioned EGF, LH and FSH is derived from mammals, preferably human or mouse EGF, LH and FSH.
本发明使用的血清替代物培养基(例如KSR)与血清培养基相比，其优势在于成分明确，一致性强。而血清培养基的批间差异大，有动物源性蛋白或病毒污染的可能。因此培养基优选无血清培养基。Compared with serum culture medium, the serum substitute culture medium (eg KSR) used in the present invention has the advantage of clear composition and strong consistency. The serum culture medium has large batch-to-batch differences, and there is a possibility of animal-derived protein or virus contamination. Therefore, the culture medium is preferably a serum-free medium.
根据本发明的一个特别优选的实施方案，本发明提供了一种睾丸类器官的制备方法，包括以下步骤：According to a particularly preferred embodiment of the present invention, the present invention provides a method for preparing testicular organoids, comprising the following steps:
(1)取雄性昆明乳鼠睾丸组织，置于预冷且加有双抗(青霉素和链霉素)的PBS中浸泡；(1) Take the testicular tissue of male Kunming suckling rats and soak it in pre-cooled PBS with double antibodies (penicillin and streptomycin);
(2)取6个无菌的9x9cm圆形细菌皿，依次加入含有双抗的PBS，编号1-6，将睾丸组织转移至编号1细菌皿中润洗1-2min，依次转移至2、3号细菌皿润洗，用无菌的手术镊轻轻戳破睾丸被膜，取出睾丸组织转移至第4号细菌皿洗涤1-2min，依次转移至第5、6号细菌皿中洗涤；(2) Take 6 sterile 9x9cm round bacterial dishes, add PBS containing double antibodies in sequence, numbered 1-6, transfer the testicular tissue to the bacterial dish numbered 1, rinse for 1-2 minutes, and transfer to 2 and 3 in sequence. Rinse the No. 4 bacterial dish, use sterile surgical forceps to gently puncture the testicular capsule, remove the testicular tissue and transfer it to the No. 4 bacterial dish for washing for 1-2 minutes, then transfer it to the No. 5 and 6 bacterial dishes for washing;
(3)将组织从6号细菌皿中取出，转移至西林瓶中，剪碎睾丸组织，持续5min；(3) Remove the tissue from the No. 6 bacterial dish, transfer it to a vial, and cut the testicular tissue into pieces for 5 minutes;
(4)加入胰酶，置于37℃恒温培养箱中消化15min，期间摇晃2-3次；(4) Add trypsin and place in a 37°C constant temperature incubator for digestion for 15 minutes, shaking 2-3 times during the period;
(5)消化后加入10％FBS中和胰酶，用玻璃吸管吹打悬液5min；(5) After digestion, add 10% FBS to neutralize the trypsin, and pipet the suspension with a glass pipette for 5 minutes;
(6)转移至15ml离心管中1500rpm离心5min；(6) Transfer to a 15ml centrifuge tube and centrifuge at 1500rpm for 5 minutes;
(7)弃去上清，用含有10体积％KSR的DMEM重悬沉淀，静置5min左右，转移上清，弃去底部沉淀，从上清中取出90ul悬液与10ul台盼蓝混匀计数；(7) Discard the supernatant, resuspend the pellet in DMEM containing 10 volume% KSR, let it stand for about 5 minutes, transfer the supernatant, discard the bottom pellet, take out 90ul of the suspension from the supernatant and mix with 10ul of trypan blue for counting. ;
(8)重悬的睾丸细胞加入到含有10体积％KSR+50ng/ml EGF的DMEM中，按照1x10 5个细胞/ml比例稀释细胞； (8) Add the resuspended testicular cells to DMEM containing 10 volume% KSR + 50ng/ml EGF, and dilute the cells at a ratio of 1x10 5 cells/ml;
(9)取20μl的细胞稀释液滴在10x10cm的方形细菌皿盖内测，将细菌皿盖倒扣在加有PBS的细菌皿上；(9) Take 20 μl of cell dilution and drop it on the inside of a 10x10cm square bacterial dish cover, and place the bacterial dish cover upside down on the bacterial dish with PBS;
(10)将细菌皿转移至34℃恒温培养箱中静置悬滴培养5天，得到初始细胞数量为约2000/粒的3D睾丸类器官。(10) Transfer the bacterial dish to a 34°C constant-temperature incubator and place it in static hanging drop culture for 5 days to obtain a 3D testicular organoid with an initial cell number of approximately 2000/cell.
还提供一种睾丸类器官的培养方法，包括以下步骤：A method for culturing testicular organoids is also provided, including the following steps:
(1)取根据上述制备的原代睾丸类器官，转移至15ml离心管中富集，1500rpm离心5min；(1) Take the primary testicular organoids prepared as above, transfer to a 15ml centrifuge tube for enrichment, and centrifuge at 1500 rpm for 5 minutes;
(2)弃去上清，用含有10体积％KSR+1ng/ml LH+1ng/ml FSH的DMEM培养基重悬睾丸类器官；(2) Discard the supernatant and resuspend the testicular organoids in DMEM medium containing 10 volume% KSR+1ng/ml LH+1ng/ml FSH;
(3)转移至50ml三角瓶中，加入20-30ml含有10体积％KSR+1ng/ml LH+1ng/ml FSH的DMEM培养基中悬浮培养；(3) Transfer to a 50ml Erlenmeyer flask, add 20-30ml of DMEM medium containing 10 volume% KSR+1ng/ml LH+1ng/ml FSH for suspension culture;
(4)将三角瓶转移至34℃恒温培养摇床上120-160rpm培养，每5天换液处理。(4) Transfer the Erlenmeyer flask to a 34°C constant-temperature culture shaker for culture at 120-160 rpm, and change the medium every 5 days.
根据本发明的一个优选实施方案，本发明还提供可通过根据本发明所述的制备睾丸类器官的方法制备的睾丸类器官。According to a preferred embodiment of the present invention, the present invention also provides testicular organoids that can be prepared by the method for preparing testicular organoids according to the present invention.
在另一方面，本发明还提供本发明所述的睾丸类器官在药物筛选或测定药物毒性中的应用。In another aspect, the present invention also provides use of the testicular organoids of the present invention in drug screening or determination of drug toxicity.
根据本发明的一个优选实施方案，将候选药物与根据本发明的睾丸类器官接触。According to a preferred embodiment of the invention, the candidate drug is contacted with the testicular organoids according to the invention.
根据本发明的一个优选实施方案，本发明还提供一种睾丸类器官的应用，其用于药物筛选测试，包括以下步骤：According to a preferred embodiment of the present invention, the present invention also provides an application of testicular organoids for drug screening testing, which includes the following steps:
(1)类器官以5粒/80ul接种96孔板，同时加不同浓度CdCl 2(1.56μM、3.12μM、6.25μM、12.5μM、25μM、50μM、100μM、200μM)，放在摇床上120-160rpm培养，3天后拍摄类器官状态及cck8(cell counting kit-8)方法测试存活率。 (1) Organoids are inoculated into a 96-well plate at 5 particles/80ul, and different concentrations of CdCl 2 (1.56μM, 3.12μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM) are added at the same time, and placed on a shaker at 120-160rpm After 3 days of culture, the organoid status was photographed and the survival rate was tested using the cck8 (cell counting kit-8) method.
在另一方面，本发明还提供一种睾丸类器官的冻存和复苏方法，包括以下步骤：On the other hand, the present invention also provides a method for cryopreservation and recovery of testicular organoids, including the following steps:
(1)冻存：类器官悬浮培养5天后，从三角瓶中收集于15ml离心管1500rpm离心5min；(1) Cryopreservation: After organoid suspension culture for 5 days, collect from the Erlenmeyer flask into a 15 ml centrifuge tube and centrifuge at 1500 rpm for 5 minutes;
(2)弃去上清，加入类器官冻存液转移至冻存管，将冻存管转移至冻存盒中-80℃过夜，第二天转移至液氮；(2) Discard the supernatant, add organoid cryopreservation solution and transfer it to a cryopreservation tube. Transfer the cryopreservation tube to a freezing box at -80°C overnight, and transfer to liquid nitrogen the next day;
(3)解冻：冻存类器官从液氮中取出后迅速37℃水浴解冻，移入事先备好的含10％FBS的15ml离心管中，1500rpm离心5min，得到睾丸类器官，按上述培养方法进行培养。(3) Thawing: Take out the frozen organoids from liquid nitrogen and quickly thaw them in a 37°C water bath. Move them into a prepared 15ml centrifuge tube containing 10% FBS. Centrifuge at 1500rpm for 5 minutes to obtain testicular organoids. Follow the above culture method. nourish.
本发明的创新点至少包括：The innovative points of the present invention at least include:
(1)通过悬滴悬浮培养方法即可获得睾丸类器官，操作简单，无需特殊设备。(1) Testicular organoids can be obtained through hanging drop suspension culture method, which is simple to operate and does not require special equipment.
(2)可以较长时间保存睾丸中支持细胞及间质细胞特定的功能，在体外模拟睾丸的功能。(2) It 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)该睾丸类器官可以进行药物测试和毒理学评价。(3) The testicular organoids can be subjected to drug testing and toxicology evaluation.
有益效果beneficial effects
本发明制备出的睾丸类器官与其来源睾丸组织遗传背景高度一致，同时培养的类器官可以较长时间保持睾丸特定功能，还可以冻存和复苏。制备和培养过程操作简便，可重复性强，药效测试时间较短，培养成本比动物模型更加经济，建立的睾丸类器官可以实现高通量药物筛选，应用前景良好。The testicular organoids prepared by the present invention are highly consistent with the genetic background of the testicular tissue from which they are derived. At the same time, the cultured organoids can maintain specific testicular functions for a long time, and can also be cryopreserved and revived. The preparation and culture process is simple and reproducible, the drug efficacy test time is short, and the culture cost is more economical than animal models. The established testicular organoids can achieve high-throughput drug screening and have good application prospects.
附图说明Description of the drawings
图1为本发明睾丸类器官悬滴制备、悬浮培养和形成的流程图。Figure 1 is a flow chart for the preparation, suspension culture and formation of hanging droplets of testicular organoids of the present invention.
图2为本发明睾丸类器官悬浮培养过程中，第5、10、15、20、25、30天睾丸类器官的面积变化曲线。Figure 2 shows the area change curve of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention.
图3为本发明睾丸类器官悬浮培养过程中，第5、10、15、20、25、30天睾丸类器官的HE染色。Figure 3 shows HE staining of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention.
图4为本发明睾丸类器官悬浮培养过程中，第15天睾丸类器官的免疫荧光染色。Figure 4 shows the immunofluorescence staining of testicular organoids on day 15 during the testicular organoid suspension culture process of the present invention.
图5为本发明睾丸类器官悬浮培养过程中，第5、10、15天睾丸类器官间质细胞、支持细胞、精原干细胞基因表达量变化。Figure 5 shows changes in gene expression of testicular organoid interstitial cells, Sertoli cells, and spermatogonial stem cells on days 5, 10, and 15 during the testicular organoid suspension culture process of the present invention.
图6为本发明睾丸类器官悬浮培养过程中，第5、10、15、20天睾丸类器官睾酮变化。Figure 6 shows the testosterone changes in testicular organoids on days 5, 10, 15 and 20 during the suspension culture process of testicular organoids of the present invention.
图7为本发明睾丸类器官悬浮培养过程中，第5、10、15、20、25、30天睾丸类器官分泌因子基因表达量变化。Figure 7 shows changes in gene expression of testicular organoid secretion factors on days 5, 10, 15, 20, 25 and 30 during the testicular organoid suspension culture process of the present invention.
图8显示了本发明睾丸类器官进行冻存和复苏处理后培养3天，冰冻切片后进行TUNEL凋亡染色实验的结果。Figure 8 shows the results of the TUNEL apoptosis staining experiment after the testicular organoids of the present invention were cryopreserved and resuscitated, cultured for 3 days, and frozen sectioned.
图9为本发明睾丸类器官悬浮培养过程中，第15天睾丸类器官经不同浓度CdCl 2处理后，测cck8。 Figure 9 shows the measurement of cck8 after the testicular organoids were treated with different concentrations of CdCl 2 on the 15th day during the suspension culture process of the testicular organoids of the present invention.
具体实施方式Detailed ways
除非另外指出，本文中所用术语具有本领域技术人员理解的一般技术含义。Unless otherwise indicated, the terms used herein have the ordinary technical meanings understood by those skilled in the art.
实施例Example
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
试剂Reagents
青霉素(MDBio),100IU/ml；Penicillin (MDBio), 100IU/ml;
链霉素(MDBio),100ug/ml；Streptomycin (MDBio), 100ug/ml;
胰酶(Gibco)；Trypsin (Gibco);
胎牛血清FBS(ExCell Bio)；Fetal bovine serum FBS (ExCell Bio);
DMEM(Gibco)；DMEM (Gibco);
血清替代物KSR(Life),10体积％KSR；Serum substitute KSR (Life), 10% KSR by volume;
台盼蓝(Sciencell)；Trypan blue (Sciencell);
黄体生成素LH(人源，Merk Millipore),1ng/ml；Luteinizing hormone LH (human source, Merk Millipore), 1ng/ml;
卵泡刺激素FSH(人源，Prospect),1ng/ml；Follicle-stimulating hormone FSH (human source, Prospect), 1ng/ml;
类器官冻存液购自创芯国际生物科技(广州)有限公司；Organoid cryopreservation solution was purchased from Chuangxin International Biotechnology (Guangzhou) Co., Ltd.;
氯化镉(Alorich),1.56-200μMCadmium chloride (Alorich), 1.56-200μM
实施例1：睾丸类器官的制备方法Example 1: Preparation method of testicular organoids
取7-10天的雄性昆明乳鼠(购自广东省实验动物中心)睾丸组织，放置在冰盒上且加含有双抗(青霉素(MDBio),100IU/ml，和链霉素(MDBio),100ug/ml)的PBS中浸泡；Take the testicular tissue of 7-10-day-old male Kunming suckling mice (purchased from Guangdong Experimental Animal Center), place it on an ice box, and add double antibodies (penicillin (MDBio), 100IU/ml, and streptomycin (MDBio)). 100ug/ml) in PBS;
取6个无菌的9x9cm圆形细菌皿，依次加入上述含有双抗的PBS，编号依次为1-6号，将睾丸组织转移至编号1细菌皿中润洗1-2min，再依次转移至2、3号细菌皿润洗，用无菌的手术镊轻轻戳破睾丸被膜，取出睾丸组织转移至第4号细菌皿洗涤1-2min，依次转移至第5、6号细菌皿中洗涤；Take 6 sterile 9x9cm round bacterial dishes, add the above PBS containing double antibodies in sequence, numbered 1-6, transfer the testicular tissue to the bacterial dish numbered 1, rinse for 1-2 minutes, and then transfer to 2 in sequence. , Rinse the No. 3 bacterial dish, use sterile surgical forceps to gently puncture the testicular capsule, remove the testicular tissue and transfer it to the No. 4 bacterial dish for washing for 1-2 minutes, and then transfer it to the No. 5 and 6 bacterial dishes for washing;
用镊子将睾丸组织从第6号细菌皿中取出，转移至西林瓶中，用手术弯剪剪碎睾丸组织，持续5min左右至组织呈匀浆状；Use tweezers to remove the testicular tissue from the No. 6 bacterial dish, transfer it to a vial, and use surgical curved scissors to cut the testicular tissue into pieces. Continue for about 5 minutes until the tissue becomes homogenized;
在西林瓶中加入10mL胰酶(Gibco)，放置于37℃恒温培养箱中消化15min，期间间隔2-3次取出，观看消化状态并适当摇晃；Add 10 mL of trypsin (Gibco) to the vial, place it in a 37°C constant temperature incubator for digestion for 15 minutes, take it out 2-3 times during this period, observe the digestion status and shake appropriately;
消化完毕后加入双倍体积的10％FBS中和胰酶，用玻璃吸管吹打悬液5min，直至无絮状物出现；After digestion, add double the volume of 10% FBS to neutralize the trypsin, and blow the suspension with a glass pipette for 5 minutes until no floc appears;
转移至15ml离心管中1500rpm离心5min；Transfer to a 15ml centrifuge tube and centrifuge at 1500rpm for 5min;
弃去上清，用含有10体积％KSR(Life)的DMEM培养基重悬沉淀，取出90ul悬液与10ul台盼蓝(Sciencell)混匀，在3min内计数；Discard the supernatant, resuspend the pellet in DMEM medium containing 10 volume% KSR (Life), take out 90ul of the suspension and mix it with 10ul of trypan blue (Sciencell), and count within 3 minutes;
重悬的睾丸细胞加入到含有10体积％KSR和50ng/ml EGF(人EGF，美国Pepro Tech公司，货号：AF-100-15)的DMEM培养基中，按照1x10 5个细胞/ml比例稀释重悬液体； The resuspended testicular cells were added to DMEM medium containing 10 volume% KSR and 50ng/ml EGF (human EGF, American Pepro Tech Company, product number: AF-100-15), and the resuspended cells were diluted according to a ratio of 1x10 5 cells/ml. suspension;
用排枪取20ul的细胞悬液转移至10x10cm的方形细菌皿盖内侧，细菌皿盖上呈红色半球状，将细菌皿盖倒扣在加有PBS的细菌皿上；Use a volley gun to take 20ul of the cell suspension and transfer it to the inside of a 10x10cm square bacterial dish cover. The bacterial dish cover will appear in the shape of a red hemisphere. Place the bacterial dish cover upside down on the bacterial dish with PBS;
将细菌皿转移至34℃、5％CO 2恒温培养箱中静置悬滴培养5天，得到初始细胞数量约为2000/粒的3D睾丸类器官。 Transfer the bacterial dish to a 34°C, 5% CO2 constant-temperature incubator for static hanging drop culture for 5 days to obtain a 3D testicular organoid with an initial cell number of approximately 2000/cell.
实施例2：睾丸类器官的培养方法Example 2: Method for culturing testicular organoids
取根据上述实施例1制备的原代睾丸类器官，转移至15ml离心管中，1500rpm离心5min；Take the primary testicular organoids prepared according to the above Example 1, transfer them to a 15 ml centrifuge tube, and centrifuge at 1500 rpm for 5 minutes;
弃去上清，用含有10体积％KSR+1ng/ml LH+1ng/ml FSH的DMEM培养基重悬睾丸类器官；Discard the supernatant and resuspend the testicular organoids in DMEM medium containing 10 volume% KSR+1ng/ml LH+1ng/ml FSH;
转移至50ml三角瓶中，根据类器官数量加入20-30ml含有10体积％KSR+1ng/ml LH+1ng/ml FSH的DMEM培养基中悬浮培养；Transfer to a 50ml Erlenmeyer flask, add 20-30ml of DMEM medium containing 10 volume% KSR+1ng/ml LH+1ng/ml FSH according to the number of organoids, and culture in suspension;
将三角瓶转移至34℃恒温5％CO 2培养摇床120-160rpm培养，每5天换液处理，由此得到睾丸类器官。 The flask was transferred to a 34°C constant-temperature 5% CO 2 culture shaker at 120-160 rpm for culture, and the medium was changed every 5 days to obtain testicular organoids.
图2为本发明睾丸类器官悬浮培养过程中，第5、10、15、20、25、30天睾丸类器官的面积变化曲线。面积变化曲线结果显示睾丸类器官随着培养时间的延长，类器官的体积在缓慢增加，表明类器官可以长时间生长并维持形态。Figure 2 shows the area change curve of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention. The area change curve results showed that the volume of testicular organoids slowly increased as the culture time increased, indicating that the organoids could grow and maintain their shape for a long time.
图3为本发明睾丸类器官悬浮培养过程中，第5、10、15、20、25、30天睾丸类器官的HE染色。HE染色表明睾丸类器官内部细胞形成一个类似睾丸组织的结构。Figure 3 shows HE staining of testicular organoids on days 5, 10, 15, 20, 25 and 30 during the suspension culture process of testicular organoids of the present invention. HE staining showed that the cells inside the testicular organoids formed a structure similar to testicular tissue.
图4为本发明睾丸类器官悬浮培养过程中，第15天睾丸类器官的免疫荧光染色。免疫荧光染色显示，类器官中的支持细胞(标记蛋白为：纤连蛋白(Fibronectin)、ZO-1、WT1)、管周肌样细胞(标记蛋白为：αSMA)、间质细胞(标记蛋白为：Star)、精原细胞(标记蛋白为：PCNA)在正常生长，并表达维持细胞生长所需要的蛋白。Figure 4 shows the immunofluorescence staining of testicular organoids on day 15 during the testicular organoid suspension culture process of the present invention. Immunofluorescence staining showed that supporting cells (marked proteins: Fibronectin, ZO-1, WT1), peritubular myoid cells (marked proteins: αSMA), and interstitial cells (marked proteins: αSMA) in organoids : Star) and spermatogonia (labeled protein: PCNA) are growing normally and expressing proteins required to maintain cell growth.
Figure PCTCN2022129770-appb-000001
Figure PCTCN2022129770-appb-000001
图5为本发明睾丸类器官悬浮培养过程中，第5、10、15天睾丸类器官间质细胞、支持细胞、精原干细胞基因表达量变化。PCR结果表明得到的睾丸类器官的基因表达量接近于成年鼠睾丸组织的基因表达量，说明制备的类器官在基因表达水平上和睾丸相似。Figure 5 shows changes in gene expression of testicular organoid interstitial cells, Sertoli cells, and spermatogonial stem cells on days 5, 10, and 15 during the testicular organoid suspension culture process of the present invention. The PCR results showed that the gene expression level of the obtained testicular organoids was close to the gene expression level of adult mouse testicular tissue, indicating that the prepared organoids were similar to testises in terms of gene expression levels.
Figure PCTCN2022129770-appb-000002
Figure PCTCN2022129770-appb-000002
引物由华大基因公司合成。Primers were synthesized by BGI.
图6为本发明睾丸类器官悬浮培养过程中，第5、10、15、20天睾丸类器官睾酮变化(睾酮放免检测试剂盒，北方生物)。结果表明，在培养的20天内，该睾丸类器官培养体系可以有效地维持睾酮分泌，在第15天状态最佳，表明类器官可以正常分泌睾酮，具有和睾丸相似的功能。Figure 6 shows the testosterone changes in testicular organoids on days 5, 10, 15, and 20 during the testicular organoid suspension culture process of the present invention (testosterone radioimmunoassay kit, Northern Biotech). The results showed that within 20 days of culture, the testicular organoid culture system could effectively maintain testosterone secretion, with the best condition on the 15th day, indicating that the organoids could secrete testosterone normally and have similar functions to testicles.
图7为本发明睾丸类器官悬浮培养过程中，第5、10、15、20、25、30天睾丸类器官分泌因子基因表达量变化。PCR结果显示睾丸类器官相关分泌因子接近于成年鼠睾丸组织，说明制备的类器官在相关分泌因子表达水平上和睾丸相似。Figure 7 shows changes in gene expression of testicular organoid secretion factors on days 5, 10, 15, 20, 25 and 30 during the testicular organoid suspension culture process of the present invention. The PCR results showed that the secreted factors related to testicular organoids were close to those of adult mouse testis tissue, indicating that the prepared organoids were similar to testis in the expression levels of related secreted factors.
Figure PCTCN2022129770-appb-000003
Figure PCTCN2022129770-appb-000003
引物由华大基因公司合成。Primers were synthesized by BGI.
实施例3：睾丸类器官的冻存和复苏Example 3: Cryopreservation and recovery of testicular organoids
冻存：类器官悬浮培养5天后，从三角瓶中收集悬液于15ml离心管1500rpm离心5min；Cryopreservation: After 5 days of organoid suspension culture, collect the suspension from the Erlenmeyer flask and centrifuge it at 1500 rpm for 5 minutes in a 15 ml centrifuge tube;
弃去上清，加入类器官冻存液(购自创芯国际生物科技(广州)有限公司)转移至冻存管，将冻存管转移至冻存盒中-80℃过夜，第二天转移至液氮；Discard the supernatant, add organoid cryopreservation solution (purchased from Chuangxin International Biotechnology (Guangzhou) Co., Ltd.) and transfer it to a cryopreservation tube. Transfer the cryopreservation tube to a cryopreservation box at -80°C overnight and transfer it the next day. to liquid nitrogen;
解冻：类器官从液氮中取出后37℃水浴迅速解冻，移入事先备好的含10％FBS的15ml离心管中，1500rpm离心5min，得到复苏后的睾丸类器官，按上述培养方法进行培养。Thaw: Take the organoids out of liquid nitrogen and quickly thaw them in a 37°C water bath. Move them into a prepared 15ml centrifuge tube containing 10% FBS. Centrifuge at 1500rpm for 5 minutes to obtain the recovered testicular organoids. Culture them according to the above culture method.
图8为本发明睾丸类器官进行冻存和复苏处理后培养3天，冰冻切片后进行TUNEL凋亡染色实验。结果显示：未冻存组基本没有凋亡(绿色表示细胞凋亡)，商品化冻存液组少量细胞凋亡，阳性对照组细胞全部凋亡(图8，A)。染色统计表明，DMEM:FBS:DMSO＝6:3:1的冻存液复苏后的活率为83.5％，商品化冻存液复苏培养后的活率为89.5％，未冻存的细胞活率在97.0％左右(图8，B)，商品化冻存液优于DMEM:FBS:DMSO＝6:3:1的冻存液。表明类器官复苏后能够维持原有状态。Figure 8 shows the testicular organoids of the present invention that were cryopreserved and resuscitated, cultured for 3 days, and then frozen sectioned for TUNEL apoptosis staining experiments. The results showed that there was basically no apoptosis in the non-cryopreserved group (green indicates cell apoptosis), a small number of cells in the commercial cryopreservation solution group were apoptotic, and all cells in the positive control group were apoptotic (Figure 8, A). Staining statistics show that the viability rate after resuscitation in DMEM:FBS:DMSO=6:3:1 cryopreservation solution is 83.5%, the viability rate after resuscitation and culture in commercial cryopreservation solution is 89.5%, and the viability rate of uncryopreserved cells is 83.5%. About 97.0% (Figure 8, B), the commercial cryopreservation solution is better than the cryopreservation solution of DMEM:FBS:DMSO=6:3:1. This shows that the organoids can maintain their original state after recovery.
实施例4：睾丸类器官的药物筛选测试Example 4: Drug Screening Test of Testicular Organoids
类器官以5粒/80ul接种96孔板(培养基为DMEM)，同时加不同浓度CdCl 2(1.56μM、3.12μM、6.25μM、12.5μM、25μM、50μM、100μM、200μM)，放在摇床上培养(34℃、5％CO 2培养箱)，3天后拍摄类器官状态及cck8方法(MCE公司，货号：HY-K0301)测试存活率。 Organoids were inoculated into a 96-well plate (the culture medium was DMEM) at 5 particles/80ul, and different concentrations of CdCl 2 (1.56μM, 3.12μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM) were added and placed on a shaker. Culture (34°C, 5% CO 2 incubator), and after 3 days, the organoid status was photographed and the survival rate was tested using the cck8 method (MCE Company, Cat. No.: HY-K0301).
结果如图9所示。结果表明，本发明的睾丸类器官用于药物毒性评价时具有良好的量效曲线关系，表明该睾丸类器官可以用做药物毒理评价模型。The results are shown in Figure 9. The results show that the testicular organoids of the present invention have a good dose-effect curve relationship when used for drug toxicity evaluation, indicating that the testicular organoids can be used as a drug toxicology evaluation model.
本领域技术人员应该理解，尽管参照上述实施例对本发明进行了具体的描述，但是本发明并不限于这些具体的实施例。基于本发明所教导的方法和技术方案，在不背离本发明的精神的前提下，本领域技术人员能够进行适当的修改或改进，由此所得的等价实施方案都在本发明的范围内。Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and equivalent embodiments obtained thereby are within the scope of the present invention.

Claims (10)

  1. 一种睾丸类器官，其包含：间质细胞、支持细胞、精原干细胞或它们的组合。A testicular organoid containing: Leydig cells, Sertoli cells, spermatogonial stem cells, or a combination thereof.
  2. 根据权利要求1所述的睾丸类器官，其为通过体外细胞培养获得的，并且能够生成睾酮，优选所述细胞是来源于啮齿动物或非人灵长类动物，小鼠或大鼠，最优选所述细胞使用的是成体细胞，优选是3天至2周大、特别优选7～10天大的动物的睾丸组织胰酶消化后得到的细胞集合。The testicular organoid according to claim 1, which is obtained by in vitro cell culture and capable of producing testosterone, preferably the cells are derived from rodents or non-human primates, mice or rats, most preferably The cells used are adult cells, preferably a collection of cells obtained by trypsin digestion of the testicular tissue of an animal that is 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
  3. 根据权利要求1或2所述的睾丸类器官，其表达以下蛋白标志物之一或它们的组合：纤连蛋白，α-SMA，ZO-1，WT1，Star，和PCNA。The testicular organoid according to claim 1 or 2, which expresses one of the following protein markers or a combination thereof: fibronectin, α-SMA, ZO-1, WT1, Star, and PCNA.
  4. 根据权利要求1-3中任一项所述的睾丸类器官，其能够表达以下基因之一或它们的组合：Hsd3b1，Hsd17b3，Star，Sox9，Wt1，Ar，Fshr，C-ret，和Crem。The testicular organoid according to any one of claims 1-3, which is capable of expressing one of the following genes or a combination thereof: Hsd3b1, Hsd17b3, Star, Sox9, Wt1, Ar, Fshr, C-ret, and Crem.
  5. 根据权利要求1-4中任一项所述的睾丸类器官，其能够分泌以下因子之一或它们的组合：Amh，Dhh，Gdnf，抑制素B，Cxcl12和Fgf2。The testicular organoid according to any one of claims 1 to 4, capable of secreting one of the following factors or a combination thereof: Amh, Dhh, Gdnf, inhibin B, Cxcl12 and Fgf2.
  6. 一种用于制备睾丸类器官的方法，其包括以下步骤：A method for preparing testicular organoids, comprising the following steps:
    1)获得幼年动物睾丸细胞，优选所述细胞是成体细胞，更优选是3天至2周大、特别优选7～10天大的动物的睾丸组织消化后得到的细胞集合；1) Obtain testicular cells of juvenile animals, preferably the cells are adult cells, more preferably a collection of cells obtained by digesting the testicular tissue of animals that are 3 days to 2 weeks old, and particularly preferably 7 to 10 days old;
    2)将获得的睾丸细胞进行悬滴培养；和任选地2) subjecting the obtained testicular cells to hanging drop culture; and optionally
    3)将步骤2)中获得的悬滴培养产物进行悬浮培养，由此获得睾丸类器官。3) Perform suspension culture on the hanging drop culture product obtained in step 2), thereby obtaining testicular organoids.
  7. 根据权利要求6所述的方法，其中：The method of claim 6, wherein:
    a)所述动物为啮齿动物或非人灵长类动物，小鼠或大鼠；a) The animal is a rodent or non-human primate, mouse or rat;
    b)在步骤1)中，取幼年动物睾丸组织，依次进行浸泡、洗涤、去被膜、剪切和细胞消化，获得幼年动物睾丸细胞，其中优选地，所述浸泡使用PBS浸泡；和/或所述洗涤中使用的洗涤液为含有青霉素和链霉素的PBS；优选地，所述消化是使用胰酶消化；b) In step 1), take the testicular tissue of young animals, and sequentially perform soaking, washing, membrane removal, shearing and cell digestion to obtain testicular cells of young animals, wherein preferably, the soaking uses PBS; and/or the The washing liquid used in the washing is PBS containing penicillin and streptomycin; preferably, the digestion is using trypsin digestion;
    c)在步骤2)中，所述悬滴培养使用的培养基是含有EGF的无血清培养基(优选DMEM，MEM、DMEM/F12培养基)，诸如含有EGF和KSR的基础培养基，并且于34℃-37℃、3％-5％CO 2浓度的培养条件下悬滴培养5-7天，优选按所述培养基的体积计，培养基中EGF的浓度为50-100ng/ml，KSR浓度为5-10体积％；和/或 c) In step 2), the medium used for the hanging drop culture is a serum-free medium containing EGF (preferably DMEM, MEM, DMEM/F12 medium), such as a basal medium containing EGF and KSR, and Hang drop culture for 5-7 days under the culture conditions of 34°C-37°C and 3%-5% CO2 concentration. Preferably, the concentration of EGF in the culture medium is 50-100ng/ml based on the volume of the culture medium. KSR The concentration is 5-10% by volume; and/or
    d)在步骤3)中，所述悬浮培养使用的培养基是含有KSR、LH和FSH的基础培养基(优选DMEM，MEM、DMEM/F12培养基)，优选按培养基的体积计，各成分的含量为：KSR，5体积％-10体积％；LH，1-10ng/ml；FSH，1-10ng/ml；和/或优选地，将步骤2)中获得的悬滴培养产物离心富集细胞，加入上述培养基，于34℃-37℃、3％-5％CO 2、120-160rpm下培养。 d) In step 3), the medium used for the suspension culture is a basic medium containing KSR, LH and FSH (preferably DMEM, MEM, DMEM/F12 medium), preferably based on the volume of the medium, each component The content is: KSR, 5%-10% by volume; LH, 1-10ng/ml; FSH, 1-10ng/ml; and/or preferably, the hanging drop culture product obtained in step 2) is centrifugally enriched For cells, add the above culture medium and culture at 34°C-37°C, 3%-5% CO 2 and 120-160 rpm.
  8. 根据权利要求1-5中任一项所述的睾丸类器官，其可通过根据权利要求6或7所述的方法制备。The testicular organoid according to any one of claims 1-5, which can be prepared by the method according to claim 6 or 7.
  9. 根据权利要求1-5和8中任一项所述的睾丸类器官在药物筛选或测定药物毒性中的应用。Use of the testicular organoid according to any one of claims 1-5 and 8 in drug screening or determination of drug toxicity.
  10. 根据权利要求9所述的应用，其中将候选药物与根据权利要求1-5和8中任一项所述的睾丸类器官接触。Use according to claim 9, wherein the candidate drug is contacted with the testicular organoid according to any one of claims 1-5 and 8.
PCT/CN2022/129770 2022-03-18 2022-11-04 Testis organoid preparation, culture and cryopreservation resuscitation methods and application WO2023173763A1 (en)

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