WO2023173763A1 - Préparation d'organoïdes testiculaires, procédés de réanimation de culture et de cryoconservation et application - Google Patents

Préparation d'organoïdes testiculaires, procédés de réanimation de culture et de cryoconservation et 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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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
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • 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

La présente invention concerne un organoïde testiculaire cultivé, ainsi que des procédés de préparation, de culture et de réanimation par cryopréservation, et une application associée. L'organoïde testiculaire peut conserver les fonctions spécifiques des testicules et réaliser un criblage de médicaments à haut débit.
PCT/CN2022/129770 2022-03-18 2022-11-04 Préparation d'organoïdes testiculaires, procédés de réanimation de culture et de cryoconservation et application WO2023173763A1 (fr)

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