CN114908032B - Preparation, culture, cryopreservation and resuscitation method and application of testicular organ - Google Patents

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

Info

Publication number
CN114908032B
CN114908032B CN202210271380.7A CN202210271380A CN114908032B CN 114908032 B CN114908032 B CN 114908032B CN 202210271380 A CN202210271380 A CN 202210271380A CN 114908032 B CN114908032 B CN 114908032B
Authority
CN
China
Prior art keywords
testis
culture
medium
cells
ksr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210271380.7A
Other languages
Chinese (zh)
Other versions
CN114908032A (en
Inventor
杨艳
黄亚东
曹振
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jinan University
Original Assignee
Jinan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jinan University filed Critical Jinan University
Priority to CN202210271380.7A priority Critical patent/CN114908032B/en
Publication of CN114908032A publication Critical patent/CN114908032A/en
Priority to PCT/CN2022/129770 priority patent/WO2023173763A1/en
Priority to ZA2023/02871A priority patent/ZA202302871B/en
Application granted granted Critical
Publication of CN114908032B publication Critical patent/CN114908032B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Reproductive Health (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a cultured testis organ and a preparation method, a culture method, a cryopreservation recovery method and application thereof. The testis organoid prepared by the invention is highly consistent with the genetic background of 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 also be frozen and resuscitated. The preparation and culture processes are simple and convenient to operate, the repeatability is high, the drug effect test time is short, the culture cost is more economical than that of an animal model, and the established testis organoids can realize high-flux drug screening and have good application prospects.

Description

Preparation, culture, cryopreservation and resuscitation method and application of testicular organ
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation, cultivation and cryopreservation recovery method and application of testis organoids.
Background
The testis is a genital organ of male animals, which is respectively positioned at the left and right sides of scrotum and takes the shape of an oval, a layer of fibrous membrane is arranged on the surface of a testosterone pill, which is called a tunica albuginea, the tunica albuginea is thickened along the rear edge of the testis, mediastinum is formed in the testis, a plurality of connective tissue compartments are emitted from the mediastinum, the testis is divided into a plurality of testis lobules, the testosterone pill lobules contain coiled seminiferous tubules, the epithelium of the seminiferous tubules can produce sperms, interstitial cells are arranged in connective tissues among the tubules, and the interstitial cells produce androgens which are closely related to male secondary characteristics, physiological functions and the like.
Organoids (Organoids) belong to (3D) cell cultures, are organ-specific cell collections derived from stem cells or precursor cells, contain some key features that represent organs, are capable of mimicking the tissue architecture, cell type composition of a real organ, and possess specific functional features, while maintaining the advantages of a simplified and readily available cell culture model. Unlike two-dimensional (2D) cell culture models, organoid culture cultures a variety of cell populations contained in a particular tissue organ in a 3D environment, with the micro-environmental system of culture being closer to in vivo. Therefore, the organoids can be used as an in vitro platform for researching interaction between cells, tissue development and toxicology, effectively supplement the existing animal and two-dimensional (2D) cell culture models, and have wide application prospects in the aspects of basic research, accurate medical treatment, drug screening and development, regenerative medicine and the like of physiological pathology of various organs.
Disclosure of Invention
The invention aims to solve the technical problems of preparation, culture and cryopreservation recovery methods and application of testis organoids, and aims to solve the problems in the prior art.
Specifically, the present invention provides a method for preparing, culturing, freezing and recovering mouse testis organoids and application thereof, wherein the method comprises the following steps: taking testis tissue of a young mouse, and sequentially carrying out soaking, washing, removing a capsule, shearing and digesting cells to obtain testis cell suspension of the mouse; performing suspension culture on the testis cells of the suckling mice by hanging drops to obtain testis organoids; testis organoids were collected by centrifugation and cultured in suspension in a specific medium.
Accordingly, in one aspect, the present invention provides a testicle organoid comprising: mesenchymal cells, supporting cells, spermatogonial stem cells, or a combination thereof.
According to a preferred embodiment of the invention, the testicle organoids are obtained by cell culture in vitro and are capable of producing testosterone, preferably the cells are derived from rodent or non-human primates, mice or rats.
According to a preferred embodiment of the invention, the testis organoids express one or any combination of the following protein markers: fibronectin, alpha-smooth muscle actin (Alpha-smooth muscle actin, alpha-SMA), desmin 1 (Tight junction protein, ZO-1), wilms tumor 1, wt1), steroid hormone synthesis acute regulatory protein (Steroidogenic acute regulatory, star), and proliferating cell nuclear antigen (Proliferating cell nuclear antigen, PCNA).
According to a preferred embodiment of the invention, the testis organoids are capable of expressing one or any combination of the following genes/proteins: 3β -hydroxysteroid dehydrogenase (3-Beta-hydroxysteroid dehydrogenase1, hsd b 1), 17β -hydroxysteroid dehydrogenase 3 (17β -hydroxysteroid dehydrogenase 3, hsd17b 3), steroid hormone synthesis acute regulatory protein (Steroidogenic acute regulatory, star), sex determining region Y box protein 9 (Sry related HMG box-9, sox 9), wilms tumor protein 1 (Wilms tumor 1, wt1), androgen receptor (Androgen Receptor, ar), follicle stimulating hormone receptor (follicle stimulating hormone receptor, fshr), proto-oncogene (C-Ret), and cyclic AMP-responsive element modulator, crem.
According to a preferred embodiment of the invention, the testicle-like organ is capable of secreting one or any combination of the following factors: anti-mullerian hormone (anti-Mullerian hormone, amh), desert hedgehog (Dhh), 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, cxcl 12) and fibroblast growth factor 2 (Fibroblast growth factor, fgf2).
Another aspect of the invention provides a method for preparing a testicle organoid comprising the steps of:
1) Obtaining testicular cells of a young animal;
2) Performing hanging drop culture on the obtained testis cells; and optionally
3) And (3) performing suspension culture on the hanging drop culture product obtained in the step (2), thereby obtaining testis-like organs.
It should be noted that in the method of the present invention, the hanging drop culture of step 2) has been able to form testosterone organoids, whereas the purpose of the suspension culture of step 3) is to provide nutrition to maintain long-term culture of testosterone 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 above step 1), the testicular tissue of the young animal is taken and sequentially subjected to soaking, washing, coating removal, shearing and cell digestion to obtain testicular cells of the young animal, wherein preferably, the soaking is performed using PBS; and/or the washing liquid used in the washing is PBS containing penicillin and streptomycin (for example, PBS and penicillin 100 IU/ml+streptomycin 100 μg/ml are mixed); preferably, the digestion is digestion with pancreatin.
According to a preferred embodiment of the invention, the invention uses a collection of cells obtained after digestion of the testis tissue of an animal, preferably 3 days to 2 weeks old, particularly preferably 7 to 10 days old.
According to a preferred embodiment of the invention, the PBS used for the soaking is ice-pre-chilled.
According to a preferred embodiment of the invention, the hanging-drop culture is performed as follows: testicular cells obtained after digestion were dropped inside a dish cover (hemispherical drop formed), and the dish cover was back-fastened to a dish with a medium such as PBS added thereto, followed by culturing in an incubator, thereby obtaining 3D testicle-like organs.
Preferably, in the above step 2), the medium used for the hanging-drop culture is a serum-free medium containing an Epidermal Growth Factor (EGF), such as a basal medium (preferably DMEM, MEM, DMEM/F12 medium) containing EGF and KSR (serum replacement, knockot serum replacement), and is fed with 3% -5% CO at 34℃to 37 ℃C 2 Suspension drop culture at concentration for 5-7 days, preferably cultureThe concentration of EGF in the medium is 50-100ng/ml. Preferably, the medium used for the hanging-drop culture is 10% by volume KSR medium containing 50ng/ml EGF, and 5% CO at 34 DEG C 2 Hanging drop culture under concentration for 5 days; and/or
In the step 3), the culture medium used in the suspension culture is a basal medium (preferably DMEM, MEM, DMEM/F12 medium) containing KSR, LH (flavoprotein) and FSH (follicle stimulating hormone), and the contents of the components are preferably as follows, based on the volume of the medium (e.g., DMEM): KSR, 5-10% by volume; LH,1-10ng/ml; FSH,1-10ng/ml. Preferably, the contents of the components are: KSR, 10% by volume; LH,1ng/ml; FSH,1ng/ml; and/or
Preferably, the hanging drop culture product obtained in the step 2) is centrifugally enriched with cells, and the culture medium is added with 3 to 5 percent CO at the temperature of between 34 and 37 DEG C 2 Culturing at 120-160 rpm; preferably, the medium is changed every 5 days, and the culture can be continued for a long period of time.
According to a particularly preferred embodiment of the present invention, each of the above EGF, LH and FSH is of mammalian, preferably human or mouse, EGF, LH and FSH.
The serum replacement media (e.g., KSR) used in the present invention have the advantage of clear composition and strong consistency compared to serum media. The serum culture medium has large batch-to-batch variation, and the possibility of animal-derived proteins or viruses is provided. 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 preparing a testicle organ, comprising the steps of:
(1) Taking testis tissue of male Kunming mice, soaking in PBS precooled and added with double antibody (penicillin and streptavidin);
(2) Taking 6 sterile 9x9 cm round bacteria dishes, sequentially adding PBS containing double antibodies, numbering 1-6, transferring testis tissues into the bacteria dishes numbered 1 for rinsing for 1-2min, sequentially transferring into bacteria dishes numbered 2 and 3 for rinsing, gently puncturing testis envelope with sterile forceps, taking out testis tissues, transferring into bacteria dishes numbered 4 for rinsing for 1-2min, and sequentially transferring into bacteria dishes numbered 5 and 6 for rinsing;
(3) Taking out tissue from the No. 6 bacteria dish, transferring to a penicillin bottle, shearing testis tissue, and lasting for 5min;
(4) Adding pancreatin, placing in a constant temperature incubator at 37deg.C, and digesting for 15min while shaking for 2-3 times;
(5) Adding 10% FBS to neutralize pancreatin after digestion, and blowing the suspension for 5min by using a glass suction tube;
(6) Transferring into a 15ml centrifuge tube, and centrifuging at 1500rpm for 5min;
(7) Discarding the supernatant, resuspending the pellet with DMEM containing 10 vol% KSR, standing for about 5min, transferring the supernatant, discarding the bottom pellet, 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% by volume KSR+50ng/ml EGF, according to 1X10 5 Diluting cells at individual cell/ml ratio;
(9) Taking 20 μl of cell dilution liquid, measuring in a square bacterial dish cover of 10×10 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 allowed to stand still for hanging drop culture for 5 days, yielding a 3D testis organoid with an initial cell number of about 2000/grain.
Also provided is a method for culturing testis organoids, comprising the steps of:
(1) Taking the primary testis-like organ prepared according to the above, transferring into a 15ml centrifuge tube for enrichment, and centrifuging at 1500rpm for 5min;
(2) The supernatant was discarded and the testis organoids were resuspended in DMEM medium containing 10% by volume KSR+1ng/ml LH+1ng/ml FSH;
(3) Transferring to a 50ml triangular flask, adding 20-30ml DMEM medium containing 10 vol% KSR+1ng/ml LH+1ng/ml FSH for suspension culture;
(4) Transferring the triangular flask to a 34 ℃ constant temperature culture shaking table for culture at 120-160rpm, and changing liquid every 5 days.
According to a preferred embodiment of the present invention, there is also provided a testicle organoid which can be prepared by the method for preparing a testicle organoid according to the present invention.
In another aspect, the invention also provides the use of the testicle organoids of the invention in drug screening or in determining drug toxicity.
According to a preferred embodiment of the invention, the candidate drug is contacted with a testicular organoid according to the invention.
According to a preferred embodiment of the present invention, there is also provided the use of a testicle organoid for a drug screening test comprising the steps of:
(1) Organoids were seeded in 96-well plates at 5 grains/80 ul with different concentrations of CdCl 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 at 120-160rpm, and the organoid state was photographed after 3 days and tested for survival by the cck8 (cell counting kit-8) method.
In another aspect, the invention also provides a method for cryopreserving and resuscitating a testis-like organ, comprising the steps of:
(1) Freezing: after the organoids are cultured for 5 days in a suspension mode, the organoids are collected from a triangular flask and centrifuged for 5min at 1500rpm in a 15ml centrifuge tube;
(2) Removing the supernatant, adding organoid frozen stock solution, transferring to a frozen stock tube, transferring the frozen stock tube to a frozen stock box at-80 ℃ overnight, and transferring to liquid nitrogen the next day;
(3) Thawing: the frozen organoids were removed from the liquid nitrogen and thawed in a water bath at 37℃rapidly, transferred to a pre-prepared 15ml centrifuge tube containing 10% FBS, centrifuged at 1500rpm for 5min to obtain the testis organoids, and cultured according to the above-described culture method.
The innovation point of the invention at least comprises:
(1) The testis organ can be obtained by a 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 testis for a long time and simulate the functions of testis in vitro.
(3) The testis organoids can be subjected to drug testing and toxicology evaluation.
Advantageous effects
The testis organoid prepared by the invention is highly consistent with the genetic background of 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 also be frozen and resuscitated. The preparation and culture processes are simple and convenient to operate, the repeatability is high, the drug effect test time is short, the culture cost is more economical than that of an animal model, and the established testis organ can realize high-flux drug screening, so that the application prospect is good.
Drawings
FIG. 1 is a flow chart of preparation, suspension culture and formation of a testicular organoid droplet of the present invention.
FIG. 2 is a graph showing the area change of testis-like organs at days 5, 10, 15, 20, 25 and 30 in the suspension culture process of testis-like organs according to the present invention.
FIG. 3 shows HE staining of testis-like organs at days 5, 10, 15, 20, 25, 30 during suspension culture of testis-like organs according to the invention.
FIG. 4 shows immunofluorescent staining of testis-like organs at day 15 during suspension culture of testis-like organs according to the invention.
FIG. 5 shows the variation of the gene expression level of testis organoid mesenchymal cells, supporting cells and spermatogonial stem cells at days 5, 10 and 15 in the suspension culture process of testis organoids according to the present invention.
FIG. 6 shows testosterone changes in testis-like organs at days 5, 10, 15 and 20 during suspension culture of testis-like organs according to the invention.
FIG. 7 shows the variation of the expression level of the testis-like organ secretion factor gene at days 5, 10, 15, 20, 25 and 30 in the suspension culture process of the testis-like organ of the present invention.
FIG. 8 shows the results of TUNEL apoptosis staining experiments performed after frozen sections of testis-like organs of the present invention were incubated for 3 days after cryopreservation and resuscitating treatment.
FIG. 9 shows the concentration of CdCl in testis-like organs at 15 days during suspension culture 2 After treatment, cck8 was measured.
Detailed Description
Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art.
Examples
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature or as per the specifications of the product. The reagents or apparatus used were conventional products available commercially without the manufacturer's knowledge.
Reagent(s)
Penicillin (MDBio), 100IU/ml;
streptomycin (MDBio), 100ug/ml;
pancreatin (Gibco);
fetal bovine serum FBS (ExCell Bio);
DMEM(Gibco)；
serum replacement KSR (Life), 10% KSR by volume;
trypan blue (scientific);
luteinizing hormone LH (human source, merk Millipore), 1ng/ml;
follicle stimulating hormone FSH (human source, proselect), 1ng/ml;
organoid frozen stock solutions were purchased from core international biotechnology (guangzhou) limited;
cadmium chloride (Alorich), 1.56-200. Mu.M
Example 1: preparation method of testis organ
7-10 days of testis tissue from male Kunming mice (purchased from Experimental animal center, guangdong province) were placed on an ice box and soaked in PBS containing double antibody (penicillin (MDBio), 100IU/ml, and streptomycin (MDBio), 100 ug/ml);
taking 6 sterile 9x9 cm round bacteria dishes, sequentially adding the PBS containing the double antibodies, sequentially transferring testis tissues into the bacteria dishes with the serial numbers of 1 to 6, rinsing for 1 to 2min, sequentially transferring the testis tissues into the bacteria dishes with the serial numbers of 2 and 3, rinsing, slightly puncturing testis membranes by using sterile forceps, taking out the testis tissues, transferring the testis tissues into the bacteria dishes with the serial numbers of 4 for rinsing for 1 to 2min, and sequentially transferring the testis tissues into the bacteria dishes with the serial numbers of 5 and 6 for rinsing;
taking out testis tissue from the No. 6 bacteria dish by forceps, transferring to a penicillin bottle, shearing the testis tissue by using surgical bending shears, and continuously stirring the testis tissue for about 5min until the testis tissue is in a uniform slurry state;
adding 10mL pancreatin (Gibco) into penicillin bottle, placing into a constant temperature incubator at 37deg.C for digestion for 15min, taking out at intervals of 2-3 times, observing digestion state, and shaking properly;
adding 10% FBS with double volume to neutralize pancreatin after digestion, and blowing the suspension with a glass suction tube for 5min until no floccule appears;
transferring into a 15ml centrifuge tube, and centrifuging at 1500rpm for 5min;
the supernatant was discarded, the pellet was resuspended in DMEM medium containing 10% by volume KSR (Life), 90ul of the suspension was removed and mixed with 10ul of trypan blue (scientific) and counted over 3 min;
the resuspended testis cells were added to DMEM medium containing 10% by volume of KSR and 50ng/ml EGF (human EGF, pepro Tech, U.S.A., cat# AF-100-15) at 1X10 5 Dilution of the resuspended fluid at individual cell/ml ratio;
transferring 20ul of cell suspension to the inner side of a square bacterial dish cover of 10x10 cm by using a row gun, enabling the bacterial dish cover to be in a red hemispherical shape, and reversely buckling the bacterial dish cover on a bacterial dish added with PBS;
transfer the bacterial dishes to 34℃with 5% CO 2 Standing in a constant temperature incubator, hanging drop culturing for 5 days to obtain 3D testis organoids with initial cell number of about 2000/granule.
Example 2: testis organ culture method
The primary testis organoids prepared according to example 1 above were transferred to a 15ml centrifuge tube and centrifuged at 1500rpm for 5min;
the supernatant was discarded and the testis organoids were resuspended in DMEM medium containing 10% by volume KSR+1ng/ml LH+1ng/ml FSH;
transferring to a 50ml triangular flask, adding 20-30ml DMEM medium containing 10 vol% KSR+1ng/ml LH+1ng/ml FSH according to the organoid quantity, and culturing in suspension;
transfer the flask to 34℃constant temperature 5% CO 2 Culturing in a culture shaker at 120-160rpm every 5And (5) performing fluid replacement treatment, thereby obtaining the testis organoids.
FIG. 2 is a graph showing the area change of testis-like organs at days 5, 10, 15, 20, 25 and 30 in the suspension culture process of testis-like organs according to the present invention. The area change curve results show that the volume of the testis organoids is slowly increased along with the extension of the culture time, which indicates that the organoids can grow for a long time and maintain the morphology.
FIG. 3 shows HE staining of testis-like organs at days 5, 10, 15, 20, 25, 30 during suspension culture of testis-like organs according to the invention. HE staining indicated that cells within the testis organoids formed a structure resembling testis tissue.
FIG. 4 shows the fluorescent staining of testis-like organs during suspension culture of testis-like organs according to the present invention, day 15. Immunofluorescent staining showed that supporting cells (labeled proteins: fibronectin, ZO-1, WT 1), peritubular myoid cells (labeled protein: aSMA), mesenchymal cells (labeled protein: star), spermatogenic cells (labeled protein: PCNA) in organoids grew normally and expressed proteins required to maintain cell growth.
FIG. 5 shows the variation of the gene expression level of testis organoid mesenchymal cells, supporting cells and spermatogonial stem cells at days 5, 10 and 15 in the suspension culture process of testis organoids according to the present invention. The PCR result shows that the gene expression quantity of the obtained testis-like organ is close to that of the testis tissue of the adult mouse, and the prepared testis-like organ is similar to the testis in the gene expression level.
Primers were synthesized by Huada gene company.
FIG. 6 shows the testosterone change of the testis-like organs (testosterone free detection kit, northern organism) at days 5, 10, 15 and 20 during the suspension culture of the testis-like organs according to the invention. The result shows that the testis-like organ culture system can effectively maintain testosterone secretion within 20 days of culture, and the state is optimal on 15 days, so that the testis-like organ can normally secrete testosterone, and has the function similar to that of a testis.
FIG. 7 shows the variation of the expression level of the testis-like organ secretion factor gene at days 5, 10, 15, 20, 25 and 30 in the suspension culture process of the testis-like organ of the present invention. The PCR result shows that the testis organoid related secretion factor is close to the testis tissue of the adult mouse, and the prepared organoid is similar to the testis at the expression level of the related secretion factor.
Primers were synthesized by Huada gene company.
Example 3: cryopreservation and resuscitation of testicle organoids
Freezing: after the organoids are cultured for 5 days in a suspension way, collecting the suspension from a triangular flask, and centrifuging the suspension in a 15ml centrifuge tube at 1500rpm for 5min;
removing supernatant, adding organoid frozen stock solution (purchased from Innovative core International Biotechnology (Guangzhou) Co., ltd.) and transferring to a frozen stock tube, transferring the frozen stock tube to a frozen stock box at-80 ℃ overnight, and transferring to liquid nitrogen the next day;
thawing: the organoids were removed from the liquid nitrogen and rapidly thawed in a 37℃water bath, transferred to a pre-prepared 15ml centrifuge tube containing 10% FBS, centrifuged at 1500rpm for 5min to obtain recovered testis organoids, and cultured according to the above-described culture method.
FIG. 8 shows TUNEL apoptosis staining experiments after 3 days of incubation after cryopreservation and resuscitating treatment of testis-like organs according to the invention, and frozen sections. The results show that: the unfrozen group had substantially no apoptosis (green indicates apoptosis), the commercial frozen solution group had a small amount of apoptosis, and the positive control group had all apoptosis (fig. 8, a). Staining statistics show that the survival rate of DMEM: FBS: DMSO=6:3:1 after resuscitation is 83.5%, the survival rate of commercial frozen solution after resuscitating and culturing is 89.5%, and the survival rate of unfrozen cells is about 97.0% (FIG. 8, B), and the commercial frozen solution is superior to the DMEM: FBS: DMSO=6:3:1 frozen solution. Indicating that the original state can be maintained after the organoid is recovered.
Example 4: drug screening test for testis organoids
Organoids were seeded in 96-well plates (DMEM medium) at 5 grains/80 ul with different concentrations of CdCl 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) and culturing on a shaker (34 ℃ C., 5% CO) 2 Incubator), the organ-like status and cck8 method (MCE company, cat No.: HY-K0301) test survival.
The results are shown in FIG. 9. The result shows that the testis-like organ has good dose-response curve relationship when being used for evaluating the drug toxicity, and the testis-like organ 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 specifically described with reference to the above embodiments, the invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments thus obtained are within the scope of the present invention.

Claims (7)

1. A method for preparing a testicle organoid comprising the steps of:
1) Obtaining testicular cells of a young animal;
2) Performing hanging drop culture on the obtained testis cells; and
3) Performing suspension culture on the hanging drop culture product obtained in the step 2), thereby obtaining testis-like organs,
wherein the testicular cells in step 1) are a collection of cells obtained after digestion of testicular tissue of an animal 7 to 10 days old, said animal being a mouse;
in step 2), the culture medium used for hanging drop culture is a serum-free medium containing EGF, and is prepared by mixing at 34-37 ℃ and 3-5% CO 2 Culturing under suspension drop culture condition for 5-7 days, wherein the concentration of EGF in the culture medium is 50-100ng/ml and the concentration of KSR is 5-10 vol% based on the volume of the culture medium; and
in the step 3), the culture medium used in the suspension culture is a basic culture medium containing KSR, LH and FSH, and the contents of the components are as follows, based on the volume of the culture medium: KSR, 5% to 10% by volume; LH,1-10ng/ml; FSH,1-10ng/ml, and wherein in step 3) the hanging-drop culture product obtained in step 2) is centrifuged to enrich cells, the above medium is added, at 34℃to 37℃with 3% -5% CO 2 Culturing at 120-160 rpm.
2. The method according to claim 1, wherein:
in the step 1), testis tissues of the young animals are taken and soaked, washed, subjected to tunica media removal, sheared and subjected to cell digestion in sequence to obtain testis cells of the young animals; and/or the washing liquid used in the washing is PBS containing penicillin and streptomycin.
3. The method of claim 2, wherein the soaking in step 1) uses PBS soaking.
4. The method of claim 1, wherein in step 1) the digestion is digestion with pancreatin.
5. The method of claim 1, wherein in step 2) the serum-free medium is DMEM, MEM or DMEM/F12 medium.
6. The method of claim 1, wherein in step 2) the serum-free medium is a basal medium comprising EGF and KSR.
7. The method according to claim 1, wherein the medium used in the suspension culture in step 3) is DMEM, MEM or DMEM/F12 medium containing KSR, LH and FSH.
CN202210271380.7A 2022-03-18 2022-03-18 Preparation, culture, cryopreservation and resuscitation method and application of testicular organ Active CN114908032B (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202210271380.7A CN114908032B (en) 2022-03-18 2022-03-18 Preparation, culture, cryopreservation and resuscitation method and application of testicular organ
PCT/CN2022/129770 WO2023173763A1 (en) 2022-03-18 2022-11-04 Testis organoid preparation, culture and cryopreservation resuscitation methods and application
ZA2023/02871A ZA202302871B (en) 2022-03-18 2023-02-27 Method for preparing, culturing, cryopreserving and reviving testicular organoids and the application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210271380.7A CN114908032B (en) 2022-03-18 2022-03-18 Preparation, culture, cryopreservation and resuscitation method and application of testicular organ

Publications (2)

Publication Number Publication Date
CN114908032A CN114908032A (en) 2022-08-16
CN114908032B true CN114908032B (en) 2024-01-09

Family

ID=82763732

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210271380.7A Active CN114908032B (en) 2022-03-18 2022-03-18 Preparation, culture, cryopreservation and resuscitation method and application of testicular organ

Country Status (3)

Country Link
CN (1) CN114908032B (en)
WO (1) WO2023173763A1 (en)
ZA (1) ZA202302871B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114908032B (en) * 2022-03-18 2024-01-09 暨南大学 Preparation, culture, cryopreservation and resuscitation method and application of testicular organ

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961822A (en) * 2021-02-25 2021-06-15 南方医科大学 Testis organoid and construction method and application thereof
WO2021113924A1 (en) * 2019-12-12 2021-06-17 The Walter And Eliza Hall Institute Of Medical Research Organoid cultures
CN114181891A (en) * 2021-12-03 2022-03-15 中国农业大学 Efficient culture method of mouse testicular organoid

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016015158A1 (en) * 2014-07-30 2016-02-04 University Health Network Organoids for drug screening and personalized medicine
US10993433B2 (en) * 2015-10-15 2021-05-04 Wake Forest University Health Sciences Method of producing in vitro testicular constructs and uses thereof
GB201611982D0 (en) * 2016-07-11 2016-08-24 Cellesce Ltd Cell culture
CN114908032B (en) * 2022-03-18 2024-01-09 暨南大学 Preparation, culture, cryopreservation and resuscitation method and application of testicular organ

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021113924A1 (en) * 2019-12-12 2021-06-17 The Walter And Eliza Hall Institute Of Medical Research Organoid cultures
CN112961822A (en) * 2021-02-25 2021-06-15 南方医科大学 Testis organoid and construction method and application thereof
CN114181891A (en) * 2021-12-03 2022-03-15 中国农业大学 Efficient culture method of mouse testicular organoid

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
3D 类器官模型的研究进展及其在化学品毒理学评价中的应用展望;刘薇 等;《生态毒理学报》;第16卷(第4期);第32-42页 *
ESTABLISHMENT AND CHARACTERIZATION OF A PORCINE TESTIS ORGANOID CULTURE SYSTEM;TAT-CHUAN CHAM;《https://harvest.usak.ca/handle/10388/13281》;第1章 *
Maxwell E Edmonds et al.Testicular organoid formation is a property of immature somatic cells, which self-assemble and exhibit long-term hormone-responsive endocrine function.《Biofabrication》.2020,第12卷(第4期),第1页摘要，第2-5节. *
Testicular organoid formation is a property of immature somatic cells, which self-assemble and exhibit long-term hormone-responsive endocrine function;Maxwell E Edmonds et al;《Biofabrication》;第12卷(第4期);第1页摘要，第2-5节 *

Also Published As

Publication number Publication date
CN114908032A (en) 2022-08-16
WO2023173763A1 (en) 2023-09-21
ZA202302871B (en) 2023-09-27

Similar Documents

Publication Publication Date Title
US9206393B2 (en) Isolated adult pluripotent stem cells and methods for isolating and cultivating thereof
CN103409368B (en) The cardiac muscle cell of stem cell and embryonic origin and the cardiac muscle cell's who infers purification process
CN114317443B (en) Breast cancer organoid culture solution, and culture reagent combination and culture method thereof
CN108103013B (en) Enzyme digestion method primary culture and identification method for smooth muscle cells of esophageal-gastric junction
CN101330935A (en) Isolation and cultivation of stem/progenitor cells from the amniotic membrane of umbilical cord and uses of cells differentiated therefrom
CN114292816B (en) Lung cancer organoid culture solution, and culture reagent combination and culture method thereof
CN114058592A (en) Immortalized yak rumen epithelial cell line and construction method thereof
WO2018086319A1 (en) Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof
CN112961822B (en) Testis organoid and construction method and application thereof
CN114908032B (en) Preparation, culture, cryopreservation and resuscitation method and application of testicular organ
CN114107173A (en) Vascularized pancreatic islet micro-organ and construction method thereof
CN114891734A (en) Immortalized yak rumen fibroblast line and construction and application thereof
CN114807034A (en) Preparation method of Muller cells derived from human pluripotent stem cells
CN105483078A (en) Isolation and primary culture methods of chicken small intestinal epithelial cells
US20070086986A1 (en) Preparation of three-dimensional mammalian ovarian follicular cell and ovarin follicle culture systems in a biocompatible matrix
CN110628709A (en) Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes
CN109628402A (en) A kind of primary organoid cultural method of gastric cancer tumor
Salem et al. Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis
CN111575227B (en) Method for establishing human-derived diabetic cardiomyopathy model
CN106244522A (en) A kind of stem cell cultivating system and cultural method thereof
CN109153963B (en) Method for changing cell culture in adhesion state
CN116024158A (en) Preparation method of 3D model for in-vitro construction of mother-child interface of endometrium organoid
CN115786247A (en) Serum-free culture medium and application thereof in maintaining hair follicle activity, hair maintenance and transplantation
CN108774630A (en) A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell
CN114276986A (en) Method for separating and purifying buffalo primary myoblasts and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant