Method for constructing organoid

Abstract

The invention discloses a method for constructing an organoid. The method comprises the following steps: (1) carrying out hanging drop culture on testicular supporting cells and primordial germ cell-like cells in an organoid culture solution according to a quantity ratio of 1: (2-6) to obtain cell aggregates; (2) culturing the cell aggregate in a gas-liquid phase culture system to obtain an organoid; wherein the content of double positive cells in the primordial germ cell-like cells is greater than 40%, the gas-liquid phase culture system comprises a solid culture medium and an organoid culture solution, and the solid culture medium is formed by the organoid culture solution and agarose gel; wherein the organoid culture solution is prepared from human bone morphogenetic protein-4, a basic fibroblast growth factor, a glial cell-derived neurotrophic factor and retinoic acid. The organoid obtained by the method has a high germ cell population proportion.

Description

Technical Field

[0001] The present invention relates to a method for constructing an organoid, and in particular to a method for constructing a testicular organoid. Background Art

[0002] With the advancement of global urbanization, the number of infertile families has further increased, which has aroused widespread concern in society. The construction of organoids in vitro provides a basis for the study of the above diseases.

[0003] CN106047800B discloses a special culture medium for directional induction and differentiation of pig pluripotent stem cells into male germ cells, including EpiLC induction medium, PGCLC induction medium and SSCLC induction medium. EpiLC induction medium contains bFGF and ActivinA. PGCLC induction medium contains BMP4, BMP8a, SCF, EGF and LIF. SSCLC induction medium contains testosterone, RA and GDNF. It does not involve the construction of organoids.

[0004] CN113881623A discloses a method for forming eggs by in vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetics. Eggs are treated with strontium ions and cytochalasin D to obtain parthenogenetic activated embryos and establish parthenogenetic embryonic stem cell lines, which are further differentiated to form primordial germ cell-like cells, and the primordial germ cell-like cells are aggregated with mouse female embryonic gonadal cells and transplanted into combined immunodeficient mice to obtain eggs. This method can obtain eggs. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for constructing an organoid, wherein the method obtains an organoid having a high proportion of germ cell populations. Further, the organoid obtained by the method has a high proportion of cells that are double positive for the supporting cell marker Sox9 and the germ cell marker Plzf.

[0006] The present invention provides a method for constructing an organoid, comprising the following steps:

[0007] (1) Sertoli cells and primordial germ cell-like cells are cultured in hanging drops in an organoid culture medium at a ratio of 1:(2-6) to obtain cell aggregates;

[0008] (2) culturing the cell aggregates in a gas-liquid phase culture system to obtain organoids;

[0009] Wherein, the content of double-positive cells in the primordial germ cell-like cells is greater than 40%, and the gas-liquid phase culture system comprises a solid culture medium and an organoid culture medium, wherein the solid culture medium is formed by an organoid culture medium and an agarose gel;

[0010] The organoid culture medium includes human bone morphogenetic protein-4 (BMP 4), basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF) and retinoic acid (RA).

[0011] According to the method of the present invention, preferably, the testicular supporting cells are from C57 mice, and the double positive cells are cells that are positive for SSEA-1 and CD61 antibodies.

[0012] According to the method of the present invention, preferably, in the organoid culture medium, the concentration of human bone morphogenetic protein-4 (BMP 4) is 20-30 ng / mL, the concentration of basic fibroblast growth factor (bFGF) is 15-25 ng / mL, the concentration of glial cell line-derived neurotrophic factor (GDNF) is 35-45 ng / mL, and the concentration of retinoic acid (RA) is 2-7 nM.

[0013] According to the method of the present invention, preferably, the organoid culture medium also includes G-MEN culture medium, KSR culture medium, penicillin-streptomycin-glutamine (Pen Strep Glutamine), MEM NEAA culture medium, GlutaMAX culture medium and 2-hydroxyethanol.

[0014] The method according to the present invention preferably further comprises the following steps:

[0015] (A) culturing mouse embryonic stem cells to be induced in a fibronectin-coated culture container in the presence of a first induction culture medium for 2.5 to 3.5 days to obtain epiblast-like cells (EpiLCs);

[0016] (B) Inducing and culturing the ectoderm-like cells in a second induction medium to obtain primordial germ cell-like cells (PGCLCs);

[0017] The first induction culture medium includes activin A and basic fibroblast growth factor (FGF2); the second induction culture medium includes human bone morphogenetic protein-4 (BMP 4), epidermal growth factor (EGF) and stem cell factor (SCF).

[0018] According to the method of the present invention, preferably, the concentration of activin A (ActivinA) in the first induction culture medium is 15-25 ng / mL, and the concentration of basic fibroblast growth factor (FGF2) is 7-17 ng / mL; the concentration of human bone morphogenetic protein-4 (BMP 4) in the second induction culture medium is 400-600 ng / mL, the concentration of epidermal growth factor (EGF) is 40-60 ng / mL, and the concentration of stem cell factor (SCF) is 90-110 ng / mL.

[0019] According to the method of the present invention, preferably, the first induction culture medium also includes N2 cell culture additives, B27 cell culture additives, KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and DMEM / F12 culture medium; the second induction culture medium also includes KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and G-MEN culture medium.

[0020] According to the method of the present invention, preferably, in step (B), the culturing time is 6 to 10 days.

[0021] According to the method of the present invention, preferably, the following steps are also included: culturing mouse embryonic stem cells in a gelatin-coated culture container in the presence of a stem cell culture medium to obtain mouse embryonic stem cells to be induced; in the stem cell culture medium, the concentration of leukemia inhibitory factor (LIF) is 900-1100 U / mL.

[0022] According to the method of the present invention, preferably, the mouse embryonic stem cells are derived from the R1S / E embryonic stem cell line derived from ICR mice, and the stem cell culture medium further comprises DMEM / F12 culture medium, fetal bovine serum, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and Sodium Prviate.

[0023] The testicular organoids constructed by the method of the present invention have a high proportion of germ cell populations and have spermatogenic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of protein immunohistochemistry experiments on organoids formed at different culture times and protein extracts from mouse testes at different days after birth.

[0025] Figure 2 The figure is a bar graph showing the expression levels of Plzf in organoids formed at different culture times and in protein extracts from mouse testes at different days after birth.

[0026] Figure 3 The bar graph shows the expression levels of Sox9 in organoids formed at different culture times and in testicular protein extracts of mice at different days after birth.

[0027] in, Figure 1-3 Here, d7 represents organoids formed after 7 days of culture; d14 represents organoids formed after 14 days of culture; d21 represents organoids formed after 21 days of culture; p6 represents the testicular protein extract of mice born on the 6th day; p10 represents the testicular protein extract of mice born on the 10th day; and p14 represents the testicular protein extract of mice born on the 14th day.

[0028] Figure 4 This is a testicular section of an azoospermia model mouse.

[0029] Figure 5 This is a testicular section of a normal adult male C57 mouse. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0031] The method for constructing an organoid of the present invention comprises the following steps: a step of obtaining a cell aggregate and a step of obtaining an organoid. In certain embodiments, it also comprises one or more steps of obtaining a testicular supporting cell, a step of obtaining a mouse embryonic stem cell to be induced, a step of obtaining an ectoderm-like cell, and a step of obtaining a primordial germ cell-like cell.

[0032] <Steps for obtaining Sertoli cells>

[0033] The testicular support cells of the present invention are derived from adult mice. According to one embodiment of the present invention, the testicular support cells are derived from C57 black mice. Specifically, they can be GFP-C57 black mice.

[0034] Specifically, the method comprises the following steps: collecting the seminiferous tubules of mice, and then digesting the seminiferous tubules to obtain flocculent tissues. Using culture medium to terminate the digestion, and then centrifuging. After the centrifugation, the supernatant is discarded, and then the precipitate is resuspended in fresh culture medium, and evenly inoculated on a culture container, and according to the principle of differential adhesion, the testicular supporting cells are obtained. The culture container can be a culture dish.

[0035] The mice can be sacrificed, the tunica albuginea removed, and the seminiferous tubules collected in PBS buffer.

[0036] Digestion can be performed by combining mechanical digestion with chemical digestion. Chemical digestion can also be performed. Collagenase and trypsin can be used for digestion. In some embodiments, the seminiferous tubules are digested by a combination of collagenase and 0.25% trypsin in a ratio of 1:1 for 2 to 8 minutes.

[0037] The centrifugal temperature may be 1 to 8° C., preferably 3 to 6° C. The centrifugal speed may be 500 to 2000 rpm, preferably 800 to 1500 rpm. The centrifugal time may be 2 to 10 min, preferably 3 to 6 min.

[0038] The culture medium includes DMEM basic culture medium, fetal bovine serum and penicillin-streptomycin.

[0039] The DMEM basic culture medium may be 38 to 50 parts by volume, preferably 42 to 46 parts by volume. The fetal bovine serum may be 2 to 8 parts by volume, preferably 4 to 6 parts by volume. The penicillin-streptomycin may be 0.1 to 0.8 parts by volume, preferably 0.3 to 0.6 parts by volume.

[0040] <Steps for obtaining mouse embryonic stem cells to be induced>

[0041] Mouse embryonic stem cells (ESC) are cultured until they form a three-dimensional dome-like cell clone morphology with neat edges, thereby obtaining mouse embryonic stem cells to be induced.

[0042] Mouse embryonic stem cell culture can be derived from the R1S / E cell line derived from ICR mice. The R1S / E cell line was purchased from the Institute of Zoology, Chinese Academy of Sciences.

[0043] Mouse embryonic stem cells are cultured in a gelatin-coated culture container containing a stem cell culture medium. The culture container may be a culture dish.

[0044] The stem cell culture medium contains leukemia inhibitory factor. Leukemia inhibitory factor can also be represented by LIF. The concentration of leukemia inhibitory factor can be 900-1100 U / mL, preferably 950-1050 U / mL, and more preferably 1000-1050 U / mL.

[0045] The stem cell culture medium also contains DMEM / F12 culture medium, fetal bovine serum, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and Sodium Prviate. In certain embodiments, the stem cell culture medium consists of DMEM / F12 culture medium, fetal bovine serum, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium, Sodium Prviate and leukemia inhibitory factor.

[0046] The volume of DMEM / F12 culture medium may be 35 to 55 parts by volume, preferably 40 to 50 parts by volume, and more preferably 44.5 to 46 parts by volume.

[0047] The amount of fetal bovine serum may be 2 to 8 parts by volume, preferably 3 to 6 parts by volume, and more preferably 4 to 5 parts by volume.

[0048] The non-essential amino acids may be present in an amount of 0.3 to 0.8 parts by volume, preferably 0.4 to 0.7 parts by volume, and more preferably 0.5 to 0.6 parts by volume.

[0049] The volume of β-mercaptoethanol may be 0.005 to 0.04 parts by volume, preferably 0.01 to 0.03 parts by volume, and more preferably 0.02 to 0.03 parts by volume.

[0050] The amount of penicillin-streptomycin may be 0.3 to 0.8 parts by volume; preferably 0.4 to 0.7 parts by volume; more preferably 0.5 to 0.6 parts by volume.

[0051] The volume of GlutaMAX culture medium may be 0.3 to 0.8 parts by volume; preferably 0.4 to 0.7 parts by volume; more preferably 0.5 to 0.6 parts by volume.

[0052] The amount of Sodium Prviate may be 0.3 to 0.8 parts by volume, preferably 0.4 to 0.7 parts by volume, and more preferably 0.5 to 0.6 parts by volume.

[0053] The culture temperature may be 35 to 40°C, preferably 36 to 39°C, and more preferably 37 to 38°C.

[0054] In the culture environment, CO 2 The content may be 2-8%, preferably 3-7%, and more preferably 4-5%.

[0055] The seeding density of mouse embryonic stem cells can be 0.5×10 per 60 mm culture dish. 5 ~1.5×10 5 ; preferably 0.8×10 5 ~1.2×10 5 More preferably, 0.9×10 5 ~1.1×10 5 indivual.

[0056] <Steps for Obtaining Ectoderm-like Cells>

[0057] The mouse embryonic stem cells to be induced are cultured in a fibronectin-coated culture container in a first induction culture medium for 2.5 to 3.5 days to obtain ectoderm-like cells. According to one embodiment of the present invention, the culture time is 3 days. Ectodermal-like cells can also be represented by EpiLC. The culture container can be a culture dish. The present invention finds that the number of days of culture of mouse embryonic stem cells to be induced and the extracellular matrix have an important influence on the proportion of double-positive cells, and subsequent culture of such ectoderm-like cells can improve the efficiency of double-positive cells in primordial germ cell-like cells and organoids.

[0058] The first induction culture medium contains activin A and basic fibroblast growth factor.

[0059] Activin A can also be represented by Activin A. The concentration of Activin A can be 15 to 25 ng / mL, preferably 18 to 23 ng / mL, and more preferably 20 to 21 ng / mL.

[0060] Basic fibroblast growth factor can also be represented by FGF2. The concentration of basic fibroblast growth factor can be 7-17 ng / mL, preferably 10-15 ng / mL, and more preferably 12-13 ng / mL.

[0061] The first induction culture medium also includes N2 cell culture additives, B27 cell culture additives, KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and DMEM / F12 culture medium. In certain embodiments, the first induction culture medium consists of N2 cell culture additives, B27 cell culture additives, KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium, DMEM / F12 culture medium, activin A and basic fibroblast growth factor.

[0062] The N2 cell culture additive may be 0.1 to 0.5 parts by volume; preferably 0.2 to 0.4 parts by volume; more preferably 0.25 to 0.3 parts by volume.

[0063] The B27 cell culture additive may be 0.2 to 0.8 parts by volume; preferably 0.3 to 0.7 parts by volume; more preferably 0.4 to 0.5 parts by volume.

[0064] KSR may be 0.2 to 0.8 parts by volume, preferably 0.3 to 0.7 parts by volume, and more preferably 0.4 to 0.5 parts by volume.

[0065] The non-essential amino acids may be present in an amount of 0.2 to 0.8 parts by volume, preferably 0.3 to 0.7 parts by volume, and more preferably 0.4 to 0.5 parts by volume.

[0066] The β-mercaptoethanol may be 0.005 to 0.04 parts by volume; preferably 0.01 to 0.03 parts by volume; more preferably 0.02 to 0.03 parts by volume

[0067] The amount of penicillin-streptomycin may be 0.3 to 0.8 parts by volume; preferably 0.4 to 0.7 parts by volume; more preferably 0.5 to 0.6 parts by volume.

[0068] The volume of GlutaMAX culture medium may be 0.3 to 0.8 parts by volume; preferably 0.4 to 0.7 parts by volume; more preferably 0.5 to 0.6 parts by volume.

[0069] The volume of DMEM / F12 culture medium may be 35 to 55 parts by volume, preferably 40 to 50 parts by volume, and more preferably 44.5 to 46 parts by volume.

[0070] The culture temperature may be 35 to 40°C, preferably 36 to 39°C, and more preferably 37 to 38°C.

[0071] In the culture environment, CO 2 The content may be 2-8%, preferably 3-7%, and more preferably 4-5%.

[0072] The seeding density of mouse embryonic stem cells to be induced can be 0.5×10 per 35 mm culture dish. 4 ~1.5×10 4 ; preferably 0.8×10 4 ~1.2×10 4 More preferably, 0.9×10 4 ~1.1×10 4 indivual.

[0073] <Steps for Obtaining Primordial Germ Cell-Like Cells>

[0074] The ectoderm-like cells are induced and cultured in the second induction culture medium to obtain primordial germ cell-like cells (PGCLCs).

[0075] The culture time may be 6 to 10 days; preferably 8 to 9 days.

[0076] The second induction medium includes human bone morphogenetic protein-4, epidermal growth factor and stem cell factor.

[0077] Human bone morphogenetic protein-4 can also be represented by BMP 4. The concentration of human bone morphogenetic protein-4 can be 400-600 ng / mL, preferably 450-550 ng / mL, and more preferably 500-520 ng / mL.

[0078] Epidermal growth factor can also be represented by EGF. The concentration of epidermal growth factor can be 40-60 ng / mL, preferably 45-55 ng / mL, and more preferably 50-52 ng / mL.

[0079] Stem cell factor can be represented by SCF. The concentration of stem cell factor can be 90 to 110 ng / mL, preferably 95 to 105 ng / mL, and more preferably 100 to 102 ng / mL.

[0080] The second induction culture medium also includes KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and G-MEN culture medium. In certain embodiments, the second induction culture medium consists of KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium, G-MEN culture medium, human bone morphogenetic protein-4, epidermal growth factor and stem cell factor.

[0081] The amount of KSR may be 0.5 to 3 parts by volume, preferably 0.8 to 2 parts by volume, and more preferably 1 to 1.5 parts by volume.

[0082] The non-essential amino acids may be present in an amount of 0.05 to 0.3 parts by volume; preferably 0.08 to 0.2 parts by volume; and more preferably 0.1 to 0.15 parts by volume.

[0083] The volume of β-mercaptoethanol may be 0.005 to 0.05 parts by volume, preferably 0.01 to 0.04 parts by volume, and more preferably 0.02 to 0.03 parts by volume.

[0084] The amount of penicillin-streptomycin may be 0.05 to 0.4 parts by volume; preferably 0.08 to 0.3 parts by volume; more preferably 0.1 to 0.2 parts by volume.

[0085] The volume of GlutaMAX culture medium may be 0.05 to 0.4 parts by volume; preferably 0.08 to 0.3 parts by volume; more preferably 0.1 to 0.2 parts by volume.

[0086] The volume of G-MEN medium can be 5 to 15 parts by volume, preferably 7 to 12 parts by volume, and more preferably 8.5 to 10 parts by volume.

[0087] The culture temperature may be 35 to 40°C, preferably 36 to 39°C, and more preferably 37 to 38°C.

[0088] In the culture environment, CO 2 The content may be 2-8%, preferably 3-7%, and more preferably 4-5%.

[0089] The seeding density of ectoderm-like cells can be 0.5×10 per 50ul culture drop. 4 ~1.5×10 4 ; preferably 0.8×10 4 ~1.2×10 4 More preferably, 0.9×10 4 ~1.1×10 4 indivual.

[0090] <Steps for obtaining cell aggregates and organoids>

[0091] The testicular support cells and primordial germ cell-like cells are cultured in hanging drops in an organoid culture medium to obtain cell aggregates. The present invention finds that the organoids formed by culturing the testicular support cells and primordial germ cell-like cells have a higher double-positive efficiency. If the testicular support cells are replaced with testicular interstitial cells, the performance of the organoids obtained is inferior to that of the present invention.

[0092] The testicular support cells can be derived from adult mice. According to one embodiment of the present invention, the testicular support cells are derived from C57 black mice. The method for obtaining the testicular support cells is as described above and will not be repeated here.

[0093] The content of double positive cells in primordial germ cell-like cells is greater than 40%; preferably, greater than 45%; more preferably, greater than 48%. Double positive cells refer to cells that are positive for both SSEA-1 and CD61 antibodies. The method for obtaining primordial germ cell-like cells is specifically described above and will not be repeated here.

[0094] The ratio of the number of testicular supporting cells to the number of primordial germ cell-like cells is 1:(2-6); preferably 1:(3-5); more preferably 1:(4-5).

[0095] The culture time may be 1 to 4 days, preferably 2 to 3 days.

[0096] The cell aggregates are cultured in a gas-liquid phase culture system to obtain organoids. The obtained organoids are testicular organoids. Cultivation in a 3D culture system is conducive to the growth of organoids.

[0097] The gas-liquid phase culture system includes a solid culture medium and an organoid culture solution, wherein the solid culture medium is formed by the organoid culture solution and agarose gel. Specifically, the solid culture medium is placed in a culture container, and then the organoid culture solution is added below the solid culture medium to form a gas-liquid phase culture system. The liquid level of the organoid culture solution is lower than that of the solid culture medium. The organoid culture solution can be mixed with the hot agarose gel and then cooled to obtain a solid culture medium.

[0098] The culture time may be 16 to 25 days, preferably 20 to 23 days, and more preferably 21 to 22 days.

[0099] During the culture process, the need to replace the culture medium is determined based on the swelling of the organoids and the color of the solid culture medium.

[0100] The organoid culture medium contains human bone morphogenetic protein-4, basic fibroblast growth factor, glial cell line-derived neurotrophic factor and retinoic acid.

[0101] Human bone morphogenetic protein-4 can be represented by BMP 4. The concentration of human bone morphogenetic protein-4 can be 20 to 30 ng / mL, preferably 22 to 28 ng / mL, and more preferably 25 to 26 ng / mL.

[0102] Basic fibroblast growth factor can be represented by bFGF. The concentration of basic fibroblast growth factor can be 15 to 25 ng / mL, preferably 18 to 23 ng / mL, and more preferably 20 to 21 ng / mL.

[0103] Glial cell line-derived neurotrophic factor can be represented by GDNF. The concentration of glial cell line-derived neurotrophic factor can be 35 to 45 ng / mL, preferably 38 to 43 ng / mL, and more preferably 40 to 41 ng / mL.

[0104] Retinoic acid can be represented by RA. The concentration of retinoic acid can be 2 to 7 nM, preferably 3 to 6 nM, and more preferably 4 to 5 nM.

[0105] The organoid culture medium also contains G-MEN medium, KSR medium, penicillin-streptomycin-glutamine, MEMNEAA medium, GlutaMAX medium and 2-hydroxyethanol. In certain embodiments, the organoid culture medium is composed of G-MEN medium, KSR medium, penicillin-streptomycin-glutamine, MEM NEAA medium, GlutaMAX medium, 2-hydroxyethanol, human bone morphogenetic protein-4, basic fibroblast growth factor, glial cell-derived neurotrophic factor and retinoic acid.

[0106] The volume of the G-MEN medium may be 35 to 50 parts by volume, preferably 38 to 45 parts by volume, and more preferably 41 to 42 parts by volume.

[0107] The volume of KSR medium can be 4 to 12 parts by volume; preferably 6 to 10 parts by volume; more preferably 7.5 to 8 parts by volume.

[0108] Penicillin-streptomycin-glutamine can be represented by Pen Strep Glutamine. The amount of penicillin-streptomycin-glutamine can be 0.1 to 0.8 parts by volume, preferably 0.3 to 0.7 parts by volume, and more preferably 0.5 to 0.6 parts by volume.

[0109] The MEM NEAA culture medium may be 0.1 to 0.8 parts by volume; preferably 0.3 to 0.7 parts by volume; more preferably 0.5 to 0.6 parts by volume.

[0110] The volume of GlutaMAX culture medium may be 0.1 to 0.8 parts by volume; preferably 0.3 to 0.7 parts by volume; more preferably 0.5 to 0.6 parts by volume.

[0111] The amount of 2-hydroxyethanol may be 0.05 to 0.4 parts by volume, preferably 0.08 to 0.3 parts by volume, and more preferably 0.1 to 0.2 parts by volume.

[0112] Example 1

[0113] GFP-C57 black mice were killed. The white membrane of the killed mice was removed under a microscope, and then the seminiferous tubules were collected in a clean PBS buffer. The seminiferous tubules were digested with collagenase and 0.25% trypsin in a 1:1 ratio for 5 minutes to obtain flocculent tissue. The digestion was terminated with culture medium, and then centrifuged at 4°C and 1000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the precipitate was resuspended with fresh culture medium and evenly inoculated on the culture dish. According to the principle of differential adhesion, testicular supporting cells were obtained.

[0114] The composition of the culture medium is: 44.5 volumes of DMEM basic culture medium, 5 volumes of fetal bovine serum, and 0.5 volumes of penicillin-streptomycin.

[0115] Example 2

[0116] The R1S / E embryonic stem cell line derived from ICR mice was cultured in gelatin-coated culture dishes in stem cell culture medium at 37°C and 5% CO. 2 The cells were cultured under the conditions of 1 × 10 cells / 60 mm culture dish until a three-dimensional dome-shaped cell clone with neat edges was formed to obtain the mouse embryonic stem cells to be induced. 5 indivual.

[0117] The stem cell culture medium consists of 44.5 volumes of DMEM / F12 culture medium, 5 volumes of fetal bovine serum, 0.5 volumes of non-essential amino acids, 0.02 volumes of β-mercaptoethanol, 0.5 volumes of penicillin-streptomycin, 0.5 volumes of GlutaMAX medium, 0.5 volumes of Sodium Prviate and leukemia inhibitory factor (LIF). The concentration of leukemia inhibitory factor is 1000U / mL.

[0118] Example 3

[0119] (A) The mouse embryonic stem cells to be induced obtained in Example 2 were placed in a 30 mm culture dish coated with fibronectin in the first induction medium at 37°C and 5% CO. 2 The inoculation density of mouse embryonic stem cells to be induced was 1×10 per 35 mm culture dish. 4 indivual.

[0120] The first induction culture medium consists of 0.25 volume parts of N2 cell culture additive, 0.5 volume parts of B27 cell culture additive, 0.5 volume parts of KSR, 0.5 volume parts of non-essential amino acids, 0.02 volume parts of β-mercaptoethanol, 0.5 volume parts of penicillin-streptomycin, 0.5 volume parts of GlutaMAX culture medium, 44.5 volume parts of DMEM / F12 culture medium, Activin A and basic fibroblast growth factor (FGF2). The concentration of Activin A is 20 ng / mL. The concentration of basic fibroblast growth factor is 12 ng / mL.

[0121] (B) Ectoderm-like cells were cultured in the second induction medium at 37°C and 5% CO 2 The primordial germ cell-like cells (PGCLCs) were obtained by culturing for 8 days under the conditions of 5% paraformaldehyde and 1% paraformaldehyde. The seeding density of ectoderm-like cells was 1×10 per 50ul culture drop. 4 indivual.

[0122] The second induction culture medium consists of 1 volume part KSR, 0.1 volume part non-essential amino acids, 0.02 volume part β-mercaptoethanol, 0.1 volume part penicillin-streptomycin, 0.1 volume part GlutaMAX medium, 8.5 volume parts G-MEN medium, human bone morphogenetic protein-4 (BMP4), epidermal growth factor (EGF) and stem cell factor (SCF). The concentration of human bone morphogenetic protein-4 is 500ng / mL. The concentration of epidermal growth factor is 50ng / mL. The concentration of stem cell factor is 100ng / mL.

[0123] Comparative Example 1

[0124] Except that the culture time in step (A) is 2 days, the rest is the same as Example 3.

[0125] Comparative Example 2

[0126] Except that in step (A), the culture dish is coated with laminin, the rest is the same as Example 3.

[0127] Example 4

[0128] (1) The Sertoli cells obtained in Example 1 and the primordial germ cell-like cells obtained in Example 3 were cultured in an organoid culture medium in a hanging drop manner at a ratio of 1:4 for 2 days to obtain cell aggregates.

[0129] (2) The organoid culture medium is mixed with 3% hot agarose gel and then cooled to obtain a solid culture medium. The solid culture medium is placed in a 24-well plate, and then 300 μL of organoid culture medium is added below the solid culture medium. The liquid level of the organoid culture medium is lower than the solid culture medium, forming a gas-liquid phase culture system.

[0130] The cell aggregates were placed on a solid culture medium for 3D culture for 21 days to obtain organoids. During the culture process, the bulge of the organoids and the color of the solid culture medium were used to determine whether the culture medium needed to be replaced.

[0131] The organoid culture medium consists of 41 volumes of G-MEN medium, 7.5 volumes of KSR medium, 0.5 volumes of penicillin-streptomycin-glutamine (Pen Strep Glutamine), 0.5 volumes of MEM NEAA medium, 0.5 volumes of GlutaMAX medium, 0.1 volumes of 2-hydroxyethanol, human bone morphogenetic protein-4 (BMP 4), basic fibroblast growth factor (bFGF), glial cell-derived neurotrophic factor (GDNF) and retinoic acid (RA). The concentration of human bone morphogenetic protein-4 is 25 ng / mL. The concentration of basic fibroblast growth factor is 20 ng / mL. The concentration of glial cell-derived neurotrophic factor is 40 ng / mL. The concentration of retinoic acid is 5 nM.

[0132] Experimental Example 1

[0133] The double positive cell population in the primordial germ cell-like cells was labeled by two antibodies, SSEA-1 and CD61. The unlabeled protein was set as a blank control group, a 7-AAD-labeled dead-live control group, and an experimental group labeled with PE-coupled SSEA-1 and FITC-coupled CD61 antibodies. The primordial germ cell-like cells formed at different times of culturing the ectoderm-like cells in Example 3 and Comparative Examples 1-2 were detected. The double positive efficiency of primordial germ cell-like cells is shown in Table 1.

[0134] Table 1

[0135]

[0136] Note: Double-positive efficiency is the percentage of double-positive labeled cell population minus compensation.

[0137] As shown in Table 1, after 8 days of culturing the ectoderm-like cells in Example 3, the double-positive efficiency of the primordial germ cell-like cells obtained was the highest, reaching 48%. After 6 days of culturing the ectoderm-like cells in Comparative Example 1, the double-positive efficiency of the primordial germ cell-like cells obtained was the highest, reaching 18%. After 6 days of culturing the ectoderm-like cells in Comparative Example 2, the double-positive efficiency of the primordial germ cell-like cells obtained was the highest, reaching 12.3%. The double-positive efficiency of the primordial germ cell-like cells obtained in Example 3 was significantly higher than that in Comparative Examples 1 and 2.

[0138] Experimental Example 2

[0139] Take the primordial germ cell-like cells with the highest double positive efficiency in Example 3 and Comparative Example 1, that is, the primordial germ cell-like cells obtained by culturing ectoderm-like cells for 8 days in Example 3 and the primordial germ cell-like cells obtained by culturing ectoderm-like cells for 6 days in Comparative Example 1, and perform immunofluorescence comparative analysis. Blimp1 was labeled with red fluorescence, c-kit was labeled with green fluorescence, and Prdm14 was labeled with yellow fluorescence. During the experiment, the primordial germ cell-like cells needed to be transferred to an adhesion slide by cell swinging operation, which destroyed their original morphology. It can be seen from the experimental results that the primordial germ cell-like cells obtained by culturing for 6 days in Comparative Example 1 expressed c-kit protein, indicating that the cell group was differentiating in the reproductive direction, and the expression of the primordial germ cell marker proteins Blimp1 and Prdm14 was very low. The primordial germ cell-like cells obtained by culturing for 8 days in Example 3 expressed significantly more cells than the primordial germ cell-like cells obtained by culturing for 6 days in Comparative Example 1, which is consistent with the results obtained in Experimental Example 1.

[0140] Experimental Example 3

[0141] The organoids formed by the cell aggregate culture on the 7th, 14th and 21st days in Example 4 and the extracts of mouse testicular proteins on the 6th, 10th and 14th days after birth were respectively used for protein immunoassay. Figure 1-3 It can be seen that the supporting cell protein Sox9 gradually increased with the increase of culture time and reached a peak on the 21st day, and the germ cell marker Plzf was not expressed significantly in the entire organoid culture. Compared with the mouse testicular protein extract, it was found that the protein expression of the organoid formed after 21 days of culture was similar to that of the mouse testicular protein extract on the 6th day after birth, while the expression of Sox9 and Plzf in the organoid formed on the 14th and 21st days of culture was low. The organoid constructed by the present invention is similar to the testicular development of normal mouse pups on the 6th day.

[0142] Experimental Example 4

[0143] The proportion of germ cell populations in organoids obtained at different culture times was identified by flow cytometry. The supporting cell marker protein Sox9 labeled with red fluorescence and the germ cell marker protein Plzf labeled with green fluorescence were used as experimental groups, and the blank control group was an organoid with its own green fluorescence. The efficiency of double positive cells is shown in Table 2. When the experimental group was cultured for 21 days, the cell population migrated upward as a whole, indicating that the cells were undergoing differentiation. On the 21st day of culture in the blank control group, the proportion of green fluorescent cells decreased, indicating that the cells began to apoptosis.

[0144] Table 2

[0145]

[0146] Note: Double-positive efficiency is the percentage of double-positive labeled cell population minus compensation.

[0147] Experimental Example 5

[0148] Azoospermia model mice were obtained by injecting busulfan into adult male C57 mice.

[0149] The azoospermia model mice obtained by the above method were raised for two weeks and then killed. The testicular microenvironment (such as Figure 4 shown). Figure 5 This is a testicular section of a normal adult male C57 mouse. Figure 5 It can be seen that the cells in the seminiferous tubules of normal adult male C57 mice are tightly arranged, and there are no gaps between the seminiferous tubules. Figure 4 It can be seen that there are fewer cells arranged in the seminiferous tubules of the azoospermia model mice, and larger gaps appear between the seminiferous tubules, which proves that the above method successfully established the azoospermia model mice.

[0150] The organoids formed by culturing the cell aggregates for 14 days in Example 4 were digested into single cell suspensions, which were then injected into the testicles of azoospermia model mice, which were then mated with normal adult black C57 female mice to produce healthy offspring with gray hair.

[0151] The mouse embryonic stem cells used in this experiment were purchased from the R1S / E cell line of the Institute of Zoology, Chinese Academy of Sciences. The mouse embryonic stem cells were established from the blastocyst of white ICR mice, and the supporting cells used in organoid culture were taken from the testicles of GFP-C57 black mice. Therefore, only the primordial germ cell-like cells induced by mouse embryonic stem cells came from ICR mice, and the mice obtained by mating had gray hair, which indicates that the organoids of the present invention have a spermatogenic effect.

[0152] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. A method for constructing an organoid, characterized in that: The steps include: (1) Sertoli cells and primordial germ cell-like cells are cultured in hanging drops in an organoid culture medium at a ratio of 1:(2-6) to obtain cell aggregates; (2) culturing the cell aggregates in a gas-liquid phase culture system to obtain organoids; Wherein, the content of double-positive cells in the primordial germ cell-like cells is greater than 40%, and the gas-liquid phase culture system comprises a solid culture medium and an organoid culture medium, wherein the solid culture medium is formed by an organoid culture medium and an agarose gel; The organoid culture medium includes human bone morphogenetic protein-4 (BMP 4), basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF) and retinoic acid (RA).

2. The method according to claim 1, characterized in that The testicular supporting cells are from C57 mice, and the double positive cells are cells that are positive for SSEA-1 and CD61 antibodies.

3. The method according to claim 1, characterized in that In the organoid culture medium, the concentration of human bone morphogenetic protein-4 (BMP 4) is 20-30 ng / mL, the concentration of basic fibroblast growth factor (bFGF) is 15-25 ng / mL, the concentration of glial cell line-derived neurotrophic factor (GDNF) is 35-45 ng / mL, and the concentration of retinoic acid (RA) is 2-7 nM.

4. The method according to claim 1, characterized in that: The organoid culture medium also includes G-MEN culture medium, KSR culture medium, penicillin-streptomycin-glutamine (Pen Strep Glutamine), MEM NEAA culture medium, GlutaMAX culture medium and 2-hydroxyethanol.

5. The method according to claim 1, characterized in that The following steps are also included: (A) culturing mouse embryonic stem cells to be induced in a fibronectin-coated culture container in the presence of a first induction culture medium for 2.5 to 3.5 days to obtain epiblast-like cells (EpiLCs); (B) Inducing and culturing the ectoderm-like cells in a second induction medium to obtain primordial germ cell-like cells (PGCLCs); The first induction culture medium includes activin A and basic fibroblast growth factor (FGF2); the second induction culture medium includes human bone morphogenetic protein-4 (BMP 4), epidermal growth factor (EGF) and stem cell factor (SCF).

6. The method according to claim 5, characterized in that The concentration of activin A (ActivinA) in the first induction culture medium is 15-25 ng / mL, and the concentration of basic fibroblast growth factor (FGF2) is 7-17 ng / mL; the concentration of human bone morphogenetic protein-4 (BMP 4) in the second induction culture medium is 400-600 ng / mL, the concentration of epidermal growth factor (EGF) is 40-60 ng / mL, and the concentration of stem cell factor (SCF) is 90-110 ng / mL.

7. The method according to claim 5, characterized in that The first induction culture medium also includes N2 cell culture additives, B27 cell culture additives, KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and DMEM / F12 culture medium; the second induction culture medium also includes KSR, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and G-MEN culture medium.

8. The method according to claim 5, characterized in that In step (B), the culture time is 6 to 10 days.

9. The method according to claim 5, characterized in that The method also includes the following steps: culturing mouse embryonic stem cells in a gelatin-coated culture container in the presence of a stem cell culture medium to obtain mouse embryonic stem cells to be induced; in the stem cell culture medium, the concentration of leukemia inhibitory factor (LIF) is 900-1100 U / mL.

10. The method according to claim 9, characterized in that The mouse embryonic stem cells are derived from the R1S / E embryonic stem cell line derived from ICR mice. The stem cell culture medium also contains DMEM / F12 culture medium, fetal bovine serum, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, GlutaMAX culture medium and Sodium Prviate.

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