Functional bovine mammary gland organoid and culture method and application thereof

Abstract

The present application belongs to the technical field of organoid culture, and particularly relates to a functional bovine mammary gland organoid as well as a culture method and application thereof. The present application overexpresses BCL2 a gene in bovine embryonic stem cells, improves early cluster culture cell cluster cluster activity and / or cell cluster survival rate; bovine embryonic stem cells overexpressing BCL2 a gene are subjected to 3D culture to obtain an organoid cell cluster; 60% Matrigel is used as a supporting matrix material to provide a 3D culture environment for the organoid cell cluster, which can not only make the organoid cell cluster sprout, but also control the elongation speed of the organoid cell cluster, form a tubular structure similar to mammary ducts, and obtain a bovine mammary gland organoid with a high mammary gland differentiation rate. The obtained bovine mammary gland organoid has the function of inducing lactation, and provides a reliable model for analyzing the mammary gland development process of dairy cattle and improving lactation of dairy cattle.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically relating to a functional bovine mammary gland organoid, its culture method, and its application. Background Technology

[0002] Fresh milk from dairy cows and other dairy animals is an important source of nutrition for humans. The mammary gland is the core organ for lactation, but in vivo research on it is costly, time-consuming, and subject to ethical restrictions. Traditional in vitro cultured mammary epithelial cells rapidly lose polarity and tissue specificity in a 2D environment, failing to accurately reflect complex physiological processes such as alveolar structure, cell-cell interactions, and lactation. 3D organoid culture, compared to conventional 2D cell culture, more closely approximates the characteristics of complex in vivo conditions and makes its behavior more similar to natural conditions, reducing the gap between cell culture systems and cell physiology, thereby simulating the physiological functions of some tissues and organs. Therefore, establishing a mammary gland organoid culture system is essential.

[0003] Currently, mammary gland organoid models, including those for mice, have been successfully established. However, as large mammals, the mammary gland structure and characteristics of cows differ significantly from those of mouse models. Furthermore, the cell sources for bovine mammary gland organoid culture are limited, and the cultured organoids lack the ability to induce lactation, making it difficult to reflect the physiological characteristics of mammary gland organoids. Successfully establishing a mammary gland organoid culture system still faces many challenges and difficulties. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a functional bovine mammary gland organoid, its culture method and application. The bovine mammary gland organoid is differentiated from bovine embryonic stem cells, breaking through the growth polarity of organoid cell clusters and realizing 3D culture of bovine mammary gland organoids. The obtained bovine mammary gland organoids have the function of inducing lactation, providing a reliable model for analyzing the mammary gland development process of dairy animals and improving lactation in dairy animals.

[0005] This invention provides a method for culturing bovine mammary gland organoids, comprising the following steps: overexpression BCL2 Genes were used to culture bovine embryonic stem cells in 3D to obtain organoid cell clusters; Add a modified matrix gel to the bottom of the cell culture dish to form a droplet; The organoid cell clusters were added to the gel drop at a density of 1 cell / drop. The cell culture vessel was inverted. After the gel drop solidified, the cell culture vessel was placed upright, mammary gland induction medium was added, and the culture was carried out at 37°C for 10-20 days to obtain bovine mammary gland organoids. The amino acid sequence of the protein encoded by the BCL2 gene is shown in SEQ ID NO:1; The modified matrix gel consists of matrix gel and N2B27 culture medium, with a volume ratio of matrix gel to N2B27 culture medium of 3:2. The mammary gland induction culture medium comprises 10-50 ng / mL EGF, 30-100 ng / mL Fgf2, 70-150 ng / mL LFgf10, 50-100 ng / mL IGF1, 30-100 ng / mL R-spondin-1, 0.5-1.5×heparin, 0.5-1.5×hydrocortisone, 10-50 μg / mL insulin, 10-20% FBS, and the balance being N2B27 medium.

[0006] Preferably, the cell culture vessel is inverted for 40-50 minutes at a temperature of 37°C.

[0007] Preferably, the volume of the adhesive droplet is 25~30μL / drop.

[0008] Preferably, the 3D culture time is 5 to 10 days; the 3D culture method is hanging drop culture.

[0009] Preferably, the hanging drop culture step includes: The first induction solution containing bovine embryonic stem cells was seeded into the inside of the cell culture vessel lid at a rate of 25-30 μL / drop, and then inverted onto the cell culture vessel tank for the first induction culture. The second induction solution was seeded into the inside of the cell culture vessel lid at a rate of 25-30 μL / drop. Bovine embryonic stem cells obtained from the first induction culture were collected, transferred to the second induction solution, and then inverted onto the cell culture vessel tank for the second induction culture to obtain organoid cell clusters. The first induction solution comprises 25-100 ng / mL BMP4, 0.5-2 μM SB431542, 10-50 ng / mL FGF2, 0.5-2 μM LDN, 10-20% FBS and the remainder N2B27 medium; The second induction solution comprises 10-50 ng / mL FGF2, 10-50 ng / mL FGF10, 5-20 μg / mL Wnt3a, 5-20 μg / mL Wnt5a, 5-20 μg / mL Wnt10b, 10-20% FBS and the remainder N2B27 culture medium.

[0010] Preferably, the temperature for the first induction culture is 37°C, and the time is 0 to 5 days; The second induction culture was conducted at a temperature of 37°C for 0 to 5 days.

[0011] Preferably, the concentration of bovine embryonic stem cells in the first induction solution containing bovine embryonic stem cells is 250-300 cells / drop.

[0012] Preferably, during the first and second induction cultures, PBS is added to the cell culture vessel.

[0013] This invention provides a bovine mammary gland organoid, prepared using the culture method described in the above technical solution.

[0014] This invention provides the application of the bovine mammary gland organoids described in the above-mentioned technical solution in one or more of the following: bovine mammary gland development, disease mechanism research for non-disease diagnosis and treatment purposes, and drug testing.

[0015] Beneficial effects: This invention provides a method for culturing bovine mammary gland organoids, and overexpressing [the organoid] in bovine embryonic stem cells. BCL2 Genes that enhance early clustering activity and / or cell cluster survival rate in organoid culture; [and] overexpression of [genes / genes]. BCL2 Bovine embryonic stem cells were cultured in 3D to obtain organoid cell clusters. Using 60% Matrigel as a supporting matrix, a 3D culture environment was provided for the organoid cell clusters, which allowed for budding and controlled elongation, forming tubular structures similar to mammary ducts, thus obtaining bovine mammary gland organoids with a high mammary gland differentiation rate. The obtained bovine mammary gland organoids possess the function of inducing lactation, providing a reliable model for elucidating the mammary gland development process in dairy animals and improving lactation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 For those carrying EGFP BCL2 A schematic diagram of overexpression of lentiviral plasmid; Figure 2 A schematic diagram of 3D clustered hanging drop culture of bovine embryonic stem cells; Figure 3 This is a graph showing the budding and growth of organoid cell clusters cultured in different concentrations of Matrices in Example 2; the scale bar is 100 μm. Figure 4 This is a diagram showing the budding and growth of organoid cell clusters in the control and treatment groups of Example 3; Figure 5 This is a diagram showing the budding and branching of organoid cell clusters in the control and treatment groups of Example 4; the overall icon scale is 100μm, and the magnified icon scale is 50μm. Figure 6This is a staining image of breast organoid marker genes in the treatment group of Example 5; the scale bar is 100 μm. Figure 7 The images show staining of mammary organoids in Example 6 with and without estrus cycle hormone induction; the overall icon size is 200 μm, and the magnified icon size is 100 μm. Detailed Implementation

[0018] This invention provides a method for culturing bovine mammary gland organoids, comprising the following steps: overexpression BCL2 Genes were used to culture bovine embryonic stem cells in 3D to obtain organoid cell clusters; Add a modified matrix gel to the bottom of the cell culture dish to form a droplet; The organoid cell clusters were added to the gel drop at a density of 1 cell / drop. The cell culture vessel was inverted. After the gel drop solidified, the cell culture vessel was placed upright, mammary gland induction medium was added, and the culture was carried out at 37°C for 10-20 days to obtain bovine mammary gland organoids. The amino acid sequence of the protein encoded by the BCL2 gene is shown in SEQ ID NO:1; The modified matrix gel consists of matrix gel and N2B27 culture medium, with a volume ratio of matrix gel to N2B27 culture medium of 3:2. The mammary gland induction culture medium comprises 10-50 ng / mL EGF, 30-100 ng / mL Fgf2, 70-150 ng / mL LFgf10, 50-100 ng / mL IGF1, 30-100 ng / mL R-spondin-1, 0.5-1.5×heparin, 0.5-1.5×hydrocortisone, 10-50 μg / mL insulin, 10-20% FBS, and the balance being N2B27 medium.

[0019] This invention addresses overexpression BCL2 Bovine embryonic stem cells containing genes were cultured in 3D to obtain organoid cell clusters. BCL2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1.

[0020] In one implementation method, bovine embryonic stem cells with a mouse feeder layer are digested and centrifuged, and the stem cells are resuspended in culture medium. The cell suspension is then returned to a culture dish and allowed to stand for 30 minutes to 1 hour, allowing the mouse feeder layer to adhere to the bottom of the dish and the embryonic stem cells to remain suspended in the culture medium. The culture medium is then recovered, and the cells are thoroughly agitated to disperse any remaining cell clumps, thus obtaining bovine embryonic stem cells. This invention provides information on the overexpression of bovine embryonic stem cells... BCL2 There are no strict requirements regarding the genetic method; any conventional method in the field can be used, such as constructing genes with... BCL2The gene was introduced into bovine embryonic stem cells using a lentiviral vector. BCL2 Gene overexpression. This invention does not specify the type of culture medium; conventional embryonic stem cell culture medium, such as N2B27 medium, is sufficient.

[0021] As one implementation method, the present invention described BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:2. In one embodiment, the 3D culture time of the present invention is 5-10 days. In another embodiment, the 3D culture time of the present invention is 7-10 days. The present invention overexpresses [gene name] in bovine embryonic stem cells. BCL2 Genes can improve the clustering activity and / or cell cluster survival rate of early organoid culture.

[0022] SEQ ID NO:1:MAHAGGTGYDNREIVMKYIHYKLSQRGYEWDAGDAGAAPPGAAPAPGILSSQPGRTPAPSRTSPPPPPAAAAGPAPSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLT PFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDSIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKALLSLALVGACITLGAYLGHK.

[0023] SEQ ID NO:2:ATGGCGCACGCGGGGGGAACAGGCTACGATAACCGAGAGATCGTGATGAAGTACATCCACTATAAGCTGTCGCAGCGGGGCTACGAGTGGGATGCCGGAGACGCGGGCGCCGCGCCCCCCGGGGCCGCTCCCGCGCCGGGCATCCTGTCCTCCCAGCCGGGCCGCACACCCGCGCCCTCCAGGACCTCCCCGCCGCCGCCCCCGGCCGCCGCCGCCGGGCCTGCGCCCAGCCCGGTGCCGCCTGTGGTGCACCTGACCCTGCGCCAGGCCGGCGATGACTTCTCTCGGCGCTACCGCCGCGACTTCGCCGAGATGTCCAGTCAGCTGCACCTGACGCCCTTCACCGCGAGGGGACGCTTCGCCACGGTGGTGGAGGAGCTCTTCAGGGACGGGGTGAACTGGGGGCGCATCGTGGCCTTCTTTGAGTTCGGAGGGGTCATGTGTGTGGAGAGCGTCAACCGGGAGATGTCGCCCCTGGTGGACAGCATCGCCCTGTGGATGACCGAGTACCTGAACCGGCACCTGCACACCTGGATCCAGGACAACGGAGGCTGGGACGCCTTTGTGGAGCTGTATGGCCCTAGCATGCGGCCCCTGTTTGATTTCTCCTGGCTGTCTCTGAAGGCACTGCTCAGTCTGGCCCTGGTGGGCGCTTGCATCACCCTGGGTGCCTATCTGGGCCATAAGTGAAGCCCACAGGCCTGCCGCCAACAAATATGCAGAAGATTCAAGAAGCAGCAGAAATAATATGCATTGTTGCTGATGCACCACTGAAGTGAAGCTGTAGTCTCT。

[0024] In one embodiment, the 3D culture method of the present invention is a hanging drop culture. In one embodiment, the hanging drop culture steps of the present invention include: seeding a first induction solution containing bovine embryonic stem cells at a rate of 25-30 μL / drop onto the inside of the cell culture dish lid, then inverting it onto the cell culture dish tank for a first induction culture; seeding a second induction solution at a rate of 25-30 μL / drop onto the inside of the cell culture dish lid; collecting the bovine embryonic stem cells obtained from the first induction culture; transferring them to the second induction solution; then inverting it onto the cell culture dish tank for a second induction culture to obtain an organoid cell cluster. In one embodiment, the present invention uses a pipette to transfer the collected bovine embryonic stem cells obtained from the first induction culture to the second induction solution.

[0025] In one embodiment, the first induction solution of the present invention comprises 25-100 ng / mL BMP4, 0.5-2 μM MSB431542, 10-50 ng / mL FGF2, 0.5-2 μM LDN, 10-20% FBS, and the balance N2B27 culture medium; in another embodiment, the first induction solution of the present invention comprises 50 ng / mL BMP4, 1 μM MSB431542, 25 ng / mL FGF2, 1 μM LDN, 15% FBS, and the balance N2B27 culture medium. In one embodiment, the temperature of the first induction culture of the present invention is 37°C. In one embodiment, the time of the first induction culture of the present invention is 0-5 days; in another embodiment, the time of the first induction culture of the present invention is 5 days. In one embodiment, the concentration of bovine embryonic stem cells in the first induction solution containing bovine embryonic stem cells of the present invention is 250-300 cells / drop; in another embodiment, the concentration of bovine embryonic stem cells in the first induction solution containing bovine embryonic stem cells of the present invention is 280 cells / drop. The present invention enables embryos to differentiate into ectoderms through a first induction culture. As one embodiment, during the first induction culture process, PBS is added to the cell culture dish to prevent excessive evaporation of the liquid.

[0026] In one embodiment, the second induction solution of the present invention comprises 10-50 ng / mL FGF2, 10-50 ng / mL LFGF10, 5-20 μg / mL Wnt3a, 5-20 μg / mL Wnt5a, 5-20 μg / mL Wnt10b, 10-20% FBS, and the balance being N2B27 culture medium; in another embodiment, the second induction solution comprises 25 ng / mL FGF2, 25 ng / mL LFGF10, 10 μg / mL Wnt3a, 10 μg / mL Wnt5a, 10 μg / mL Wnt10b, 15% FBS, and the balance being N2B27 culture medium. In one embodiment, the temperature of the second induction culture of the present invention is 37°C. In one embodiment, the time of the second induction culture of the present invention is 0-5 days; in another embodiment, the time of the second induction culture of the present invention is 5 days. The second induction culture of the present invention enables differentiation of ectoderm into non-neuroectoderm. In one embodiment, during the second induction culture process of the present invention, PBS is added to the cell culture vessel to prevent excessive evaporation of the liquid.

[0027] After obtaining the organoid cell clusters, this invention adds a modified matrix gel to the bottom of a cell culture dish to form droplets; the organoid cell clusters are added to the droplets at a density of 1 cell / drop, the cell culture dish is inverted, and after the droplets solidify, the cell culture dish is placed upright, mammary gland induction medium is added, and the dish is cultured at 37°C for 10-20 days to obtain bovine mammary gland organoids. As one embodiment, this invention involves culturing at 37°C for 20 days.

[0028] The modified matrix gel of this invention consists of matrix gel and N2B27 culture medium in a volume ratio of 3:2. This invention limits the volume ratio of matrix gel to N2B27 culture medium to allow organoid cell clusters to grow in 3D while simultaneously enabling them to sprout, controlling their elongation rate, and forming tubular structures similar to mammary ducts.

[0029] The breast cancer induction culture medium of this invention comprises 10-50 ng / mL EGF, 30-100 ng / mL Fgf2, 70-150 ng / mL Fgf10, 50-100 ng / mL IGF1, 30-100 ng / mL R-spondin-1, 0.5-1.5×heparin, 0.5-1.5×hydrocortisone, 10-50 μg / mL insulin, 10-20% FBS, and the balance being N2B27 culture medium. As one embodiment, the breast cancer induction culture medium of this invention comprises 25 ng / mL EGF, 50 ng / mL Fgf2, 100 ng / mL Fgf10, 75 ng / mL IGF1, 50 ng / mL R-spondin-1, 1×heparin, 1×hydrocortisone, 25 μg / mL insulin, 15% FBS, and the balance being N2B27 culture medium.

[0030] In one embodiment, the N2B27 culture medium of the present invention comprises 40-50 mL DF12, 40-50 mL Neurobasal, 300-600 μL N2, 0.5-1 mL B27, 0.5-1 mL L-glutamine, 0.5-1 mL penicillin-streptomycin, 0.5-1 mL NEAA, and 50-100 μL β-mercaptoethanol. In another embodiment, the N2B27 culture medium of the present invention comprises 48 mL DF12, 48 mL Neurobasal, 500 μL N2, 1 mL B27, 1 mL L-glutamine, 1 mL penicillin-streptomycin, 1 mL NEAA, and 100 μL β-mercaptoethanol. In one embodiment, the penicillin-streptomycin penicillin-streptomycin mixture is used, with a penicillin concentration of 10000 U / mL and a streptomycin concentration of 10000 µg / mL.

[0031] In one embodiment, the cell culture vessel of the present invention is inverted for 40-50 minutes at a temperature of 37°C. The present invention limits the inversion time and temperature of the cell culture vessel, which can improve the germination rate of organoid cell clusters.

[0032] This invention provides a bovine mammary gland organoid, prepared using the culture method described in the above technical solution.

[0033] This invention provides the application of the bovine mammary gland organoids described in the above-mentioned technical solution in one or more of the following: bovine mammary gland development, disease mechanism research for non-disease diagnosis and treatment purposes, and drug testing.

[0034] To further illustrate the present invention, a functional bovine mammary gland organoid, its culture method, and its application are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Preparation of organoid cell clusters 1. Construction of lentiviral plasmids Using pLVX-EF1α-IRES-Puro as the backbone vector, bovine needles were inserted between its EF-1α site and P2A site. BCL2 Genes, and in cattle BCL2 A segment of EGFP, a fluorescent protein labeled with green, was added to the posterior end of the gene to obtain a recombinant vector containing EGFP. BCL2 The overexpression lentiviral plasmid is designated pLVX-EF1α-IRES-Puro-EGFP- BCL2 The diagram is as follows Figure 1 As shown.

[0036] 2. Cow BCL2 overexpression of genes Following existing techniques (Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells), purchased bovine embryonic stem cells were cultured with a feeder layer to obtain bovine embryonic stem cells with a mouse feeder layer. Using a feeder layer removal kit (purchased from Miltenyi, product number 130-095-531), the bovine embryonic stem cells with the mouse feeder layer were digested and centrifuged to remove the feeder layer. The bovine embryonic stem cells were resuspended in N2B27 medium, and the cell suspension was re-layered into culture dishes and incubated for 30 min–1 h. At this point, the mouse feeder layer adhered to the bottom of the dish, and the embryonic stem cells remained suspended in the culture medium. Following existing techniques (Construction of a LILRA2 overexpression lentiviral vector and establishment of a stable THP-1-LILRA2 transfected cell line), bovine embryonic stem cells were overexpressed with bovine... BCL2 Genes were used to select cells exhibiting green fluorescence as positive cells, which were then verified to prove... BCL2 Overexpression was successfully achieved in bovine embryonic stem cells, yielding overexpressed... BCL2 Bovine embryonic stem cells containing genes.

[0037] 3. Preparation of culture medium (1) Prepare N2B27 medium according to the following ratio: 48mL DF12, 48mL Neurobasal, 500μL N2, 1mL B27, 1mL L-glutamine, 1mL penicillin (10000U / mL, streptomycin (10000µg / mL)), 1mL NEAA and 100μL β-mercaptoethanol. Add 15% fetal bovine serum to N2B27 medium to obtain organoid culture medium.

[0038] (2) Add 50 ng / mL BMP4, 1 μM SB, 25 ng / mL FGF2 and 1 μM LDN to the organoid culture medium to obtain the first induction solution.

[0039] (3) Add 25 ng / mL FGF2, 25 ng / mL FGF10, 10 μg / mL Wnt3a, 10 μg / mL Wnt5a and 10 μg / mL Wnt10b to the organoid culture medium to obtain the second induction solution.

[0040] 4. Three-dimensional (3D) cluster culture of bovine embryonic stem cells (1) Resuspend the overexpression using the first induction solution BCL2 Bovine embryonic stem cells were thoroughly blown apart to disperse any remaining cell clusters, and then cell counting was performed.

[0041] (2) Differentiation of embryos into ectoderm: When performing 3D cluster culture, the hanging drop culture method is adopted. The culture medium containing cells is dropped at 25 μL / drop, with each hanging drop containing about 250 cells. The culture medium is then flipped over and placed on the bottom of the dish containing PBS to prevent large amounts of liquid from evaporating. The first induction culture is performed at a temperature of 37°C.

[0042] (3) Differentiation of ectoderm into non-neural ectoderm: The second induction solution was dispensed into the inside of the dish lid at a rate of 25 μL / drop. Embryonic stem cells from step (2) after 5 days of first induction culture were aspirated using a pipette and directly transferred to the second induction solution. The dish lid was then inverted and placed on the bottom of the dish containing PBS to prevent excessive evaporation. Second induction culture was then performed to obtain organoid cell clusters. The second induction culture lasted for 5 days under the following conditions: 37°C, 5% CO2. Overexpression BCL2 Genes enable stem cell clusters to differentiate into ectoderm and ectoderm into neuroectoderm.

[0043] Example 2 Add 15% fetal bovine serum to N2B27 medium to obtain a premix solution. Mix the matrix gel and the premix solution at a certain volume ratio to obtain modified matrix gels with certain matrix gel concentrations (40%, 50%, 60%, 70%, and 80%).

[0044] Different concentrations of modified matrix gel were added to the bottom of the culture dish at a rate of 25-30 μL / drop. Then, using a pipette, one organoid cell cluster obtained from 10 days of clump culture in Example 1 was added to each gel droplet (recorded as day 0 after embedding). The culture dish was then inverted and placed in a 37°C incubator. After the gel had completely solidified, the dish was upright, mammary gland induction medium was added, and the dish was continued to be incubated at 37°C. The budding and growth of the organoids were observed, and the results are as follows: Figure 3As shown in the figure. The mammary gland induction medium consisted of 25 ng / mL EGF, 50 ng / mL Fgf2, 100 ng / mL Fgf10, 75 ng / mL IGF1, 50 ng / mL LR-spondin-1, 1× heparin, 1× hydrocortisone, 25 μg / mL insulin, 15% FBS and the remainder N2B27 medium.

[0045] according to Figure 3 It can be seen that the ability and state of organoid cell clusters to grow in different concentrations of modified matrix gel are not the same. At a gel concentration of 40%, the gel was too thin and too soft, resulting in insufficient support and collapse. This prevented the organoids from forming a 3D culture environment, causing uncontrolled growth of the cell clusters during budding. Instead of forming tubular structures resembling mammary ducts, the cell clusters formed thin, filamentous structures (day 7). At a gel concentration of 50%, while budding was not as rapid as at 40%, tubular structures still could not be formed. At concentrations of 70% or even 80%, the excessive concentration caused the mammary organoid cell clusters to only bud (as seen in the bubble-like structures around the cell clusters on day 2), but they could not elongate (no significant difference in appearance between day 5 and day 7). A 60% gel concentration, with its moderate firmness, allowed the mammary organoid cell clusters to bud (day 5) while controlling their elongation, resulting in tubular structures resembling mammary ducts (day 7). Therefore, a 60% glue concentration is the optimal glue concentration for the budding and growth of bovine mammary gland organoids.

[0046] Example 3 A premix was prepared by adding 15% fetal bovine serum to N2B27 medium. The matrix gel and the premix were then mixed at a volume ratio of 3:2 to obtain a modified matrix gel with a concentration of 60%. The modified matrix gel was added to the bottom of a culture dish at a rate of 25–30 μL / drop. Then, using a pipette, organoid cell clusters obtained from 10 days of clump culture in Example 1 were dispensed into each droplet at a rate of 1 cell / drop. The culture dishes were then randomly divided into a control group and a treatment group. The control group (not inverted) had the culture dishes placed upright in a 37°C incubator; the treatment group (inverted) had the culture dishes placed upside down in a 37°C incubator.

[0047] After the gel in both the control and treatment groups completely solidified, the treatment group was placed upright in a culture dish, and the mammary gland induction medium from Example 2 was added to both the treatment and control group culture dishes. The dishes were then incubated at 37°C, and the organoid budding and growth were observed. The results are as follows: Figure 4 As shown.

[0048] according to Figure 4 It can be seen that the control group lacked the inversion step, the matrix gel could not provide support, and the organoid cell clusters could not sprout and elongate.

[0049] Example 4 A premix was prepared by adding 15% fetal bovine serum to N2B27 medium. The matrix gel and the premix were then mixed at a volume ratio of 3:2 to obtain a modified matrix gel with a concentration of 60%. The modified matrix gel was added to the bottom of a culture dish at a rate of 25-30 μL / drop. Then, using a pipette, organoid cell clusters obtained from 10 days of clump culture in Example 1 were added to each droplet at a rate of 1 cell / drop. The culture dishes were then inverted and placed in a 37°C incubator for 10-50 minutes. Afterward, the dishes were upright, and mammary gland induction medium from Example 2 was added. The dishes were then incubated at 37°C for another 10 days. The budding and growth of organoids under different inversion conditions were recorded, and the results are shown in Table 1.

[0050] Table 1. Organoid budding rate at different inversion times

[0051] As can be seen from Table 1, the solidification effect varies with different inversion times, and the germination effect also varies. The longer the inversion time, the better the germination effect.

[0052] Example 5 Adding 15% fetal bovine serum to N2B27 medium yielded a premix. The matrix gel and premix were then mixed at a volume ratio of 3:2 to obtain a modified matrix gel with a concentration of 60%. The modified matrix gel was added to the bottom of a culture dish at a rate of 25–30 μL / drop. Then, using a pipette, organoid cell clusters obtained from 10 days of clump culture in Example 1 were dispensed into each droplet at a rate of 1 cell / drop. The culture dishes were then inverted and placed in a 37°C incubator for 40 minutes. Afterward, the dishes were inverted and randomly divided into a control group and a treatment group for the following treatments: Treatment group: Add the mammary gland induction medium from Example 2 to the culture dish and continue to culture in a 37°C incubator for 10 days to obtain organoids.

[0053] Control group: The difference from the treatment group is that the mammary gland induction culture medium does not contain Fgf2 and Fgf10.

[0054] 1. Observe the budding and branching of mammary gland organoids in the control and treatment groups during the culture period. The results are as follows: Figure 5 As shown, the treatment group with added Fgf2 and Fgf10 was able to form buds and thicker tubular structures; while the treatment group without added Fgf2 and Fgf10, although the organoids could bud, it had a significant impact on their subsequent elongation growth. That is, some buds could not elongate further after growing to a certain extent, and even if some could elongate, they lost the tubular structure and formed filamentous vessels.

[0055] 2. The organs expressed the mammary primordium marker protein Krt4 and the mammary duct marker protein Krt8, demonstrating that these organoids not only differentiated into mammary primordia (i.e., mammary stem cells) but also further differentiated into ductal tissue based on the primordia. Based on this principle, organoids from different treatment times in Example 5 were used to detect mammary organoid marker proteins, and the differentiation rate of organoids at different time points was statistically analyzed. The results are as follows: Figure 6 As shown in Table 2.

[0056] Table 2. Differentiation rate of mammary gland organoids at different times

[0057] Note: The differentiation rate in Table 2 represents the proportion of mammary gland organoids that did not differentiate into mammary gland organoids.

[0058] according to Figure 6 As shown in Table 2, the organoids expressed the Krt4 marker protein of mammary primordia at their center, demonstrating that the organoids differentiated gradually from non-neuroectoderm towards the mammary gland. The expression and aggregation of mammary primordia became more pronounced with increasing post-embedding culture time. Furthermore, mammary ducts (Krt8) differentiated further from the mammary primordia. On day 10 post-embedding, Krt4 specific expression of the mammary primordia was clearly observed at the center of the organoid, while the ducts sprouted and branched from the primordia, simultaneously expressing their specific ductal epithelial marker protein, Krt8. With increasing culture time, the expression ratio of mammary gland-related marker proteins in the control group (without mammary organoid directional induction) gradually decreased, while the experimental group maintained a high level of mammary differentiation induction.

[0059] Example 6 Validation of organoid lactation performance After a complete estrus cycle, bovine mammary glands enter the lactation period, at which time the mammary glands begin a high-lactation phase. Organoids from the control and treatment groups in Example 5 were added to a second induction medium and then cultured at 37°C for 10 days. The second induction medium consisted of 1 nM β-estradiol, 75 ng / mL Fgf2, 50 ng / mL Fgf10, 1 μg / mL Prolactin, 50 nM Progesterone, 25 μg / mL insulin, 15% FBS, and the remainder being N2B27 medium.

[0060] Five days after treatment, milk protein was detected in the mammary organoids. The results showed that the treated group of mammary organoids secreted milk protein. Figure 7 ).

[0061] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for culturing bovine mammary gland organoids, characterized in that, Includes the following steps: overexpression BCL2 Genes were used to culture bovine embryonic stem cells in 3D to obtain organoid cell clusters; Add a modified matrix gel to the bottom of the cell culture dish to form a droplet; The organoid cell clusters were added to the gel drop at a density of 1 cell / drop. The cell culture vessel was inverted. After the gel drop solidified, the cell culture vessel was placed upright, mammary gland induction medium was added, and the culture was carried out at 37°C for 10-20 days to obtain bovine mammary gland organoids. The BCL2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1; The modified matrix gel consists of matrix gel and N2B27 culture medium, with a volume ratio of matrix gel to N2B27 culture medium of 3:

2. The mammary gland induction culture medium comprises 10-50 ng / mL EGF, 30-100 ng / mL Fgf2, 70-150 ng / mL Fgf10, 50-100 ng / mL IGF1, 30-100 ng / mL R-spondin-1, 0.5-1.5×heparin, 0.5-1.5×hydrocortisone, 10-50 μg / mL insulin, 10-20% FBS, and the balance being N2B27 medium; The 3D culture steps include: first inducing bovine embryonic stem cells to undergo first induction culture using a first induction solution, and then using a second induction solution to undergo second induction culture to obtain organoid cell clusters. The first induction solution consists of 25-100 ng / mL BMP4, 0.5-2 μM SB431542, 10-50 ng / mL FGF2, 0.5-2 μM DN, 10-20% FBS and the remainder N2B27 medium; The second induction solution consists of 10-50 ng / mL FGF2, 10-50 ng / mL FGF10, 5-20 μg / mL Wnt3a, 5-20 μg / mL Wnt5a, 5-20 μg / mL Wnt10b, 10-20% FBS and the remainder N2B27 culture medium; The first induction culture time is 5 days, and the second induction culture time is 5 days.

2. The cultivation method according to claim 1, characterized in that, The cell culture vessel is inverted for 40-50 minutes at a temperature of 37°C.

3. The cultivation method according to claim 1, characterized in that, The volume of the gel droplets is 25~30μL / drop.

4. The cultivation method according to claim 1, characterized in that, The 3D culture method is hanging drop culture.

5. The cultivation method according to claim 4, characterized in that, The steps of the hanging drop culture include: The first induction solution containing bovine embryonic stem cells was seeded into the inside of the cell culture vessel lid at a rate of 25-30 μL / drop, and then inverted onto the cell culture vessel tank for the first induction culture. The second induction solution was seeded into the inside of the cell culture vessel lid at a rate of 25-30 μL / drop. Bovine embryonic stem cells obtained from the first induction culture were collected, transferred to the second induction solution, and then inverted onto the cell culture vessel tank for the second induction culture to obtain organoid cell clusters.

6. The cultivation method according to claim 5, characterized in that, The temperature for the first induction culture was 37°C; the temperature for the second induction culture was 37°C.

7. The cultivation method according to claim 5, characterized in that, The concentration of bovine embryonic stem cells in the first induction solution containing bovine embryonic stem cells is 250-300 cells / drop.

8. The cultivation method according to claim 5, characterized in that, During the first and second induction cultures, PBS was added to the cell culture dish.

9. A bovine mammary gland organoid, prepared by the culture method according to any one of claims 1 to 8.

10. The use of the bovine mammary gland organoid of claim 9 in pharmaceutical testing for bovine mammary gland development and / or for non-disease diagnostic and therapeutic purposes.

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