Culture medium and culture method of breast cancer organoid

Abstract

The invention discloses a culture medium and a culture method of a breast cancer organoid. The culture medium comprises an Advanced DMEM / F12 basal culture medium and an additive, the additive is prepared from penicillin streptomycin / amphotericin B, a B-27 additive, L-glutamine amide, HEPES (Hydroxyethyl PES), N-Acetylcysteine, nicotinamide, SB202190, A83-01, FGF10 (Fibroblast Growth Factor 10), EGF (Epidermal Growth Factor), human Hergulin beta-1, Y27632, WNT-3A, R-spondin 1, NOGGIN (Nitrogen Growth Factor IN), FGF7 (Fibroblast Growth Factor 7) and beta-estradiol. The culture method of the breast cancer organoid comprises the step of using the breast cancer organoid culture medium, and the method specifically comprises the steps of tissue sample taking, primary tissue construction, breast cancer organoid liquid changing and breast cancer organoid subculture. According to the prepared breast cancer organoid culture medium, tumor cells taken from breast cancer tumors grow fast, the cell speed is high, and follow-up medicine screening experiments can be facilitated; after the breast cancer organoid culture medium is used for culturing the breast cancer organoid, the cellular morphology and the protein expression condition of the organoid are identified through HE and immunohistochemistry, and it can be seen that the organoid can well reduce the pathological characteristics of the tissue.

Description

Technical Field

[0001] The patent of this invention belongs to the field of biotechnology, and specifically relates to the culture medium and culture method of breast cancer organoids. Background Art

[0002] Organoids are preclinical models established on the basis of three-dimensional culture. They are miniature organs formed by in vitro three-dimensional culture of normal tissue or tumor tissue from patients. They highly restore and maintain the biological characteristics, pathological characteristics and drug responsiveness of the original tumor tissue, which greatly meets the needs of current precision medicine. Although the breast cancer organoid model appeared earlier and the research is more mature, there are many molecular types of breast cancer, and the exploration of culture medium suitable for the growth of breast cancer organoids has never stopped. The existing breast cancer organoid culture medium has the following problems: 1. Tumor cells obtained from breast cancer tumors grow relatively slowly in breast cancer organoid culture medium, and cell proliferation is slow. The average diameter of the organoids is less than 100 μm within 7 to 14 days, which is not conducive to subsequent drug screening experiments; 2. The main reason why organoids can be used in precision medicine is that they can highly restore the biological characteristics and pathological features of the original tumor tissue, but the existing organoid culture medium cannot maintain the pathological characteristics of the original tissue well.

[0003] Therefore, this paper proposes a culture medium and culture method for breast cancer organoids. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a culture medium and a culture method for breast cancer organoids that can more quickly obtain a large number of high-fidelity breast cancer organoids.

[0005] In order to achieve the above technical effects, the present invention provides a culture medium for breast cancer organoids, including Advanced DMEM / F12 basal culture medium and additives; The additives include penicillin / amphotericin B, B-27™ additive, L-glutamine, HEPES, N-Acetylcysteine, nicotinamide, SB202190, A83-01, FGF10, EGF, human Heregulinβ-1, Y27632, WNT-3A, R-spondin 1, NOGGIN, FGF7, and β-estradiol.

[0006] Preferably, according to the present invention, the final concentration of the components of the additive includes 0.5-1.5X penicillin / amphotericin B, 0.5-1.5X B-27™ additive, 0.5-1.5X L-glutamine, 9-12mM HEPES, 1-1.5mM N-Acetylcysteine, 4-6mM nicotinamide, 450-550nM SB202190, 450-550nM A83-01, 45-55ng / ml FGF10, 4-6ng / ml EGF, 4-6nM human Heregulinβ-1, 9-12uM Y27632, 0.4-0.6ug / ml WNT-3A, 0.4-0.6ug / ml R-spondin 1. 0.05-0.12ug / ml NOGGIN, 18-22ng / ml FGF7, 180-220nM β-estradiol.

[0007] Preferably according to the present invention, the final concentrations of the components of the additive include 1X penicillin-streptomycin, amphotericin B, 1X B-27™ additive, 1X L-glutamine, 10mM HEPES, 1.25mM N-Acetylcysteine, 5mM nicotinamide, 500nM SB202190, 500nM A83-01, 50ng / ml FGF10, 5ng / ml EGF, 5nM human Heregulinβ-1, 10uM Y27632, 0.5ug / ml WNT-3A, 0.5ug / ml R-spondin 1, 0.1ug / ml NOGGIN, 20ng / ml FGF7, and 200nM β-estradiol.

[0008] The present invention also provides a method for culturing breast cancer organoids, comprising using the breast cancer organoid culture medium of the present invention, and the method specifically comprises the following steps: S1. Collection of tissue samples: Use sampling tools to accurately collect breast cancer tumor tissue, and the tumor cells must be concentrated and the cell vitality is good. Then quickly put the removed tumor tissue block into a pre-cooled centrifuge tube containing preservation solution and transport it to the laboratory; Furthermore, the material collection method in S1 specifically includes the following steps: S101, prepare tissue preservation and transport solution in advance, add preservation solution into centrifuge tubes, put them into transport boxes with a temperature of 4-8°C, and put them into the operating room; S102. Use sampling tools to accurately collect breast cancer tumor tissue from areas where tumor cells are relatively concentrated and cell vitality is good. Each tumor tissue piece should be the size of a red bean, not too large. Only 2 to 3 tissue samples are needed. S103, the removed tumor tissue block is quickly placed into a pre-cooled centrifuge tube containing a preservation solution, and then placed into a transport box. This process is controlled within 1 minute; S104. Ensure that the sample is completely covered with tissue preservation transport fluid and that the tissue sample is still in a low-temperature environment and immersed in tissue preservation fluid when it arrives at the laboratory. The entire transportation process is controlled in a low-temperature environment of 2~8℃, and the transportation time is less than 72h.

[0009] S2. Construction of primary tissue: The tumor tissue is transferred to a clean laboratory, and is repeatedly washed and digested until tumor cells are obtained. Then, it is filtered and centrifuged, and the supernatant is discarded for later use. Then, the corresponding matrix gel is added to the cell pellet, and finally, it is placed in an organoid plate and cultured with breast cancer organoid culture medium; Furthermore, the method for constructing the primary tissue in S2 specifically comprises the following steps: S201. After registering the patient information, transfer the tumor tissue to the clean laboratory, sterilize it and place it in the clean bench. Prepare three culture dishes and pour PBS with 1% antibiotics into them. S202, soak the tumor tissue in the first dish, shake it thoroughly to wash it, use tweezers to remove dead cells, fat tissue, muscle tissue, and some blood cells, and after removing them, transfer the sample to the second dish and cut it into pieces the size of rice grains, and thoroughly wash the tissue with PBS containing double antibodies, then transfer the small tumor tissue pieces to the third culture dish and continue to thoroughly wash the tissue pieces, and finally put them into a centrifuge tube; S203, the tissue blocks in the centrifuge tube were cut into pieces without large particles, and then transferred to the tumor tissue digestion solution, and the sample was resuspended by blowing, and quickly transferred to the shaker, and digested at 37°C for 50 minutes. Every 10 minutes, the sample was beaten with a pipette and 10ul was taken out to observe the digestion status, until the tumor cells were largely digested and there were no large tumor tissue blocks, and then the next step could be performed; S204, after digestion is completed, FBS is quickly added to the digestion solution to stop digestion, the liquid is mixed, and then filtered with a rinsed filter, and then the filtered suspension is collected for centrifugation and the supernatant is discarded, PBS is continued to be added to wash the precipitate, centrifuged and the supernatant is discarded, and then PBS is added to resuspend the cells, and a small portion is taken for cell counting and death rate counting, and the remaining liquid is centrifuged and the supernatant is discarded for standby use; S205, Matrigel 3D encapsulation: This experimental step is performed on ice. Add the corresponding Matrigel to the cell pellet according to the amount of organoids and cells, and mix slowly on ice; S206. Organoid plate culture: Use the pipette tip to suck up the mixture of extracellular matrix and cells and move it to the center of the bottom of the cell culture plate. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 for incubation. After the extracellular matrix gel solidifies and no longer flows, add breast cancer organoid culture medium and place the plate in a 37°C and 5% CO2 incubator for culture.

[0010] S3. Change the medium of breast cancer organoids: Take out the organoid culture cell plate from the cell culture incubator, aspirate and discard the original culture medium in the clean bench, add new breast cancer organoid culture medium, and perform this operation every 2-3 days; Furthermore, the breast cancer organoid medium replacement method in S3 specifically comprises the following steps: S301. Take out the breast cancer organoid culture medium from the 4°C refrigerator and rewarm it at room temperature or in a CO2 incubator. S302, take out the organoid culture cell plate from the cell culture incubator, use a pipette to suck out the original culture medium in the clean bench and discard it into the waste liquid tank, then add the newly warmed breast cancer organoid culture medium; S303. After the liquid is replaced, the cells are observed and photographed under an inverted microscope, and then placed in a CO2 incubator for further culture.

[0011] S4. Subculture of breast cancer organoids: When breast cancer organoids are cultured to a diameter of 100-500 uM or when the density of the organoids is particularly dense, generally 7-14 days, remove the original preservation solution in the clean bench, then centrifuge and wash, re-wrap with matrix gel and place on the organoid plate for culture. Change the solution every 3-4 days and repeat the subculture process.

[0012] Furthermore, the breast cancer organoid subculture method in S4 specifically comprises the following steps: S401. When the breast cancer organoids are cultured to a diameter of 100-500 uM or the density of the organoids is particularly dense, generally 7-14 days, remove the original preservation solution in the clean bench, add DMEM basal culture medium, scrape the mixture of matrix gel and organoids with a pipette tip, transfer to a centrifuge tube, pipette several times and then centrifuge; S402. After centrifugation, discard the supernatant and add organoid digestion solution 5 times the volume of the extracellular matrix and organoid mixture. Mix well with a pipette and digest at 37°C for 3 min to 6 min. S403, cleaning of organoids: After digestion, add 4-5 times the volume of the digestion solution of DMEM basal medium to terminate digestion and mix by pipetting and centrifugation. Remove the supernatant and add DMEM basal medium again to clean 1-2 times. S404, 3D encapsulation of breast cancer organoids with matrix gel: This experimental step is performed on ice or in an ice box. According to the amount of organoids and cells, add the corresponding matrix gel to the cell pellet and mix well by blowing on ice to avoid the generation of bubbles and gel coagulation; S405, organoid seed plate culture: move the mixed cell mixture to the center of the bottom of the cell culture well plate, add matrix gel to the wells of the culture plate, and then place the culture plate in a cell culture incubator at 37°C and 5% CO2 for incubation for 20 minutes; S406. After the extracellular matrix gel solidifies and no longer flows, add breast cancer organoid culture medium. Return the plate to a 37°C, 5% CO2 incubator for culture; change the medium every 3-4 days, and subculture when the breast cancer organoids are cultured to a diameter of 100-500 uM or when the organoid density is particularly dense.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly: The breast cancer organoid culture medium prepared by the present invention enables the tumor cells obtained from breast cancer tumors to grow quickly, and the cell speed is fast. Within 7 to 14 days, the average diameter of the organoids is greater than 100 μm, which can be beneficial to subsequent drug screening experiments; and after culturing breast cancer organoids with the breast cancer organoid culture medium configured by the present invention, the organoid cell morphology and protein expression are identified by HE and immunohistochemistry, and it can be seen that the organoids can well restore the pathological characteristics of the tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for describing the embodiment are briefly introduced below. Those skilled in the art can also obtain other drawings based on these drawings without paying any creative work.

[0015] Figure 1 The bright field pictures of the first day of culturing breast cancer organoids of the present invention and the bright field pictures of the first day of culturing breast cancer organoids of a certain brand 1; Figure 2 This is a bright field picture of the breast cancer organoid cultured on the 7th day of the present invention and a bright field picture of the breast cancer organoid cultured on the 7th day of a certain brand 1; Figure 3 The bright field pictures of the breast cancer organoids cultured on the second day of the present invention and the bright field pictures of the breast cancer organoids cultured on the second day of a certain brand 2; Figure 4 This is a bright field picture of the breast cancer organoid cultured on the 6th day of the present invention and a bright field picture of the breast cancer organoid cultured on the 6th day of a certain brand 2; Figure 5 These are pictures of the first day of culture with the breast cancer organoid culture medium of the present invention and the first day of culture with a certain brand 3 after the primary bank was established; Figure 6These are pictures of the breast cancer organoid culture medium of the present invention and a certain brand 3 on the 6th day after the primary bank was established; Figure 7 These are the immunohistochemical experimental pictures of HE, ER, PR, and HER2; DETAILED DESCRIPTION

[0016] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0017] The prior art in the present invention has the following problems: because the existing TIL cell culture method mainly uses IL-2 as the main stimulating factor, and is supplemented with human AB serum as a nutritional supplement for co-culture, but under such culture conditions, the cells cannot maintain a low level of differentiation, and their proliferation ability and long-term persistence in the body are low, and cannot meet the number of immune cells required for effective clinical treatment.

[0018] Therefore, the inventors provide a culture medium for breast cancer organoids, including Advanced DMEM / F12 basal culture medium and additives; The additives include penicillin / amphotericin B, B-27™ additive, L-glutamine, HEPES, N-Acetylcysteine, nicotinamide, SB202190, A83-01, FGF10, EGF, human Heregulinβ-1, Y27632, WNT-3A, R-spondin 1, NOGGIN, FGF7, and β-estradiol.

[0019] Preferably, according to the present invention, the final concentration of the components of the additive includes 0.5-1.5X penicillin / amphotericin B, 0.5-1.5X B-27™ additive, 0.5-1.5X L-glutamine, 9-12mM HEPES, 1-1.5mM N-Acetylcysteine, 4-6mM nicotinamide, 450-550nM SB202190, 450-550nM A83-01, 45-55ng / ml FGF10, 4-6ng / ml EGF, 4-6nM human Heregulinβ-1, 9-12uM Y27632, 0.4-0.6ug / ml WNT-3A, 0.4-0.6ug / ml R-spondin 1. 0.05-0.12ug / ml NOGGIN, 18-22ng / ml FGF7, 180-220nM β-estradiol.

[0020] Preferably according to the present invention, the final concentrations of the components of the additive include 1X penicillin-streptomycin, amphotericin B, 1X B-27™ additive, 1X L-glutamine, 10mM HEPES, 1.25mM N-Acetylcysteine, 5mM nicotinamide, 500nM SB202190, 500nM A83-01, 50ng / ml FGF10, 5ng / ml EGF, 5nM human Heregulinβ-1, 10uM Y27632, 0.5ug / ml WNT-3A, 0.5ug / ml R-spondin 1, 0.1ug / ml NOGGIN, 20ng / ml FGF7, and 200nM β-estradiol.

[0021] The present invention also provides a method for culturing breast cancer organoids, comprising using the breast cancer organoid culture medium of the present invention, and the method specifically comprises the following steps: S1. Collection of tissue samples: Use sampling tools to accurately collect breast cancer tumor tissue, and the tumor cells must be concentrated and the cell vitality is good. Then quickly put the removed tumor tissue block into a pre-cooled centrifuge tube containing preservation solution and transport it to the laboratory; Furthermore, the material collection method in S1 specifically includes the following steps: S101, prepare tissue preservation and transport solution in advance, add preservation solution into centrifuge tubes, put them into transport boxes with a temperature of 4-8°C, and put them into the operating room; S102. Use a scalpel or other sampling tool to accurately collect breast cancer tumor tissue from areas where tumor cells are concentrated and cell vitality is good. Each tumor tissue piece should be the size of a red bean, not too large. Only 2 to 3 tissue samples are needed. S103, the removed tumor tissue block is quickly placed into a pre-cooled centrifuge tube containing a preservation solution, and then placed into a transport box. This process is controlled within 1 minute; S104. Ensure that the sample is completely covered with tissue preservation transport fluid and that the tissue sample is still in a low-temperature environment and immersed in tissue preservation fluid when it arrives at the laboratory. The entire transportation process is controlled in a low-temperature environment of 2~8℃, and the transportation time shall not exceed 72h.

[0022] S2. Construction of primary tissue: The tumor tissue is transferred to a clean laboratory, and is repeatedly washed and digested until tumor cells are obtained. Then, it is filtered and centrifuged, and the supernatant is discarded for later use. Then, the corresponding matrix gel is added to the cell pellet, and finally, it is placed in an organoid plate and cultured with breast cancer organoid culture medium; Furthermore, the method for constructing the primary tissue in S2 specifically comprises the following steps: S201. After registering the patient information, transfer the tumor tissue to the clean laboratory, sterilize it and place it in the clean bench. Prepare three 6-cm culture dishes and pour in PBS with 1% antibiotics. S202, 1cm 3 Immerse the tumor tissue in the first dish, shake it thoroughly to wash it, use tweezers to remove dead cells, fat tissue, muscle tissue, and some blood cells. After removing them, transfer the sample to the second dish and cut it into pieces the size of rice grains. Wash the tissue thoroughly with PBS containing double antibodies. Then transfer the small tumor tissue pieces to the third culture dish and continue to wash the tissue pieces thoroughly. Finally, put them into a 1.5ml centrifuge tube. S203, the tissue blocks in the centrifuge tube were cut into pieces and then transferred to the tumor tissue digestion solution, and the samples were resuspended by blowing, and then quickly transferred to the shaker, and digested at 37°C for 50 minutes. Every 10 minutes, the sample was beaten with a pipette and 10ul was taken out to observe the digestion status until the tumor cells were largely digested and there were no large amounts of tumor tissue blocks, and then the next step could be performed; S204, after digestion, FBS was quickly added to the digestion solution to stop digestion, the liquid was mixed, and then filtered with a 100 μm filter after rinsing, and then the filtered suspension was collected and centrifuged for 3 minutes and the supernatant was discarded, and 5 ml of PBS was added to wash the precipitate, centrifuged for 3 minutes and the supernatant was discarded, and then 1 ml of PBS was added to resuspend the cells, and a small portion was taken for cell counting and death rate counting, and the remaining liquid was centrifuged for 3 minutes and the supernatant was discarded for later use; S205, Matrigel 3D encapsulation: This experimental step is performed on ice. According to the amount of organoids and cells, add the corresponding Matrigel (e.g., 25 μl of Matrigel per well in a 24-well culture plate) to the cell pellet and mix well by slowly pipetting on ice. S206. Organoid plate culture: Use the pipette tip to suck up the mixture of extracellular matrix and cells and move it to the center of the bottom of the cell culture plate (24-well plate, 205μl matrix gel / well). Place the culture plate in a cell culture incubator at 37°C and 5% CO2 for incubation. After the extracellular matrix gel solidifies and no longer flows, add breast cancer organoid culture medium and place the plate in a 37°C, 5% CO2 incubator for culture.

[0023] S3. Change the medium of breast cancer organoids: Take out the organoid culture cell plate from the cell culture incubator, aspirate and discard the original culture medium in the clean bench, add new breast cancer organoid culture medium, and perform this operation every 2-3 days; Furthermore, the breast cancer organoid medium replacement method in S3 specifically comprises the following steps: S301. Take out the breast cancer organoid culture medium from the 4°C refrigerator and rewarm it at room temperature or in a CO2 incubator. S302, take out the organoid culture cell plate from the cell culture incubator, use a 1 ml pipette to aspirate the original culture medium in the clean bench and discard it into the waste tank, then add about 500 ul of newly warmed breast cancer organoid culture medium; S303. After the liquid is replaced, the cells are observed and photographed under an inverted microscope, and then placed in a CO2 incubator for further culture.

[0024] S4. Subculture of breast cancer organoids: When breast cancer organoids are cultured to a diameter of 100-500 uM or when the density of the organoids is particularly dense, generally 7-14 days, remove the original preservation solution in the clean bench, then centrifuge and wash, re-wrap with matrix gel and place on the organoid plate for culture. Change the solution every 3-4 days and repeat the subculture process.

[0025] Furthermore, the breast cancer organoid subculture method in S4 specifically comprises the following steps: S401. When the breast cancer organoids are cultured to a diameter of 100-500 uM or the density of the organoids is particularly dense, generally 7-14 days, remove the original preservation solution in the clean bench, add DMEM basal culture medium, scrape the mixture of matrix gel and organoids with a pipette tip, transfer to a 1.5 ml centrifuge tube, pipette several times, and centrifuge at 100-300 g for 3 minutes; S402. After centrifugation, discard the supernatant and add organoid digestion solution 5 times the volume of the extracellular matrix and organoid mixture. Mix well with a pipette and digest at 37°C for 3 min to 6 min. S403, cleaning of organoids: After digestion, add 4-5 times the volume of DMEM basal medium of the digestion solution to terminate digestion and mix by pipetting, centrifuge at 300g for 3 min, remove the supernatant, add DMEM basal medium again once or twice, centrifuge at 300g for 3 min, remove the supernatant, and wash in this way; S404, 3D encapsulation of breast cancer organoids with matrix gel: This experimental step is performed on ice or in an ice box. In the cell pellet, according to the amount of organoids and cells (generally, transfer from 1 well to 2 wells or from 1 well to 3 wells), add the corresponding matrix gel (e.g., 24-well culture plate, 25uL matrix gel per well), and mix well by blowing on ice to avoid the generation of bubbles and gel coagulation; S405, Organoid plate culture: Move the mixed cell mixture to the center of the bottom of the cell culture plate, 24-well culture plate, 25uL of matrix gel in each well. Then put the culture plate in a 37℃, 5% CO2 cell culture incubator and incubate for 20 minutes; S406. After the extracellular matrix gel solidifies and no longer flows, add breast cancer organoid culture medium. Return the plate to a 37°C, 5% CO2 incubator for culture, change the medium every 3-4 days, and subculture when the breast cancer organoids are cultured to a diameter of 100-500 uM or when the organoid density is particularly dense.

[0026] The functions of each reagent in the above technical solution are as follows: 1. Penicillin-streptomycin / amphotericin B: Penicillin is a β-lactam antibiotic that kills bacteria by blocking the synthesis of bacterial cell walls, causing them to swell, rupture, and autolyze; streptomycin is an aminoglycoside antibiotic that can inhibit bacterial protein synthesis; amphotericin B is a polyene antifungal drug that can irreversibly bind to the sterols in the fungal cell membrane to form a transmembrane channel that destroys the integrity of the membrane, causing small molecules or nucleotides, amino acids, etc. in the cell to leak out of the membrane, destroying the normal metabolism of the fungus and causing death, and is therefore used to inhibit fungal and yeast contamination. The combination of the three ingredients can effectively inhibit the reproduction of bacteria, fungi, and yeast and the contamination of tumor cells; 2. L-Glutamine: It is a conditionally essential amino acid that provides important energy and nitrogen source for cell growth and metabolism. It not only participates in the biosynthesis of cells, but also participates in the energy metabolism of cells during cell culture. It is an important part of cell culture. 3. B-27: It is a common culture medium supplement that can replace serum and does not cause differentiation of cells in the system in three-dimensional organoid culture; 4.HEPES: A non-ionic amphoteric buffer with good buffering capacity, no toxicity to cells, and the ability to maintain a constant pH range for a long time, which can maintain the stability of the solution in organoid culture; 5. N-Acetylcysteine: NAC is a powerful antioxidant that can remove reactive oxygen species (ROS) and reduce oxidative stress damage to cells. During organoid culture, the production of ROS may affect cell growth and differentiation, so the use of NAC helps protect cells from oxidative stress damage; 6. Nicotinamide: Nicotinamide is a B3 vitamin that participates in a variety of enzymatic redox reactions and is a nutrient required for long-term culture of organoids. Nicotinamide, together with cytokines and other biochemical reagents, can exert anti-inflammatory properties, inhibit the activity of Sirtuins, promote the formation of organoids, and prolong the life of organoids. 7.SB202190: A highly effective p38 MAPK kinase inhibitor that targets p38α / β, inhibits cell death, and promotes organoid formation; 8.A83-01: Activin / NODAL / TGF-β pathway inhibitor, inhibits ALK5 / 4 / 7 kinase activity, maintains in vitro cell self-renewal, and prolongs the in vitro culture time of organoids; 9. FGF10: Fibroblast growth factor-10, which plays an important role in regulating the proliferation and differentiation of various stem cells in organoids, and shows a synergistic effect with FGF7 in maintaining organoid growth; 10. EGF: It is a potent growth factor that can stimulate a variety of epithelial development, angiogenesis, fibroblast proliferation and colony formation of epidermal cells in culture. In organoid culture, EGF binds to EGF receptors to activate signaling pathways such as PI3K / AKT, RAS / ERK and JAK / STAT, thereby driving cell proliferation and differentiation; 11. Human Heregulinβ-1: Heregulinβ-1 is a ligand of epidermal growth factor (EGF) and can bind to EGF receptors to promote the proliferation and differentiation of epithelial cells. Adding Heregulinβ-1 to organoid culture can increase the formation rate of organoids. At the same time, the addition of Heregulinβ-1 can make organoids heal quickly after passage and promote organoid growth; 12.Y27632: A small molecule inhibitor of Rho-associated protein kinase ROCK, which helps maintain the primary tumor characteristics of organoids, inhibits stem cell apoptosis, and improves cloning efficiency; 13.WNT-3A: It is an activator of the Wnt / β-catenin pathway, promoting stem cell growth, proliferation, and differentiation, while inhibiting cell apoptosis; 14. R-spondin 1: As an activator of the Wnt signaling pathway, it provides the necessary environmental conditions for the formation and maintenance of organoids by enhancing Wnt signals, especially in proliferative stem cells. It is a key factor in the in vitro expansion culture of various adult stem cells (including intestine, stomach and liver); 15.NOGGIN: Noggin has become an important factor in developmental biology and regenerative medicine research. Noggin regulates the proliferation and differentiation of stem cells by inhibiting the bone morphogenetic protein (BMP) signaling pathway and maintains the growth and function of organoids. 16. FGF7: Fibroblast growth factor 7, a mitogenic factor that helps these organs form and develop, and FGF7 and FGF10 show synergistic effects in maintaining organoid growth; 17.β-estradiol: It is a commonly used cytokine in breast cancer organoids. β-estradiol can promote cell proliferation by binding to estrogen receptors and activating downstream signaling pathways. At the same time, there are many subtypes of breast cancer, and estradiol helps guide stem cells to differentiate into specific cell types. Example 1

[0027] Based on the above technical solution, in the process of studying breast cancer organoid culture, in order to experiment the effect of the breast cancer organoid culture medium in the technical solution in the process of breast cancer organoid culture, the inventor collected brand 1, brand 2, and brand 3 to conduct the following comparative experiments for verification: like Figure 1 to Figure 2 As shown, these are photos of breast cancer organoids cultured on the 1st day and the 7th day using a certain brand 1 and a breast cancer organoid culture medium of the present invention respectively after passage; like Figure 3 to Figure 4 As shown, these are photos of breast cancer organoids cultured on the 2nd and 6th days after passage using a certain brand 2 and the breast cancer organoid culture medium of the present invention, respectively; like Figure 5 to Figure 6 As shown, these are pictures of the first and sixth days of culture using a certain brand 3 and the breast cancer organoid culture medium of the present invention after the primary library was established; As can be seen from the above figure, the growth rate of breast cancer organoids cultured using the present invention is significantly faster than that of other brands of breast cancer organoid culture media. The present invention can obtain a large number of organoids in a short period of time for subsequent drug screening experiments. Example 2

[0028] Based on the above experimental results, the inventors conducted chemical pathological experiments on the cultured organoids and original tumor tissues, including immunohistochemical experimental results of HE and ER, PR, and HER2, to verify whether breast cancer organoids can reproduce the pathological characteristics of original breast cancer in vitro.

[0029] like Figure 7 As shown, after culturing breast cancer organoids with the breast cancer organoid culture medium configured by the present invention, the cell morphology and protein expression of the organoids were identified by HE and immunohistochemistry, and it can be seen that the organoids can well restore the pathological characteristics of the tissue.

[0030] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0031] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A culture medium for breast cancer organoids, characterized in that: Including Advanced DMEM / F12 basal medium and additives; The additives include penicillin / amphotericin B, B-27™ additive, L-glutamine, HEPES, N-Acetylcysteine, nicotinamide, SB202190, A83-01, FGF10, EGF, human Heregulinβ-1, Y27632, WNT-3A, R-spondin 1, NOGGIN, FGF7, and β-estradiol.

2. The culture medium for breast cancer organoids according to claim 1, characterized in that: The final concentrations of the additive components include 0.5-1.5X penicillin / amphotericin B, 0.5-1.5X B-27™ additive, 0.5-1.5X L-glutamine, 9-12mM HEPES, 1-1.5mM N-Acetylcysteine, 4-6mM nicotinamide, 450-550nM SB202190, 450-550nM A83-01, 45-55ng / ml FGF10, 4-6ng / ml EGF, 4-6nM humanHeregulinβ-1, 9-12uM Y27632, 0.4-0.6ug / ml WNT-3A, 0.4-0.6ug / ml R-spondin 1. 0.05-0.12ug / ml NOGGIN, 18-22ng / ml FGF7, 180-220nM β-estradiol.

3. The culture medium for breast cancer organoids according to claim 1, characterized in that: The final concentrations of the components of the additive include 1X penicillin-streptomycin, amphotericin B, 1X B-27™ additive, 1X L-glutamine, 10mM HEPES, 1.25mM N-Acetylcysteine, 5mM nicotinamide, 500nM SB202190, 500nM A83-01, 50ng / ml FGF10, 5ng / ml EGF, 5nM human Heregulinβ-1, 10uM Y27632, 0.5ug / ml WNT-3A, 0.5ug / ml R-spondin 1, 0.1ug / ml NOGGIN, 20ng / ml FGF7, and 200nM β-estradiol.

4. A method for culturing breast cancer organoids, comprising using the breast cancer organoid culture medium of the present invention, characterized in that: The method specifically comprises the following steps: S1. Collection of tissue samples: Use sampling tools to accurately collect breast cancer tumor tissue, and the tumor cells must be concentrated and the cell vitality is good. Then put the removed tumor tissue block into a pre-cooled centrifuge tube containing preservation solution and transport it to the laboratory; S2. Construction of primary tissue: The tumor tissue is transferred to a clean laboratory, washed and digested until tumor cells are obtained, then filtered and centrifuged, the supernatant is discarded for later use, and then the corresponding matrix gel is added to the cell sediment, and finally it is placed in an organoid plate and cultured with breast cancer organoid culture medium; S3. Change the medium of breast cancer organoids: Take out the organoid culture cell plate from the cell culture incubator, aspirate and discard the original culture medium in the clean bench, add new breast cancer organoid culture medium, and perform this operation every 2-3 days; S4. Subculture of breast cancer organoids: When the breast cancer organoids are cultured to a diameter of 100-500 uM, which takes 7-14 days, remove the original preservation solution in the clean bench, centrifuge and wash, re-wrap with matrix gel and place on the organoid plate for culture. Change the solution every 3-4 days for subculture.

5. The method for culturing breast cancer organoids according to claim 4, characterized in that: The material collection method in S1 specifically comprises the following steps: S101, prepare tissue preservation and transport solution in advance, add preservation solution into centrifuge tubes, put them into transport boxes with a temperature of 4-8°C, and place them in the operating room; S102. Use a sampling tool to accurately collect breast cancer tumor tissue from the area where tumor cells are concentrated and cell vitality is good. Each tumor tissue block is the size of a red bean, and 2 to 3 tissue samples are sufficient; S103, the removed tumor tissue block is placed in a pre-cooled centrifuge tube containing a preservation solution, and then placed in a transport box. This process is controlled within 1 minute; S104. Ensure that the sample is completely covered with tissue preservation transport fluid and that the tissue sample is still in a low-temperature environment and immersed in tissue preservation fluid when it arrives at the laboratory. The entire transportation process is controlled in a low-temperature environment of 2~8℃, and the transportation time is less than 72h.

6. The method for culturing breast cancer organoids according to claim 4, characterized in that: The method for constructing the primary tissue in S2 specifically comprises the following steps: S201. After registering the patient information, transfer the tumor tissue to the clean laboratory, sterilize it and place it in the clean bench. Prepare three culture dishes and pour PBS with 1% antibiotics into them. S202, soak the tumor tissue in the first dish, shake and wash, remove dead cells, fat tissue, muscle tissue, and some blood cells with tweezers, transfer the sample to the second dish after removal, cut into pieces the size of rice grains, and wash the tissue with PBS containing double antibodies, then transfer the small tumor tissue pieces to the third culture dish to continue washing the tissue pieces, and finally put them into a centrifuge tube; S203, the tissue blocks in the centrifuge tube were cut into viscous pieces, and then transferred to the tumor tissue digestion solution, and the samples were resuspended by blowing, and then transferred to the shaker, and digested at 37°C for 50 minutes. Every 10 minutes, the sample was beaten with a pipette and 10ul was taken out to observe the digestion status, until the tumor cells were digested out and no tumor tissue blocks were left, and then the next step could be performed; S204, after digestion, add FBS to the digestion solution to stop digestion, mix the liquid, and then filter it with a rinsed filter, then collect the filtered suspension, centrifuge and discard the supernatant, continue to add PBS to wash the precipitate, centrifuge and discard the supernatant, then add PBS to resuspend the cells, and take a portion of it for cell counting and death rate counting, and centrifuge the remaining liquid and discard the supernatant for later use; S205, Matrigel 3D encapsulation: This experimental step is performed on ice. Add the corresponding Matrigel to the cell pellet according to the amount of organoids and cells, and mix well by pipetting on ice; S206. Organoid plate culture: Use the pipette tip to suck up the mixture of extracellular matrix and cells and move it to the center of the bottom of the cell culture plate. Place the culture plate in a cell culture incubator at 37°C and 5% CO2 for incubation. After the extracellular matrix gel solidifies and no longer flows, add breast cancer organoid culture medium and place the plate in a 37°C and 5% CO2 incubator for culture.

7. The method for culturing breast cancer organoids according to claim 4, characterized in that: The method for replacing the fluid in breast cancer organoids in S3 specifically comprises the following steps: S301. Take out the breast cancer organoid culture medium from the 4°C refrigerator and rewarm it at room temperature or in a CO2 incubator. S302, take out the organoid culture cell plate from the cell culture incubator, use a pipette to suck out the original culture medium in the clean bench and discard it into the waste liquid tank, then add the newly warmed breast cancer organoid culture medium; S303. After the liquid is replaced, the cells are observed and photographed under an inverted microscope, and then placed in a CO2 incubator for further culture.

8. The method for culturing breast cancer organoids according to claim 4, characterized in that: The breast cancer organoid subculture method in S4 specifically comprises the following steps: S401. When the breast cancer organoids grow to a diameter of 100-500 uM, which takes 7-14 days, remove the original preservation solution in the clean bench, add DMEM basal culture medium, scrape the mixture of matrix gel and organoids with a pipette tip, transfer to a centrifuge tube, pipette several times, and then centrifuge; S402. After centrifugation, discard the supernatant and add organoid digestion solution 5 times the volume of the extracellular matrix and organoid mixture. Mix well with a pipette and digest at 37°C for 3 min to 6 min. S403, cleaning of organoids: After digestion, add 4-5 times the volume of DMEM basal medium of the digestion solution to terminate digestion, mix by pipetting and centrifugation, remove the supernatant, and wash 1-2 times in this way; S404, 3D encapsulation of breast cancer organoids with matrix gel: This experimental step is performed on ice or in an ice box. According to the amount of organoids and cells, add the corresponding matrix gel to the cell pellet and mix well by blowing on ice to avoid the generation of bubbles and gel coagulation; S405, organoid seed plate culture: move the mixed cell mixture to the center of the bottom of the cell culture well plate, add matrix gel to the wells of the culture plate, and then place the culture plate in a cell culture incubator at 37°C and 5% CO2 for incubation for 20 minutes; S406. After the extracellular matrix gel solidifies and no longer flows, add breast cancer organoid culture medium and place the plate back into the incubator at 37°C and 5% CO2 for culture; change the medium every 3-4 days and subculture when the breast cancer organoids are cultured to a diameter of 100-500 uM.

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