Ovarian cancer organoid-macrophage co-culture system as well as preparation method and application thereof

Abstract

The invention discloses an ovarian cancer organoid-macrophage co-culture system as well as a preparation method and application thereof. The preparation method comprises the following steps: providing ovarian cancer cells; the method comprises the following steps: resuspending ovarian cancer cells in matrigel, then inoculating the matrigel on which the ovarian cancer cells are resuspended into a culture container, after the matrigel is completely solidified, adding the matrigel into an ovarian cancer organoid complete culture medium, culturing to obtain an ovarian cancer organoid wrapped in the matrigel, and separating to obtain the ovarian cancer organoid; providing macrophage M2, mixing the macrophage M2 and the ovarian cancer organoid, and culturing by using a co-culture medium. In combination with a specific embodiment, according to the preparation method of the ovarian cancer organoid-macrophage co-culture system, macrophages M2 and ovarian cancer organoid are mixed and then cultured with a co-culture medium, the obtained ovarian cancer organoid-macrophage co-culture system contains the macrophages M2, and the functions of the macrophages can be researched.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an ovarian cancer organoid-macrophage co-culture system, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is a malignant tumor of the ovary, referring to malignant tumors that grow on the ovary. Because early symptoms are often subtle, early detection and diagnosis of ovarian cancer are difficult, and treatment outcomes are often poor in advanced stages. Therefore, ovarian cancer is one of the most deadly gynecological cancers in women, with a high recurrence rate and poor prognosis, seriously threatening women's health. The immune microenvironment of ovarian cancer exhibits significant immunosuppressive characteristics, especially with tumor-associated macrophages (TAMs) accounting for 30%–50% of the tumor, directly impacting its immune system.

[0003] Organoid technology can cultivate miniature tissues that retain the characteristics of the primary tumor in a three-dimensional matrix using patient tumor tissue. It has the following advantages: it preserves the genomic characteristics of the patient's tumor (such as TP53 mutation); it maintains glandular structure and intercellular interactions; it can reflect individualized drug response and is close to clinical practice. Therefore, organoids are considered to be the most valuable in vitro models for translation between clinical and laboratory settings.

[0004] However, existing ovarian cancer organoid models are mainly composed of tumor epithelial cells and lack key immune cell components in the tumor microenvironment, especially macrophages. Therefore, it is difficult to simulate the role of tumor-associated macrophages (TAMs) in the development and progression of ovarian cancer in vitro.

[0005] Therefore, it is necessary to provide an ovarian cancer organoid-macrophage co-culture system and its preparation method that can reconstruct macrophage components in the ovarian cancer tumor microenvironment in vitro, in order to overcome the technical deficiency of existing ovarian cancer organoid models that lack macrophages. Summary of the Invention

[0006] Therefore, it is necessary to provide an ovarian cancer organoid-macrophage co-culture system and its preparation method that can solve the above problems.

[0007] In addition, it is necessary to provide the application of the above-mentioned ovarian cancer organoid-macrophage co-culture system.

[0008] A method for preparing an ovarian cancer organoid-macrophage co-culture system includes the following steps: Ovarian cancer tissue was provided, cleaned with a washing solution, and then cut to a thickness of 0.5 mm. 3 ~1mm 3 The cells were then digested with tissue digestion fluid, and after digestion, they were separated to obtain ovarian cancer cells. The ovarian cancer cells were resuspended in a matrix gel, and then the matrix gel containing the ovarian cancer cells was seeded into a culture container. After the matrix gel was completely solidified, ovarian cancer organoid complete culture medium was added. The medium was completely or partially changed every 2 to 3 days. After 7 to 14 days of culture, ovarian cancer organoids encapsulated in the matrix gel were obtained. The ovarian cancer organoids were then separated. Macrophages were provided and mixed with ovarian cancer organoids at a cell ratio of 2-5:1. The mixture was then cultured in a co-culture medium with complete or partial medium changes every 1-3 days for 5-7 days to obtain the desired ovarian cancer organoid-macrophage co-culture system. The co-culture medium consisted of 25-100 ng / mL EGF, 0.1-1 μM A83-01, 5-10 mM nicotinamide, 5-10 μM Y-27632, 10-40 ng / mL NRG1, and 1%-4% FBS.

[0009] In one embodiment, the complete culture medium for ovarian cancer organoids consists of 25 ng / mL to 100 ng / mL EGF, 10% to 30% R-Spondin1 conditioned medium, 10% to 40% Wnt3a conditioned medium, 0.1 μM to 1 μM A83-01, 5 mM to 10 mM nicotinamide, 5 μM to 10 μM Y-27632, and 10 ng / mL to 40 ng / mL NRG1.

[0010] In one embodiment, the operation of digesting with tissue digestion solution and separating the cells after digestion to obtain ovarian cancer cells is as follows: digesting sequentially with tissue digestion solution I and tissue digestion solution II, centrifuging the digestion solution to separate the cells, filtering the obtained cells through a 100μm sieve, and centrifuging the filtrate again to obtain the ovarian cancer cells.

[0011] In one embodiment, the washing solution is DPBS + 1% Pen-Strep Solution, and the matrix gel is Matrigel, BME, or a synthetic hydrogel.

[0012] In one embodiment, the method further includes staining the ovarian cancer organoids with a first fluorescent dye; And / or, the macrophages are cells stained with a second fluorescent cell dye, wherein the fluorescence colors of the first fluorescent dye and the second fluorescent dye are different.

[0013] In one embodiment, the macrophages are peripheral blood macrophages, TIL-derived macrophages, ascites macrophages, or THP-1-derived macrophages.

[0014] In one embodiment, the macrophage is macrophage M2; The procedure for providing macrophages is as follows: peripheral blood is provided, and mononuclear cells are isolated from it. The mononuclear cells are stimulated with M-CSF to obtain macrophages M0. Macrophages M0 are induced with LPS+IFN-γ to obtain macrophages M1. Then, macrophages M1 are induced with IL-4+IL-13 to obtain macrophages M2.

[0015] In one embodiment, in the process of inducing macrophage M0 with LPS+IFN-γ to obtain macrophage M1, and then inducing macrophage M1 with IL-4+IL-13 to obtain macrophage M2, the concentration of LPS is 100 ng / mL, the concentration of IFN-γ is 10 g / mL to 20 ng / mL, the concentration of IL-4 is 10 ng / mL to 20 ng / mL, and the concentration of IL-13 is 10 ng / mL to 20 ng / mL.

[0016] An ovarian cancer organoid-macrophage co-culture system was prepared by the above-described method for preparing an ovarian cancer organoid-macrophage co-culture system.

[0017] Application of an ovarian cancer organoid-macrophage co-culture system in cancer drug screening, immunomodulator screening, cancer drug efficacy prediction, and immunomodulator screening.

[0018] In conjunction with specific embodiments, the preparation method of the ovarian cancer organoid-macrophage co-culture system of the present invention involves mixing macrophages and ovarian cancer organoids and culturing them in a co-culture medium. The resulting ovarian cancer organoid-macrophage co-culture system contains macrophages and can reconstruct the macrophage component in the ovarian cancer tumor microenvironment in vitro. This overcomes the technical deficiency of existing ovarian cancer organoid models that lack macrophages. It can also be used to study macrophage function (tumor-associated macrophage (TAM) formation mechanism, molecular mechanism of macrophage promoting tumor growth, and macrophage polarization sensitivity to tumors).

[0019] Furthermore, compared with traditional ovarian cancer organoids, the ovarian cancer organoid-macrophage co-culture system prepared by the method of the present invention contains macrophages, which can simulate a more realistic microenvironment, quantify macrophage behavior, and improve its accuracy for drug evaluation. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] in: Figure 1 This is a flowchart illustrating a method for preparing an ovarian cancer organoid-macrophage co-culture system according to one embodiment.

[0022] Figure 2a and Figure 2b A photomicrograph of ovarian cancer cells cultured in ovarian cancer organoid complete culture medium on day 2.

[0023] Figure 3a and Figure 3b A photomicrograph of ovarian cancer cells cultured in ovarian cancer organoid complete culture medium on day 6.

[0024] Figure 4a and Figure 4b A photomicrograph of ovarian cancer cells cultured in ovarian cancer organoid complete culture medium on day 9.

[0025] Figure 5a and Figure 5b This is a pathological image of an ovarian cancer organoid after HE staining.

[0026] Figure 6a and Figure 6b The image shows the CK7 detection results after IHC staining of ovarian cancer organoids.

[0027] Figure 7a and Figure 7b Image showing CA125 detection results after IHC staining of ovarian cancer organoids.

[0028] Figure 8a and Figure 8b The image shows the P53 detection results after IHC staining of ovarian cancer organoids.

[0029] Figure 9a and Figure 9b Image showing the Ki67 detection results after IHC staining of ovarian cancer organoids.

[0030] Figure 10a and Figure 10b Micrograph of ovarian cancer cell line A2780 after administration of KPT9274 (50 μM).

[0031] Figure 11a and Figure 11bMicrograph of ovarian cancer cell line A2780 after administration of KPT9274 (5 μM).

[0032] Figure 12 Cell viability graph of ovarian cancer cell line A2780 after being treated with KPT9274.

[0033] Figure 13a and Figure 13b Micrograph of KPT9274 (100 μM) administered to ovarian cancer organoids.

[0034] Figure 14a and Figure 14b Micrograph of KPT9274 (25 μM) administered to ovarian cancer organoid A2780.

[0035] Figure 15 Cell viability graph of KPT9274 administered to ovarian cancer organoids.

[0036] Figure 16a and Figure 16b Photomicrograph of cisplatin (100 μM) administered to ovarian cancer organoids.

[0037] Figure 17a and Figure 17b Photomicrograph of cisplatin (6.25 μM) administered to ovarian cancer organoid A2780.

[0038] Figure 18 Cell viability graph of cisplatin administered to ovarian cancer organoids.

[0039] Figure 19 Fluorescence micrographs of ovarian cancer organoids cultured on day 1 after the addition of caspase 3 / 7 fluorescent dye.

[0040] Figure 20 Fluorescence micrographs of ovarian cancer organoids cultured on day 2 after the addition of caspase 3 / 7 fluorescent dye.

[0041] Figure 21 Fluorescence micrographs of ovarian cancer organoids cultured on day 3 after the addition of caspase 3 / 7 fluorescent dye.

[0042] Figure 22 The image shows the flow cytometry results of macrophage M1 cells from group BC.

[0043] Figure 23 The image shows the flow cytometry results of macrophage M1 cells in the experimental group.

[0044] Figure 24 The image shows the flow cytometry results of macrophage M2 cells from group BC.

[0045] Figure 25The image shows the flow cytometry results of macrophage M2 cells in the experimental group.

[0046] Figure 26 Fluorescence micrograph of ovarian cancer organoids and macrophage M2 cells on day 1 after co-culture.

[0047] Figure 27 Fluorescence micrograph of ovarian cancer organoids and macrophage M2 cells on day 2 after co-culture.

[0048] Figure 28 Fluorescence micrograph of ovarian cancer organoids and macrophage M2 cells on day 3 after co-culture. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Combination Figure 1 The present invention discloses a method for preparing an ovarian cancer organoid-macrophage co-culture system according to one embodiment, comprising the following steps: S10. Provide ovarian cancer tissue, clean it thoroughly with a cleaning solution, and then cut it to 0.5mm. 3 ~1mm 3 The cells were then digested with tissue digestion fluid, and after digestion, they were separated to obtain ovarian cancer cells.

[0051] Specifically, ovarian cancer tissue is provided to the patient. After the ovarian cancer tissue is obtained, fatty tissue and normal tissue are removed, leaving only the lesion.

[0052] Preferably, in this embodiment, the procedure of digesting with tissue digestion solution and separating after digestion to obtain ovarian cancer cells is as follows: digesting sequentially with tissue digestion solution I and tissue digestion solution II, centrifuging the digestion solution to separate the cells, filtering the obtained cells through a 100μm sieve, and centrifuging the filtrate again to obtain ovarian cancer cells.

[0053] Tissue digestion solution I is a tissue digestion solution containing collagenase and / or neutral protease, while tissue digestion solution II is a tissue digestion solution containing trypsin and / or DNase. The two solutions differ in digestion intensity and duration and are used for staged digestion of tumor tissue.

[0054] Specifically, tissue digestion solution I is mainly used for preliminary digestion of tissues to loosen their structure, while tissue digestion solution II is used for further digestion to obtain single cells or small cell clusters. The sequential digestion of these two solutions helps to improve the efficiency of ovarian cancer cell isolation and reduce cell damage.

[0055] Specifically, the ovarian cancer tissue was digested sequentially with tissue digestion solution I and tissue digestion solution II in a shaker at 37°C.

[0056] Specifically, if the obtained ovarian cancer cells contain a large number of red blood cells (the cell precipitate is red), red blood cell lysis buffer can be added to perform red blood cell lysis (refer to the red blood cell lysis buffer product instructions for specific experimental methods).

[0057] Preferably, in this embodiment, the cleaning solution is DPBS + 1% Pen-Strep Solution. In other embodiments, other types of cleaning solutions may also be selected.

[0058] S20. The ovarian cancer cells obtained in S10 are resuspended in a matrix gel. Then, the matrix gel containing the resuspended ovarian cancer cells is seeded into a culture container. After the matrix gel has completely solidified, ovarian cancer organoid complete culture medium is added. The medium is completely or partially changed every 2 to 3 days. After culturing for 7 to 14 days, ovarian cancer organoids encapsulated in the matrix gel are obtained. After isolation, ovarian cancer organoids are obtained.

[0059] Preferably, in this embodiment, the complete culture medium for ovarian cancer organoids consists of 25 ng / mL to 100 ng / mL EGF, 10% to 30% R-Spondin1 conditioned medium, 10% to 40% Wnt3a conditioned medium, 0.1 μM to 1 μM A83-01, 5 mM to 10 mM nicotinamide, 5 μM to 10 μM Y-27632, and 10 ng / mL to 40 ng / mL NRG1.

[0060] Preferably, in this embodiment, the matrix gel is Matrigel, BME, or a synthetic hydrogel.

[0061] Specifically, in this embodiment, it can be calculated as 1*10 4 ~2*10 4 Ovarian cancer cells were resuspended in matrix gel at a density of cells / mL.

[0062] In other implementations, the cell density can be adjusted according to actual needs.

[0063] The procedure for seeding ovarian cancer cell-resuspended matrix gel into a culture vessel is as follows: Place the matrix gel resuspended with ovarian cancer cells into a cell culture plate preheated to 37°C (ensure the pipette tip does not reach the bottom of the culture plate), and seed the gel droplet in the center of each well. If the cell culture plate is a 24-well plate, seed 50 μL per well.

[0064] Preferably, this embodiment also includes the operation of staining ovarian cancer organoids with a first fluorescent dye.

[0065] Specifically, the procedure for staining ovarian cancer organoids with the first fluorescent dye is as follows: remove the matrix gel with DPBS, collect the ovarian cancer organoids from the gel droplets, add the first fluorescent dye to the complete culture medium for ovarian cancer organoids, resuspend the ovarian cancer organoids, and complete the staining.

[0066] The first fluorescent dye can be a caspase 3 / 7 fluorescent dye.

[0067] S30. Provide macrophages and mix them with the ovarian cancer organoids obtained in S20 at a cell ratio of 2-5:1. Then, culture them in a co-culture medium and perform a complete or partial medium change every 1-3 days. After culturing for 5-7 days, the desired ovarian cancer organoid-macrophage co-culture system is obtained.

[0068] The co-culture medium consisted of 25 ng / mL to 100 ng / mL EGF, 0.1 μM to 1 μM A83-01, 5 mM to 10 mM nicotine amide, 5 μM to 10 μM Y-27632, 10 ng / mL to 40 ng / mL NRG1, and 1% to 4% FBS.

[0069] It should be noted that, to promote the interaction between immune cells and ovarian cancer organoids, the co-culture medium used was an organoid basal medium with Wnt3a and / or R-spondin1 removed, further supplemented with 1%–4% FBS. Removing Wnt3a and / or R-spondin1 helps reduce the excessive stemness maintenance state of the organoids, thereby enhancing their response to exogenous immune cell signals. The addition of FBS provides basic nutrition and supports the survival and functional maintenance of immune cells under co-culture conditions.

[0070] Preferably, in this embodiment, the macrophages are peripheral blood macrophages, TIL-derived macrophages, ascites macrophages, or THP-1-derived macrophages.

[0071] More preferably, in this embodiment, the macrophage is macrophage M2. The operation of providing macrophage M2 is as follows: peripheral blood is provided and mononuclear cells are isolated from it. The mononuclear cells are stimulated with M-CSF to obtain macrophage M0. Macrophage M0 is induced with LPS+IFN-γ to obtain macrophage M1. Then, macrophage M1 is induced with IL-4+IL-13 to obtain macrophage M2.

[0072] Specifically, in this embodiment, macrophage M0 is induced with LPS+IFN-γ to obtain macrophage M1, and then macrophage M1 is induced with IL-4+IL-13 to obtain macrophage M2. The concentration of LPS is 100 ng / mL, the concentration of IFN-γ is 10 g / mL~20 ng / mL, the concentration of IL-4 is 10 ng / mL~20 ng / mL, and the concentration of IL-13 is 10 ng / mL~20 ng / mL.

[0073] Obtaining macrophages M0 may include: obtaining peripheral blood mononuclear cells (PBMCs) from peripheral blood samples by density gradient centrifugation, followed by enriching the monocyte population using immunomagnetic bead sorting, for example, using immunomagnetic beads targeting CD14 and / or CD16 for positive selection of monocytes (or using negative selection methods that remove CD3 / CD19 / CD56, etc.) to obtain monocytes; seeding the monocytes in macrophage differentiation medium and adding macrophage differentiation factors (e.g., M-CSF, with a final concentration of 25–100 ng / mL), and culturing for 5–7 days to obtain unpolarized M0 macrophages.

[0074] Based on this, macrophages in different polarization states can be further induced: M0 macrophages are added to M1 polarization-inducing factors (e.g., IFN-γ and / or LPS, cultured for 24–72 h) to obtain M1-like macrophages; or M2 polarization-inducing factors (e.g., IL-4 and / or IL-13, cultured for 24–96 h) are added to obtain M2-like macrophages. To improve the purity of different polarization populations, in some embodiments, immunomagnetic beads can be used for further enrichment of the corresponding cell populations after induction, for example, using immunomagnetic beads targeting M1-related surface markers (e.g., CD80 / CD86) or M2-related surface markers (e.g., CD163 / CD206) for positive selection or negative removal, thereby obtaining purified M1-like or M2-like macrophage populations.

[0075] In other implementations, alternative methods such as GM-CSF and IL-10 can be used to induce macrophage M0 to polarize into macrophage M2.

[0076] Preferably, in this embodiment, the macrophages are cells stained with the second fluorescent cell dye, and the fluorescence colors of the first and second fluorescent dyes are different.

[0077] Specifically, in this embodiment, red fluorescent dye can be added to the culture medium to resuspend macrophages M0 and complete the staining. After staining, macrophages M0 can continue to undergo subsequent induction of polarization.

[0078] The second fluorescent dye can be CellTracker Green / Red.

[0079] In other embodiments, the first fluorescent dye and the second fluorescent dye may also be DiO, DiI, DiD, etc.

[0080] In conjunction with specific embodiments, the preparation method of the ovarian cancer organoid-macrophage co-culture system of the present invention involves mixing macrophages and ovarian cancer organoids and culturing them in a co-culture medium. The resulting ovarian cancer organoid-macrophage co-culture system contains macrophages and can reconstruct the macrophage component in the ovarian cancer tumor microenvironment in vitro. This overcomes the technical deficiency of existing ovarian cancer organoid models that lack macrophages. It can also be used to study macrophage function (tumor-associated macrophage (TAM) formation mechanism, molecular mechanism of macrophage promoting tumor growth, and macrophage polarization sensitivity to tumors).

[0081] Furthermore, compared with traditional ovarian cancer organoids, the ovarian cancer organoid-macrophage co-culture system prepared by the method of the present invention contains macrophages, which can simulate a more realistic microenvironment, quantify macrophage behavior, and improve its accuracy for drug evaluation.

[0082] The ovarian cancer organoid-macrophage co-culture system of the present invention can also be used for 1. Observe the infiltration / aggregation behavior of macrophages into organoids; 2. Simulating the effects of M1 / M2 polarization on the tumor microenvironment; 3. Detect macrophage-induced organoid proliferation, apoptosis, and migration changes.

[0083] The present invention also discloses an embodiment of an ovarian cancer organoid-macrophage co-culture system prepared by the above-described method for preparing an ovarian cancer organoid-macrophage co-culture system.

[0084] This invention also discloses the application of the above-mentioned ovarian cancer organoid-macrophage co-culture system in the fields of cancer drug screening, immunomodulator screening, cancer drug efficacy prediction, and immunomodulator screening.

[0085] Specifically, the above-mentioned ovarian cancer organoid-macrophage co-culture system can be used for screening and efficacy prediction of CSF1R inhibitors (blocking TAM recruitment), screening and efficacy prediction of PI3Kγ inhibitors (inhibiting immunosuppressive M2), screening and efficacy prediction of JAK / STAT inhibitors (regulating inflammatory responses), and screening and efficacy prediction of anti-IL-6 inhibitors.

[0086] The experimental procedure is roughly as follows: drug treatment → co-culture → imaging → throughput analysis, which can be used for drug screening and efficacy prediction.

[0087] The following are specific examples.

[0088] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0089] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0090] The ovarian cancer tissue and peripheral blood used in the following examples were all obtained from a tertiary hospital that has obtained ethical approval.

[0091] MasterAim® ovarian cancer organoid complete culture medium consists of 60 ng / mL EGF, 20% R-Spondin1 conditioned medium, 25% Wnt3a conditioned medium, 0.5 μM A83-01, 7.5 mM nicotinamide, 7.5 μM Y-27632, and 25 ng / mL NRG1.

[0092] The co-culture medium consisted of 60 ng / mL EGF, 0.5 μM A83-01, 7.5 mM nicotine amide, 7.5 μM Y-27632, 25 ng / mL NRG1, and 2.5% FBS.

[0093] Example 1: Construction of Ovarian Cancer Organoids 1) Transport of ovarian cancer tissue from patients Ovarian cancer tissue was obtained from the patient during surgery; no chemotherapy had been administered prior to the procedure. The fresh tissue was trimmed to a volume of approximately 0.5 cm. 3 Then place it in a specimen storage tube. (Note: If the tissue block is too large, it will affect the exchange of nutrients in the tissue transport and preservation solution, thus affecting the specimen's viability. The tissue specimen needs to be trimmed to a volume of approximately 0.5 cm.) 3 Add sufficient MasterAim® tissue transport and preservation solution (#100-049) to immerse the tissue, transport it in an ice box, and transport it to the laboratory for processing as soon as possible.

[0094] 2) Construction of ovarian cancer organoids from patients 1. Transfer the tissue specimen to a 3.5cm culture dish, place the culture dish on an ice box, add 2 mL of washing solution (DPBS + 1% Pen-Strep Solution) to remove adipose tissue and normal tissue, leaving only the lesion.

[0095] 2. Wash the specimen with the cleaning solution, rinsing repeatedly until the solution is clear, approximately 5-10 times. Remove the cleaning solution, add MasterAim® Tissue Digestion Solution I (#100-050) (approximately 200 μL), and cut the specimen to approximately 0.5 mm. 3 ~1 mm 3 Size.

[0096] 3. Transfer the chopped tissue to a 15 mL centrifuge tube, add 4 mL of MasterAim® Tissue Digestion Solution I, and digest on a shaker at 37°C for 30-40 min. Observe every 15 min to see if there are large areas of cells leaking out. The digestion time of this step may be adjusted according to the condition of the specimen.

[0097] 4. After digestion with digestion solution I is complete, add 8 mL of washing solution to stop digestion, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 2 mL of MasterAim® tissue digestion solution II (#100-051), and digest at 37℃ on a shaker for 10 min.

[0098] 5. After digestion, add 4 mL of washing solution to stop digestion, centrifuge at 1200 rpm for 5 min, and collect the cells.

[0099] 6. Discard the supernatant, add 2 mL of washing buffer to resuspend the cells, pre-wet a 100 μm cell sieve with 2 mL of washing buffer, filter the cell suspension through the cell sieve, and centrifuge the filtrate at 1200 rpm for 5 min.

[0100] 7. Cell counting: Count the number of cell clusters using an automated cell counter, according to a 1*10⁻⁶ cell count. 4 Resuspend the cells in Matrigel at a density of 50 μL / mL. Seed the resuspended cells into 24-well cell culture plates preheated to 37°C, ensuring the pipette tip does not reach the bottom of the plate and the droplet is placed in the center of each well.

[0101] 8. Let the culture plate stand at 37°C for 30 min to allow the matrix gel to completely solidify, and gently add 500 μL of MasterAim® ovarian cancer organoid complete culture medium preheated at 37°C to each well.

[0102] 9. Add sterile DPBS to the other uninoculated droplets in the wells to maintain humidity during incubation, cover the culture plate, and incubate at 37°C and 5% CO2.

[0103] 10. Observe the culture of organoids under a microscope, completely changing the medium every 3 days. Take pictures on the 2nd, 6th, and 9th days, and the resulting micrographs are shown below. Figure 2a , Figure 2b , Figure 3a , Figure 3b , Figure 4a and Figure 4b As shown. Before passage, ensure that most organoids are larger than 100 μm in size.

[0104] Figure 2a , Figure 2b , Figure 3a , Figure 3b , Figure 4a and Figure 4b Organoid formation was observed 3 days after inoculation, and structurally complete ovarian cancer organoids were formed 9 days after inoculation, which retained the glandular structure and mutation characteristics of the primary tumor.

[0105] 3) Cryopreservation of ovarian cancer organoids derived from patients 1. Observe the growth of organoids. When the organoids are of good activity, the number of live cells per milliliter should not be less than 5*10. 5 Cryopreservation can be carried out at this time, with each tube of organoids containing approximately 5*10 cells. 5 If there are many cells, care should be taken to freeze them separately in individual tubes.

[0106] 2. Carefully aspirate the culture medium from the wells, add an appropriate amount of TrypLE Express Enzyme (1X), disperse the droplets, and incubate at 37°C for 3 min. Observe the organoid digestion under a microscope. When the organoids have digested to a size of 40-60 μm, add 2-3 times the volume of digestion solution (DPBS) to stop the digestion. Centrifuge at 1200 rpm for 5 min and discard the supernatant. Resuspend the cells in 3 mL of DPBS, centrifuge at 1200 rpm for 5 min, and discard the supernatant.

[0107] 3. Add organoid cryopreservation solution (#100-006) to resuspend the precipitate, mix well, and dispense 1 mL into labeled cryovials. Place the cryovials in a programmed cooling box and perform gradient cooling: 4℃ (20 min), -20℃ (2 h), -80℃ (overnight), and finally transfer to a liquid nitrogen tank.

[0108] 4) Passaging of ovarian cancer organoids derived from patients The growth and morphological changes of organoids were observed regularly using an inverted microscope (DMi1, Leica, USA), and the size and area of ​​organoids were measured using ImageJ software. When the density exceeded 80%, passage was required. Cells were dissociated using digestion solution, centrifuged to remove the supernatant, and digestion was terminated by adding an equal proportion of culture medium. After counting, cells were processed according to a 1*10⁻⁶ ratio. 4 Cells were resuspended in a matrix gel (BME or Matrigel) at a density of 1 / mL and then cultured in fresh culture medium.

[0109] 5) Identification of organoids: HE and IHC staining of ovarian cancer organoids. 1. Paraffin-embedded sections of ovarian cancer organoids Organoids in the gel droplets were fixed with 4% paraformaldehyde, and the fixed organoids were collected and embedded in agarose. The solidified agarose was then placed in a dehydrator for dehydration and paraffin impregnation. The paraffin-impregnated agarose blocks were then placed in an embedding machine for paraffin embedding, and after cooling and solidification, they were sectioned using a microtome.

[0110] 2. HE and IHC staining of ovarian cancer organoids First, dewax the paraffin sections: place the sections in a 70℃ oven and bake until the paraffin melts. Then, immerse the organoid sections in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol in sequence, and then rinse them with ultrapure water.

[0111] HE staining: After dewaxing, the sections were stained sequentially with hematoxylin for nuclear staining and eosin for cytoplasmic staining. Then, the sections were sequentially immersed in 85% ethanol, 95% ethanol, anhydrous ethanol, xylene I, and xylene II. After air drying, the sections were mounted, photographed, and stored. Figure 5a and Figure 5b .

[0112] IHC staining: After dewaxing, the sections underwent antigen retrieval, peroxidase blocking, blocking, incubation with primary antibody, incubation with secondary antibody, DAB staining, hematoxylin counterstaining of cell nuclei, washing, and sequential immersion in 85% ethanol-95% ethanol-anhydrous ethanol-xylene. After air drying, the sections were mounted, photographed, and stored. Figure 6a , Figure 6b , Figure 7a , Figure 7b , Figure 8a , Figure 8b , Figure 9a and Figure 9b The tested substances were CK7, CA125, P53, and Ki67.

[0113] Combination Figure 6a , Figure 6b , Figure 7a ,Figure 7b , Figure 8a , Figure 8b , Figure 9a and Figure 9b As can be seen, the structurally intact ovarian cancer organoids prepared in this embodiment retain the glandular structure and mutation characteristics of the primary tumor.

[0114] 6) Primary drug susceptibility testing of ovarian cancer organoids 1. Experimental Grouping Ovarian cancer cell line A2780 is generally considered to have high sensitivity to cisplatin, with an IC50 of 1–5 μM (this conclusion has been validated in multiple publicly available drug screening databases and literature). In this invention, the above cell line was selected as a drug sensitivity control model. Ovarian cancer cell line A2780 is a commonly used human ovarian cancer cell line and was purchased from Guangzhou Magic Biotechnology Co., Ltd.

[0115] Experimental group 1: Ovarian cancer organoid complete culture medium + ovarian cancer organoids + KPT9274 (concentrations: 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3 replicates per concentration); Experimental group 2: Ovarian cancer organoid complete culture medium + ovarian cancer organoids + cisplatin (concentrations: 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3 replicates per concentration); Control group: Ovarian cancer cell culture medium + ovarian cancer cell line A2780 + KPT9274 (concentrations: 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3 replicates per concentration); Negative control group: culture medium + organoids; blank control group: culture medium.

[0116] 2. ATP detection The collected primary cells were grouped according to the experimental groupings described above, at a ratio of 3*102. 4 Cells were seeded at a density of 200 μL of ovarian cancer organoid complete culture medium into each well of a 96-well plate. After 72 h of culture, the experimental group was given the drug. After 72 h of drug treatment, ATP was added to detect the data, and the results were analyzed using a microplate reader.

[0117] The test results of the control group are as follows Figure 10a , Figure 10b , Figure 11a , Figure 11b , Figure 12 As shown, the test results of experimental group 1 Figure 13a , Figure 13b , Figure 14a , Figure 14b ,Figure 15 The test results of experimental group 2 Figure 16a , Figure 16b , Figure 17a , Figure 17b and Figure 18 As shown.

[0118] Combination Figure 19 , Figure 20 , Figure 21 and Figure 19 It can be seen that as the concentration of KPT9274 increases, the cell density of the ovarian cancer cell line A2780 gradually decreases. The cells, which were originally adherent and morphologically intact, gradually exhibited phenomena such as cell shrinkage, rounding, detachment, and increased fragmentation, indicating a significant decrease in cell viability. Furthermore, this change showed a dose-dependent trend. Combined with… Figure 20 The cell viability assay results showed that KPT9274 had a significant inhibitory effect on A2780 cells, and the cell survival rate gradually decreased with increasing drug concentration. According to the dose-response curve fitting analysis, the optimal drug concentration of KPT9274 for the ovarian cancer cell line A2780 was 4.233 μM.

[0119] Combination Figure 21 , Figure 22 , Figure 23 and Figure 24 It can be seen that the overall structure of ovarian cancer organoids changed significantly after KPT9274 treatment. With increasing drug concentration, the organoids, which were initially relatively intact and well-defined, gradually became smaller and more loosely structured, with some organoids showing collapse or disintegration, indicating that organoid growth was significantly inhibited. Combined with… Figure 25 Cell viability assay results showed that KPT9274 had a significant inhibitory effect on ovarian cancer organoids, and the inhibitory effect increased with increasing drug concentration. Dose-response curve analysis indicated that the optimal dosage concentration of KPT9274 for ovarian cancer organoids was 32.32 μM.

[0120] Combination Figure 26 , Figure 27 , Figure 28 and ​ It can be seen that the morphology and structure of ovarian cancer organoids changed significantly after cisplatin treatment. With increasing cisplatin concentration, the number of organoids gradually decreased, and the structure changed from dense and intact to loose and fragmented; some organoids showed obvious disintegration, suggesting that cisplatin has a significant growth inhibitory effect on ovarian cancer organoids. ​ The cell viability assay results showed that the inhibitory effect of cisplatin on ovarian cancer organoids was dose-dependent. According to dose-response curve analysis, the optimal concentration of cisplatin for ovarian cancer organoids was 5.563 μM.

[0121] The above results indicate that drug treatment can cause morphological changes and decreased cell viability in both two-dimensional ovarian cancer cell lines and three-dimensional ovarian cancer organoid models. Furthermore, the morphological observation results are consistent with the quantitative detection results, verifying the feasibility and stability of the model in drug sensitivity evaluation.

[0122] Example 2: Co-culture of ovarian cancer organoids and macrophages 1) Culture, polarization, expansion, and fluorescent labeling of macrophages After peripheral blood anticoagulation, PBMCs were separated by density gradient centrifugation. PBMCs were washed 1–2 times, counted, and their concentration adjusted. Monocytes were seeded into 24-well plates, and macrophage culture medium containing M-CSF was added. The plates were incubated at 37°C and 5% CO2, with partial or complete medium changes every 1–3 days. PBMCs / monocytes were cultured in the presence of M-CSF for 4–7 days to obtain M0 cells. Cells were collected for subsequent polarization or co-culture (using a gentle cell scraper / enzyme digestion to avoid high damage).

[0123] M1 polarization: After M0 cells have adhered and stabilized, discard the original culture medium. Add polarization medium containing LPS and IFN-γ. Continue culturing for approximately 24–48 h (depending on experimental requirements).

[0124] M2 Polarization: After M0 cells have adhered and stabilized, discard the original culture medium. Add polarization medium containing IL-4 and IL-13. Continue culturing for approximately 24–48 h. The concentrations are: LPS 100 ng / mL, IFN-γ 10–20 ng / mL, IL-4 10–20 ng / mL, and IL-13 10–20 ng / mL.

[0125] After centrifugation to collect cultured macrophages, the supernatant was discarded, and the macrophages were resuspended in a culture medium containing a fluorescent tracer dye, CellTracker Red. The resuspended cells were incubated at 37 ℃ in the dark for 30 min to complete the fluorescent labeling. After incubation, the cells were collected by centrifugation, the supernatant was discarded, and the cells were washed 4–5 times with 1 mL of DPBS to remove unbound fluorescent dye, finally obtaining fluorescently labeled macrophages.

[0126] 2) Fluorescent labeling of ovarian cancer organoids The matrix gel was removed with cold DPBS, and the ovarian cancer organoids in the gel droplets were collected. The precipitate was collected by centrifugation. In some embodiments, to facilitate the differentiation of different cell populations in the co-culture system, the fluorescent labeling can be achieved by adding a cell-tracing fluorescent dye to the M cell culture system. The final concentration of the fluorescent dye can be 0.5 μM–5 μM, and the labeling time can be 10 min–40 min; the labeling process can be carried out under light-protected conditions. After resuspending the ovarian cancer organoid precipitate, the ovarian cancer organoids were added to a 96-well low-adsorption culture plate at a density of approximately 2*104 cells per well. Images were taken on days 1, 2, and 3 during the culture process to obtain... ​ , ​ and ​ .

[0127] Combination ​ , ​ and ​ It can be seen that the ovarian cancer organoids labeled with CellTracker Green fluorescent dye maintained a stable fluorescence signal during the culture process. The fluorescence signal could be clearly observed on the first, second and third days of culture, and the fluorescence distribution was consistent with the spatial position of the organoids. No obvious fluorescence diffusion or background increase was observed.

[0128] As the culture time was extended, the overall morphology of the ovarian cancer organoids remained intact, without large-area structural damage or rapid disappearance, indicating that the fluorescent labeling process did not have a significant adverse effect on the survival and structural stability of the organoids.

[0129] The above results demonstrate that the fluorescent labeling method used in this invention can achieve effective labeling and continuous tracking of organoids without significantly affecting the activity of ovarian cancer organoids, and is suitable for subsequent co-culture observation and dynamic analysis with immune cells.

[0130] 3) PBMC-induced macrophages and flow cytometry identification PBMCs separated by Ficoll separation solution were processed at a ratio of 1*10 6 Macrophages were resuspended in X-VIVO medium at a density of 100 ng / mL and collected after stimulation with 100 ng / mL M-CSF for 6 days. They were then induced with 100 ng / mL LPS and 20 ng / mL IFN-γ for 48 h to obtain the M1 genotype, and induced with 20 ng / mL IL-4 to obtain the M2 genotype.

[0131] Flow cytometry experiments were conducted in two groups: a blank group (unstained with antibody, BC group) and an experimental group (containing antibody M1 for CD80 typing and antibody M1 for CD206 typing). Cells were incubated at 4°C for 30 minutes in the dark. After centrifugation (300-400g, 5 min) with buffer added, the supernatant was discarded, and this process was repeated 1-2 times. Cells were resuspended in 500 μL of buffer and immediately analyzed by flow cytometry. ​ , ​ , ​ and ​ The gatening strategy involves using FSC / SSC to gate lymphocytes → CD86. + M1 type or CD206 + M2 classification.

[0132] Figure 22 shows the blank control (BC group) without antibody. After FSC / SSC gating, only background fluorescence signal was detected, and no specific expression of CD86 or CD206 was observed, indicating that the flow cytometry detection system has low background and less non-specific binding.

[0133] As shown in Figure 23, after induction with LPS and IFN-γ, the proportion of CD86 positive cells in macrophages increased significantly, while the expression level of CD206 was low, suggesting that PBMC-derived monocytes had been successfully polarized into M1 type macrophages.

[0134] Figure 24 shows the control group for M2 flow cytometry detection. No obvious positive signal was observed in the CD206 detection channel, further verifying that the antibody detection has good specificity.

[0135] As can be seen from Figures 22, 23, 24 and 25, after IL-4 induction, the proportion of CD206 positive cells in macrophages increased significantly, while the expression level of CD86 was low. This indicates that PBMC-derived monocytes can be stably induced and polarized into M2 type macrophages, and the flow cytometry identification results are clear and reliable.

[0136] 4) Co-culture of ovarian cancer organoids and macrophages Labels: M2 (red), organoids (green). Inoculate the organoids into a fixed number per well on a low-absorption plate; add co-culture medium.

[0137] Mix M2 and organoids (based on cell count) at a ratio of 3:1, gently stir, and let stand for 20 minutes to promote contact before returning to the incubator. Perform a complete medium change every two days. Take photos on days 1, 2, and 3 during the culture process to obtain... ​ , ​ and ​ Among them, green fluorescence represents ovarian cancer organoids, and red represents macrophage M2.

[0138] As shown in Figure 26, red fluorescently labeled M2 macrophages and green fluorescently labeled ovarian cancer organoids were observed to coexist on the first day of co-culture, and the two were spatially adjacent, indicating that the co-culture system was successfully established.

[0139] Figure 27 shows that on day 2 of co-culture, M2 macrophages remained viable and continued to be distributed around the organoids, indicating that the co-culture conditions can support the co-existence of the two cell types. Combined with Figures 26 and 27, it can be seen that during the three consecutive days of co-culture, both the fluorescently labeled ovarian cancer organoids and M2 macrophages remained stable and coexisted spatially without significant cell shedding or mass death, demonstrating that the established ovarian cancer organoid-M2 macrophage co-culture system has good stability and reproducibility.

[0140] In the description of the embodiments of the present invention, it should be noted that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of the present invention. In addition, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0141] In the description of the embodiments of the present invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention based on the specific circumstances.

[0142] In embodiments of the present invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0143] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an ovarian cancer organoid-macrophage co-culture system, characterized in that, Includes the following steps: Ovarian cancer tissue was provided, cleaned with a washing solution, and then cut to a thickness of 0.5 mm. 3 ~1mm 3 The cells were then digested with tissue digestion fluid, and after digestion, they were separated to obtain ovarian cancer cells. The ovarian cancer cells were resuspended in a matrix gel, and then the matrix gel containing the ovarian cancer cells was seeded into a culture container. After the matrix gel was completely solidified, ovarian cancer organoid complete culture medium was added. The medium was completely or partially changed every 2 to 3 days. After 7 to 14 days of culture, ovarian cancer organoids encapsulated in the matrix gel were obtained. The ovarian cancer organoids were then separated. Macrophages were provided and mixed with ovarian cancer organoids at a cell ratio of 2-5:

1. The mixture was then cultured in a co-culture medium with complete or partial medium changes every 1-3 days for 5-7 days to obtain the desired ovarian cancer organoid-macrophage co-culture system. The co-culture medium consisted of 25-100 ng / mL EGF, 0.1-1 μM A83-01, 5-10 mM nicotinamide, 5-10 μM Y-27632, 10-40 ng / mL NRG1, and 1-4% FBS.

2. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to claim 1, characterized in that, The complete culture medium for ovarian cancer organoids consisted of 25 ng / mL to 100 ng / mL EGF, 10% to 30% R-Spondin1 conditioned medium, 10% to 40% Wnt3a conditioned medium, 0.1 μM to 1 μM A83-01, 5 mM to 10 mM nicotinamide, 5 μM to 10 μM Y-27632, and 10 ng / mL to 40 ng / mL NRG1.

3. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to claim 2, characterized in that, The procedure for digesting with tissue digestion solution and separating the cells after digestion to obtain ovarian cancer cells is as follows: digesting sequentially with tissue digestion solution I and tissue digestion solution II, centrifuging the digestion solution to separate the cells, filtering the obtained cells through a 100μm sieve, and centrifuging the filtrate again to obtain the ovarian cancer cells.

4. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to claim 3, characterized in that, The cleaning solution is DPBS + 1% Pen-Strep Solution, and the matrix gel is Matrigel, BME, or a synthetic hydrogel.

5. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to claim 3, characterized in that, It also includes the operation of staining the ovarian cancer organoids with a first fluorescent dye; And / or, the macrophages are cells stained with a second fluorescent cell dye, wherein the fluorescence colors of the first fluorescent dye and the second fluorescent dye are different.

6. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to any one of claims 1 to 5, characterized in that, The macrophages are macrophages derived from peripheral blood, TILs, ascites, or THP-1.

7. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to claim 6, characterized in that, The macrophages are macrophages M2; The procedure for providing macrophages is as follows: peripheral blood is provided, and mononuclear cells are isolated from it. The mononuclear cells are stimulated with M-CSF to obtain macrophages M0. Macrophages M0 are induced with LPS+IFN-γ to obtain macrophages M1. Then, macrophages M1 are induced with IL-4+IL-13 to obtain macrophages M2.

8. The method for preparing the ovarian cancer organoid-macrophage co-culture system according to claim 7, characterized in that, In the process of inducing macrophage M0 with LPS+IFN-γ to obtain macrophage M1, and then inducing macrophage M1 with IL-4+IL-13 to obtain macrophage M2, the concentration of LPS was 100 ng / mL, the concentration of IFN-γ was 10 g / mL~20 ng / mL, the concentration of IL-4 was 10 ng / mL~20 ng / mL, and the concentration of IL-13 was 10 ng / mL~20 ng / mL.

9. An ovarian cancer organoid-macrophage co-culture system, characterized in that, It was prepared by the method for preparing the ovarian cancer organoid-macrophage co-culture system according to any one of claims 1 to 8.

10. The application of the ovarian cancer organoid-macrophage co-culture system according to claim 9 in the fields of cancer drug screening, immunomodulator screening, cancer drug efficacy prediction, and immunomodulator screening.

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