Culture medium and method for culturing malignant effusion specimen organ of epithelioid vascular endothelial tumor

Abstract

The invention provides a culture medium and a method for culturing malignant effusion specimen organs of epithelioid vascular endothelial tumor. The culture medium is added with 10-50ng / ml of CTGF for the first time. The culture method comprises the following steps: filtering a malignant effusion sample by using a 100 [mu] m filter screen, centrifugally collecting cells, embedding by using matrigel in a volume ratio of 1: 1, adjusting the cell density to 4 * 10 < 5 > / ml, and culturing for 12-16 days under the conditions of 37 DEG C and 5% CO2. The concentration of CTGF is crucial to the formation and growth of organoids, the optimal concentration value of 20ng / mL is obtained according to tests, the number of the organoids is large, the diameter of the organoids is large, the establishment rate of the organoids is successfully increased to 70% or above from less than 30%, the diameter of the obtained organoids reaches 120 + / -20 microns, the obtained organoids can be stably passed to P3 generations or above, and the gene characteristics and pathological phenotypes of primary tumors are reserved. The method is easy and convenient to operate, the modeling period is short (14 days), a reliable in-vitro model is provided for individualized drug sensitivity detection, drug screening and pathogenesis research of epithelioid vascular endothelioma, and the technical blank in the rare tumor research field is filled.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoids, and in particular relates to a culture medium and method for culturing organoids of malignant effusion specimens of epithelial hemangioendothelioma. Background Art

[0002] Epithelioid hemangioendothelioma (EHE) is a rare, low- to intermediate-grade malignant vascular tumor originating from the vascular endothelium. It can affect multiple organs throughout the body, often arising in soft tissues, the liver, lungs, and bones. Its malignancy lies between completely benign hemangiomas and highly malignant angiosarcomas. In 2015, the World Health Organization classified it as a low- to intermediate-grade malignant vascular tumor with the potential for metastasis. EHE, as a subtype, has an incidence of less than one in a million. As a rare disease, EHE lacks effective treatment options confirmed by large-scale clinical studies. Consequently, there is no standardized treatment regimen. Current treatment options primarily include watchful waiting, surgery, chemotherapy, radiotherapy, and organ transplantation. For inoperable, extensively advanced EHE, cytotoxic agents, targeted therapies, and biologic response (immune) modifiers have been reported, but these studies are primarily based on individual cases, small sample sizes, or retrospective analyses. Current research on hemangioendothelioma faces many challenges in many aspects. There is still a lack of patient-derived models that can reproduce the complexity of the tumor genome, and drug research on EHE is also at a stage of stagnation or slow development.

[0003] The primary characteristic of EHE is the presence of fusion genes, such as WWTR1-CAMTA1 or YAP1-TFE3, with a minority of cases displaying other rare fusion genes. WTR1-CAMTA1 and YAP1-TFE3 fusion EHE account for ≥90% and ≤10% of EHE cases, respectively. WWTR1-CAMTA1 and YAP1-TFE3 contain WWTR1 (TAZ) and YAP1, key components of the Hippo signaling pathway. This pathway plays an important role in organ growth, regeneration, and tissue repair. The core of the Hippo pathway is regulated by kinases such as MST1, MST2, LATS1, and LAT2; adaptor proteins such as SAV1, MOB1A, and MOB1B; transcriptional coactivators such as YAP1 and WWTR1; and TEAD transcription factors. Connective tissue growth factor (CTGF), also known as cysteine-rich growth factor, is a downstream effector of the WWTR1-CAMTA1 fusion gene. It plays a key role in the sustained anchorage-independent proliferation of NIH3T3 cells transformed by the WWTR1-CAMTA1 fusion gene by interacting with integrin αIIbβ3.

[0004] Malignant peritoneal effusions often represent advanced stages of hemangioendothelioma. These effusions contain tumor cells and related cellular components, enabling the construction of in vitro models that provide unique specimens for studying the biological characteristics, metastatic mechanisms, and drug sensitivity of hemangioendothelioma. Among the novel in vitro models currently being developed are patient-derived organoids (PDOs), three-dimensional micro-organs cultivated in vitro in a matrix gel-supported environment. These PDOs are self-organized from the patient's own tumor cells. They possess genetic and phenotypic characteristics highly consistent with their source tissue, more closely resembling the actual patient tumor and reflecting the drug sensitivity of the original tissue. Therefore, they can serve as "surrogates" for in vivo tumors for in vitro clinical drug screening. Tumors are highly heterogeneous, with significant variations in tumor cells within the same patient, and even more pronounced between different patients. This genetic, phenotypic, and functional heterogeneity in tumor cells across patients leads to significant inter-individual variability in treatment approaches. Even patients with the same genetic mutation may experience varying responses to treatment. Ignoring individual variability and relying solely on empirical treatment is inherently unwise, resulting in poor overall efficacy and even treatment failure. Therefore, using effective models (such as tumor organoid models) for drug sensitivity testing can effectively improve the accuracy of treatment methods and greatly reduce the failure rate of clinical treatment. It can provide guidance for individualized clinical treatment, avoid the loss of effective treatment time and effective treatment opportunities due to ineffective treatment, and thus improve patient survival rate.

[0005] However, there are currently no reports on methods for culturing epithelial hemangioendothelioma organoids. Traditional organoid culture media usually contain basal culture media (such as DMEM / F12), growth factors (such as EGF, FGF), serum replacements, etc., but these components are not ideal for the culture of epithelial hemangioendothelioma organoids. The organoid culture method for epithelial hemangioendothelioma malignant effusion cells is also unclear. This is the trend and focus of future research models. Developing a culture medium that can significantly improve the efficiency of epithelial hemangioendothelioma organoid culture has important scientific and clinical significance. Summary of the Invention

[0006] The present invention provides a culture medium and method for culturing organoids from malignant effusion specimens of epithelial hemangioendothelioma, aiming to solve the technical problem of poor culture effect of epithelial hemangioendothelioma organoids in the prior art.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a culture medium for culturing organoids from malignant effusion specimens of epithelial hemangioendothelioma, comprising a basal culture medium, specific additive factors, and connective tissue growth factor at a concentration of 10-50 ng / mL;

[0009] The specific additional factors include 8-12 mM HEPES, 1.5-2.5 mM Glutamax, 1× B27, 1× N2, 90-110 U / ml penicillin or streptomycin, 0.4-0.6 μM A83-01, 45-55 ng / ml EGF, 90-110 ng / ml Noggin, 8-12 μM Y-27632, 4-6 μM Forskolin, 8-12 ng / ml FGF-10, 8-12 ng / ml FGF-basic, 450-550 ng / ml R-Spondin1, 90-110 ng / ml Wnt-3A and 3-5 mM nicotinamide.

[0010] In a preferred embodiment, the concentration of the connective tissue growth factor is 20 ng / ml.

[0011] Based on the above scheme, experiments have shown that when the CTGF concentration is 20ng / ml, the diameter of the organoids increases significantly (120-150μm on average), and the structure is dense, which is consistent with the typical morphology of EHE tumors. 20ng / ml CTGF can increase the organoid formation rate to more than 70% (compared to <30% in the group without CTGF), and the number is stable (≥50 organoids per well). When CTGF is at a low concentration of 10ng / ml, although organoids are generated, the diameter of the organoids is small. When CTGF is at a high concentration of 50ng / ml, the number and size of the organoids do not increase but decrease. Therefore, it is preferably selected that the concentration of the connective tissue growth factor is 20ng / ml.

[0012] Preferably, the basal culture medium is Advanced DMEM / F-12 culture medium.

[0013] Based on the above scheme, the amino acids, vitamins and HEPES buffer system contained in the Advanced DMEM / F-12 culture medium can stably maintain pH and provide the metabolic support required for organoid growth, which is particularly suitable for tumor cells derived from malignant effusions; the serum-free formula combined with the B27 / N2 additive can eliminate batch differences and accurately regulate the effects of CTGF exogenous growth factor.

[0014] In a second aspect, the present invention provides a method for culturing epithelial hemangioendothelioma organoids, using any culture medium described in the first aspect, comprising the following steps:

[0015] S1. Process the peritoneal malignant effusion sample of epithelioid hemangioendothelioma and collect the cells after centrifugation;

[0016] S2. Resuspend the cells in Matrigel and embed them in a culture plate.

[0017] S3. Add the culture medium described in claims 1-3, culture at 37°C and 5% CO2, replace the culture medium every 2-3 days, and culture for 12-16 days to obtain organoids.

[0018] In a preferred embodiment, in step S1, the malignant effusion sample is filtered through a 100 μm cell sieve and then centrifuged to collect cells;

[0019] If the red blood cell content in the cell pellet exceeds the threshold, use red blood cell lysis buffer and then wash.

[0020] Based on the above protocol, malignant effusion samples were filtered through a 100 μm cell sieve to remove tissue fragments and cell clumps, ensuring that a single-cell suspension was obtained for subsequent culture; the cell pellet was collected after centrifugation to enrich tumor cells and key stromal cells.

[0021] When the red blood cell content exceeds the threshold, treatment with red blood cell lysis buffer can effectively remove red blood cell contamination, preventing the release of hemoglobin and other substances from interfering with organoid growth, while retaining active tumor cell populations, thereby improving the purity and success rate of organoid culture.

[0022] In the preferred scheme, in step S2, the volume ratio of matrix gel to culture medium is 1:1, and the cell density is 4×10 5 / ml.

[0023] Based on the above scheme, a 1:1 volume ratio of matrix gel and culture medium was used to form a suitable three-dimensional culture microenvironment. The matrix gel provided the scaffold structure and extracellular matrix signals required for organoid growth, while the culture medium ensured nutrient supply and growth factor diffusion. The balance between the two can maintain the stability of cell aggregates while avoiding excessive hardening that affects nutrient penetration. At the same time, the cell density was controlled at 4×10 5 / ml, which can not only ensure sufficient cell-to-cell interactions to promote organoid self-assembly, but also avoid nutrient competition or hypoxic necrosis caused by excessive density, thereby significantly improving the formation efficiency, size uniformity and structural integrity of epithelial hemangioendothelioma organoids.

[0024] In a third aspect, the present invention provides an application of epithelial hemangioendothelioma organoids prepared by any of the methods for culturing epithelial hemangioendothelioma organoids described in the second aspect in the field of drug screening.

[0025] In a fourth aspect, the present invention provides an application of epithelial hemangioendothelioma organoids prepared by any of the epithelial hemangioendothelioma organoid culture methods described in the second aspect in the preparation of personalized tumor vaccines.

[0026] In a fifth aspect, the present invention provides an application of epithelial hemangioendothelioma organoids prepared by any of the epithelial hemangioendothelioma organoid culture methods described in the second aspect in evaluating the efficacy of cell therapy.

[0027] In a sixth aspect, the present invention provides an application of epithelial hemangioendothelioma organoids prepared by any of the epithelial hemangioendothelioma organoid culture methods described in the second aspect in the field of sensitivity detection of chemotherapy drugs and targeted drugs.

[0028] The beneficial effects of the present invention are:

[0029] The present invention provides a culture medium and method for culturing organoids from malignant effusion specimens of epithelial hemangioendothelioma. Cells are collected by centrifugation from the malignant effusion sample and then embedded in Matrigel. A specialized culture medium containing 10-50 ng / ml of CTGF growth factor is added. Culture is maintained at 37°C in a CO2 incubator, with the medium changed every three and a half days. Within two weeks, epithelial hemangioendothelioma organoids can be obtained. This culture system, by adding CTGF for the first time, significantly improves the growth rate, morphological integrity, and functionality of organoids, increasing the establishment rate from less than 30% with traditional methods to over 70%. The resulting organoids reach a diameter of 120±20 μm and fully retain the genetic characteristics and pathological phenotype of the primary tumor.

[0030] In addition, the organoid model established by the present invention can be stably passaged to P3 generation or above, supports low-temperature storage and recovery, breaks through the time limit of primary cell culture, and provides technical support for the establishment of a tumor biological sample library. The method is easy to operate, and the survival rate and quantity of the obtained organoids are excellent. It can accurately reflect the biological characteristics of the patient's tumor and can be used to guide clinical drug selection and new drug development. It has the advantages of a short modeling cycle (only 14 days, a high success rate, and easy operation. It effectively solves the problem of the lack of existing in vitro research models for epithelial hemangioendothelioma, and provides a scientific, reliable and clinically suitable in vitro model for basic research, drug development and personalized treatment of such rare tumors, filling the technical gap in this field and having important significance for promoting precision diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 This is the first bright field image of organoids cultured at a CTGF concentration of 0 ng / mL.

[0033] Figure 2 This is the second bright field image of the organoid cultured at a CTGF concentration of 10 ng / mL.

[0034] Figure 3 This is the third bright field image of the organoid cultured at a CTGF concentration of 20 ng / mL.

[0035] Figure 4 This is the fourth bright field image of organoids cultured at a CTGF concentration of 50 ng / mL.

[0036] Figure 5 This is a graph showing the diameter and number of epithelial hemangioendothelioma organoids after culture at different CTGF concentrations.

[0037] Figure 6 This is a picture of the results of epithelial hemangioendothelioma organoid passaging.

[0038] Figure 7 This is the HE staining result of epithelial hemangioendothelioma organoid. DETAILED DESCRIPTION

[0039] 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. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit 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.

[0040] Example 1:

[0041] This embodiment provides a culture medium for culturing organoids from malignant effusion specimens of epithelial hemangioendothelioma, comprising a basal culture medium, specific additive factors, and connective tissue growth factor at a concentration of 10-50 ng / mL;

[0042] The specific additional factors include 8-12mM HEPES, 1.5-2.5mM Glutamax, 1× B27, 1× N2, 90-110U / ml penicillin or streptomycin, 0.4-0.6μM A83-01, 45-55ng / ml EGF, 90-110ng / ml Noggin, 8-12μM Y-27632, 4-6μM Forskolin, 8-12ng / ml FGF-10, 8-12ng / ml FGF-basic, 450-550ng / ml R-Spondin1, 90-110ng / ml Wnt-3A and 3-5mM nicotinamide.

[0043] Specifically, the specific additional factors include 10mM HEPES, 2mM Glutamax, 1x B27, 1x N2, 100units / ml penicillin / streptomycin, 0.5μM A83-01, 50ng / ml EGF, 100ng / ml Noggin, 10μM Y-27632, 5μM Forskolin, 10ng / ml FGF-10, 10ng / ml FGF-basic, 500ng / ml R-Spondin 1, 100ng / ml Wnt-3A, and 4mM Nicotinamide.

[0044] Wherein, the concentration of the connective tissue growth factor is 20 ng / ml.

[0045] Specifically, the concentration of the connective tissue growth factor can be in the range of 10-50. Among them, a better solution is: when the concentration of the connective tissue growth factor is 20ng / ml, the organoid diameter and number perform better.

[0046] In order to provide the metabolic support required for organoid growth, based on any of the above schemes, the basal culture medium is Advanced DMEM / F-12 culture medium.

[0047] Example 2:

[0048] This embodiment provides a method for culturing epithelial hemangioendothelioma organoids, using the culture medium described in any one of the embodiments, comprising the following steps:

[0049] S1. Process the peritoneal malignant effusion sample of epithelioid hemangioendothelioma and collect the cells after centrifugation;

[0050] S2. Resuspend the cells in Matrigel and embed them in a culture plate.

[0051] S3. Add the culture medium described in claims 1-3, culture at 37°C and 5% CO2, replace the culture medium every 2-3 days, and culture for 12-16 days to obtain organoids.

[0052] Specifically, S1. Filter a freshly obtained malignant effusion sample through a 100 μm cell sieve to remove tissue debris and cell clumps, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect the cell pellet; if the red blood cell content in the cell pellet exceeds 20% (volume ratio), add 2-3 volumes of red blood cell lysis buffer, lyse at room temperature for 2-3 minutes, add 3 volumes of PBS buffer to terminate the reaction, centrifuge at 1500 rpm for 5 minutes, and repeat washing 1-2 times;

[0053] S2. Resuspend the cell pellet in Matrigel and epithelial hemangioendothelioma organoid culture medium (volume ratio 1:1) on ice and gently pipette to mix thoroughly to avoid bubbles. The cell density is 4 × 10 5 / mL to prepare a cell suspension; then, plate 2w cells / 50μL / droplet in the center of each well of a preheated 24-well plate, so that the sample forms a dome structure in the center of each well. Plate three wells per group. Place the inoculated culture dish in a CO2 incubator at 37°C for 5 minutes. Gently shake the gel droplet until no noticeable flow is observed. Carefully invert the plate and allow it to fully solidify for 25 minutes.

[0054] S3. Add 1000 μL of epithelial hemangioendothelioma organoid culture medium to each well. Replace 50% of the volume of fresh culture medium every 2-3 days during the culture period. After microscopic examination and photography, place the cells in an incubator at 37°C and 5% CO2. Mature organoids with a diameter of 100-150 μm can be obtained after 12-16 days of culture.

[0055] Example 3:

[0056] This embodiment provides an application of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids described in any one of Example 2 in the field of drug screening.

[0057] Example 4:

[0058] This embodiment provides an application of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids described in any one of Example 2 in the preparation of personalized tumor vaccines.

[0059] Embodiment 5:

[0060] This embodiment provides an application of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids described in any one of Example 2 in evaluating the efficacy of cell therapy.

[0061] Example 6:

[0062] This embodiment provides an application of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids described in any one of Example 2 in the field of chemotherapy drug and targeted drug sensitivity detection.

[0063] The present invention will be further explained below in conjunction with specific experiments:

[0064] 1. Cell Treatment

[0065] 1. Cell Sieving and Centrifugation: Filter the epithelial hemangioendothelioma malignant effusion using a 100 μm cell sieve, then centrifuge at 1500 rpm for 5 minutes and remove the supernatant. Resuspend the cell pellet in PBS buffer containing antibiotics, gently pipette to mix, and centrifuge again under the same conditions for 5 minutes. Discard the supernatant. Repeat this washing process twice.

[0066] 2. Red blood cell lysis: If there are a large number of red blood cells in the cell pellet, add 2 mL of red blood cell lysis buffer, gently pipette to mix, and lyse for 1-3 minutes at room temperature. Then, add 3 volumes of PBS buffer containing antibiotics, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and wash once. If red blood cell lysis is incomplete, repeat this step once.

[0067] 2. Preparation of Cell Suspension

[0068] On ice, the cell pellet was resuspended in Matrigel (Corning, 356231) and epithelial hemangioendothelioma organoid culture medium at a volume ratio of 1:1. The cell density was adjusted to 4 × 10 5 / mL.

[0069] III. Organoid Plating and Culture

[0070] 2 weeks of cells were plated at 50 μL / droplet in the center of each well of a preheated 24-well plate, forming a dome-shaped structure in the center of the well. Three wells were plated per group. The inoculated culture dish was then placed in a CO2 incubator at 37°C for 5 minutes. After gently shaking the gel droplet and no noticeable flow was observed, the plate was carefully inverted and allowed to solidify for 25 minutes.

[0071] After solidification, 1000 μL of epithelial hemangioendothelioma organoid culture medium was added to each well. After microscopic examination and photography, the cells were placed in an incubator and cultured at 37°C and 5% CO2.

[0072] 4. Culture medium composition

[0073] Epithelial hemangioendothelioma organoid culture medium contains basal medium, specific supplemental factors, and connective tissue growth factor (CTGF). The final concentrations of the specific supplemental factors are: 10 mM HEPES, 2 mM Glutamax, 1x B27, 1x N2, 100 units / ml penicillin / streptomycin, 0.5 μM A83-01, 50 ng / ml EGF, 100 ng / ml Noggin, 10 μM Y-27632, 5 μM Forskolin, 10 ng / ml FGF-10, 10 ng / ml FGF-basic, 500 ng / ml R-Spondin 1, 100 ng / ml Wnt-3A, and 4 mM Nicotinamide.

[0074] Among them, CTGF was set at four concentrations, namely 0 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL.

[0075] The growth of organoids was observed every day, the culture medium was replaced every 2-3 days, and photos were taken under a microscope.

[0076] 5. Test results

[0077] 1. Organoid Culture Results: Organoids cultured in the medium of the present invention containing CTGF had already formed a distinct three-dimensional structure on day 7. In contrast, organoids cultured in the medium without CTGF grew slowly, with a large number of cells adhering to the wall and gradually dying. By day 14, the diameter of the organoids in the medium of the present invention was significantly larger than that in the medium without CTGF. Figure 1-Figure 5 The arrows in the figure point to organoids. The number and diameter of organoids at different CTGF concentrations are as follows:

[0078]

[0079]

[0080] This shows that the formation and growth of epithelial hemangioendothelioma organoids are closely related to the concentration of connective tissue growth factor (CTGF). In the absence of CTGF, no organoid formation was observed. As the CTGF concentration increased from 0 ng / mL to 10 ng / mL, the number of organoids increased significantly to 58, with a diameter between 20 and 50 microns. When the CTGF concentration was further increased to 20 ng / mL, the number of organoids reached a maximum of 113, and the diameter also increased significantly to 30-120 microns, showing the best promoting effect. However, when the CTGF concentration was increased to 50 ng / mL, although the number of organoids was large, reaching 109, the diameter decreased to between 30 and 80 microns, indicating that excessive CTGF concentration inhibited the growth of organoids.

[0081] 2. Organoid passage results: After the organoids were successfully constructed, they were passaged using TRYPLE. After passage, they were cultured in epithelial hemangioendothelioma organoid medium containing 20 ng / mL CTGF. The results are shown in Figure 6 .

[0082] P0: Cells can be seen aggregated into large clumps, but the clumps are relatively irregular in shape and vary greatly in size. Some areas have a high cell density and are tightly connected to each other, indicating the initial growth of cells. There is a lot of cell debris and some free cells in the background. This is because some cells have not yet fully aggregated to form organoids during the initial culture process, or some cells have detached during processing.

[0083] Passage P1: After passaging, the morphology of the cell clumps gradually becomes more regular, and the outlines of the organoids become clearly visible. The number of cell clumps increases, but there is still some variation in size. The cells are relatively densely packed, indicating good growth. Some organoids have a small number of scattered cells around them, representing newly formed cell clumps after passaging. The background is improved compared to Passage P0, with less cell debris, indicating a successful passaging process, high cell viability, and that the culture system supports good cell growth.

[0084] P2: The organoids have a more regular morphology, exhibiting a distinctly round or oval shape, are more uniform in size, and have tightly packed cells, displaying a well-defined three-dimensional structure. The number of organoids has further increased, indicating strong cell proliferation during passage and that the culture medium is able to effectively support organoid growth and maintain their stability. The clear background, with minimal cell debris and free cells, indicates that during the P2 culture process, the cell growth state is stable, the culture environment is good, and there is no significant cell death or shedding.

[0085] P3: Organoids maintain excellent morphological characteristics, with regular form, uniform size, and tightly packed cells, demonstrating good growth and stability. The number of organoids is comparable to or slightly increased compared to P2, indicating that organoids maintain robust proliferation and maintenance capacity at P3, demonstrating that this culture system can support long-term, stable organoid culture. The background remains clear, with no obvious signs of cell debris or bacterial contamination, further demonstrating the success of the culture process and the effectiveness of the culture medium.

[0086] Overall, from P0 to P3, the morphology of the organoids gradually became regular, the size tended to be uniform, the cells were more densely arranged, and the cell debris in the background gradually decreased, indicating that the organoids grew well during the passaging process. The culture medium and method provided by the present invention can effectively support their growth and maintain their stability.

[0087] See Figure 7 The cell nuclei in the image vary in size and morphology, with clearly visible nucleoli. This indicates the presence of different cell types within the organoid, demonstrating a degree of heterogeneity. Some cells are in an actively proliferating state, displaying characteristics of tumor cells. The uneven distribution and density of the cytoplasm reflect differences in the functional state and metabolic activity of the cells. Some cells are rich in cytoplasm, indicating strong metabolic and synthetic capabilities, consistent with the high metabolic capacity of tumor cells. This demonstrates the successful construction of an epithelial hemangioendothelioma organoid model.

[0088] In summary, the present invention provides a new culture system for establishing an in vitro model of epithelial hemangioendothelioma. The culture medium provided by the present invention significantly improves the culture success rate of epithelial hemangioendothelioma organoids by adding CTGF, and can stably passage to at least P3. This provides new tools and strategies for the research and treatment of epithelial hemangioendothelioma, and has important scientific research and clinical application value.

[0089] The present invention provides a new culture system for establishing an in vitro model of epithelial hemangioendothelioma. By adding CTGF to the culture medium, the culture success rate of epithelial hemangioendothelioma organoids is significantly improved, and the organoids can be stably passaged to at least P3, opening up a new path for the research and treatment of epithelial hemangioendothelioma and having extremely important scientific research and clinical application value.

[0090] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that falls within the scope defined by the claims of the present invention falls within the scope of protection of the present invention.

Claims

1. A culture medium for culturing organoids from malignant effusion specimens of epithelial hemangioendothelioma, characterized in that: Includes basal medium, specific supplemental factors, and connective tissue growth factor at a concentration of 10-50 ng / mL; The specific additional factors include 8-12 mM HEPES, 1.5-2.5 mM Glutamax, 1× B27, 1× N2, 90-110 U / ml penicillin or streptomycin, 0.4-0.6 μM A83-01, 45-55 ng / ml EGF, 90-110 ng / ml Noggin, 8-12 μM Y-27632, 4-6 μM Forskolin, 8-12 ng / ml FGF-10, 8-12 ng / ml FGF-basic, 450-550 ng / ml R-Spondin1, 90-110 ng / ml Wnt-3A and 3-5 mM nicotinamide.

2. The epithelial hemangioendothelioma organoid culture medium according to claim 1, characterized in that The concentration of the connective tissue growth factor was 20 ng / ml.

3. The epithelial hemangioendothelioma organoid culture medium according to claim 1, characterized in that The basic culture medium is Advanced DMEM / F-12 culture medium.

4. A method for culturing epithelial hemangioendothelioma organoids, characterized in that: Using the culture medium according to any one of claims 1 to 3, comprising the following steps: S1. Process the peritoneal malignant effusion sample of epithelioid hemangioendothelioma and collect the cells after centrifugation; S2. Resuspend the cells in Matrigel and embed them in a culture plate. S3. Add the culture medium described in claims 1-3, culture at 37°C and 5% CC2, replace the culture medium every 2-3 days, and culture for 12-16 days to obtain organoids.

5. The method for culturing epithelial hemangioendothelioma organoids according to claim 4, wherein: In step S1, the malignant effusion sample is filtered through a 100 μm cell sieve and then centrifuged to collect cells; If the red blood cell content in the cell pellet exceeds the threshold, use red blood cell lysis buffer and then wash.

6. The method for culturing epithelial hemangioendothelioma organoids according to claim 4, characterized in that: In step S2, the volume ratio of Matrigel to culture medium was 1:1 and the cell density was 4 × 10 5 / ml.

7. Use of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids according to any one of claims 4 to 6 in the field of drug screening.

8. Use of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids according to any one of claims 4 to 6 in the preparation of personalized tumor vaccines.

9. Use of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids according to any one of claims 4 to 6 in evaluating the effect of cell therapy.

10. Use of epithelial hemangioendothelioma organoids prepared by the culture method of epithelial hemangioendothelioma organoids according to any one of claims 4 to 6 in the field of sensitivity detection of chemotherapeutic drugs and targeted drugs.

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