Isolation method of ovarian cancer related mesenchymal stem cells and its application in anti-tumor

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a method for isolating ovarian cancer-related mesenchymal stem cells and their application in anti-tumor treatment. The isolation method includes the following steps: (S1) extracting and isolating mesenchymal stem cells from fresh tumor samples from ovarian cancer patients using the direct tissue block method; (S2) sequentially screening and confirming the mesenchymal stem cells as tumor-derived through cell morphology identification, surface marker analysis, iterative stemness detection, and tumor and / or fibroblast marker exclusion experiments. Experiments have demonstrated that ovarian cancer-related mesenchymal stem cells can be used to evaluate the anti-tumor efficacy of drugs, their anti-invasive ability against tumor matrix support, and / or their anti-globulinization ability against tumor matrix support.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for isolating ovarian cancer-related mesenchymal stem cells and their application in anti-tumor therapy. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell derived from the mesoderm, possessing self-renewal capacity and multi-lineage differentiation potential. They originate from diverse sources, primarily bone marrow, umbilical cord, placenta, and adipose tissue. The tumor microenvironment is widely considered a key factor in tumor growth and spread. It not only provides cancer cells with essential nutrients and growth signals but also promotes tumor immune escape and drug resistance through complex cell communication mechanisms. Cancer cells construct an ecological niche supporting their survival and proliferation by recruiting and reprogramming non-cancer host cells, such as immune cells and pericytes, and by remodeling the vascular system and extracellular matrix. Within the tumor microenvironment, MSCs influence tumor cell behavior by secreting various cytokines and migrate to the tumor site under the influence of various growth factors and chemokines, participating in and coordinating the formation of the tumor matrix. Due to the heterogeneity of MSCs, those from different tissue sources may exhibit different functional characteristics. Currently, there are no specific methods for isolating and identifying ovarian cancer CA-MSCs, leading to a lack of consistent standards in research. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for isolating ovarian cancer-associated mesenchymal stem cells and their application in anti-tumor therapy.

[0004] This invention is achieved through the following technical solution:

[0005] This invention protects a method for isolating ovarian cancer-related mesenchymal stem cells, comprising the following steps:

[0006] (S1) Mesenchymal stem cells were extracted and isolated from fresh tumor samples from ovarian cancer patients using the direct tissue block separation method.

[0007] (S2) Mesenchymal stem cells were screened and confirmed to be tumor-derived through sequential cell morphology identification, surface marker analysis, iterative stemness detection, and tumor and / or fibroblast marker exclusion experiments; among which,

[0008] The cell morphology identification is to screen for cells that are tripolar or spindle-shaped, with a diameter of 280–350 μm, have 2–3 cell processes, a nucleus diameter of 25–30 μm, and abundant cytoplasm.

[0009] The surface marker analysis was performed by screening cells using flow cytometry to identify cells that were positive for CD105, CD73, CD90, and CD44, but negative for CD45, CD34, CD11b, CD19, and HLA-DR.

[0010] The iterative stemness test is used to screen for CA-MSC cells with good morphology and stable expression levels of stem cell surface markers during continuous passage culture from primary to Nth generation; where N is a natural number ≥10.

[0011] The exclusion of tumor cell markers refers to the exclusion of individuals with positive cell markers for epithelial cells and primary epithelial malignant tumors of the ovary.

[0012] The exclusion of fibroblast markers refers to the exclusion of ovarian tumor-associated fibroblasts and populations with positive fibroblast markers.

[0013] Furthermore, the extraction and separation of mesenchymal stem cells using the tissue block separation method specifically includes the following steps:

[0014] Fresh tumor samples from patients clinically diagnosed with ovarian cancer were washed under sterile conditions with a balanced salt solution containing dual antibodies, cut into small pieces, and cultured in a mesenchymal stem cell-specific culture medium for 24 hours. Subsequently, the tissue pieces were removed, and the cells were purified using the adhesion difference method, which utilizes the difference in the speed of cell adhesion to remove epithelial cells, and mesenchymal stem cells were extracted and isolated.

[0015] Preferably, the bispecific antibodies are 300 U / mL penicillin and 300 μg / mL streptomycin.

[0016] Preferably, the cell marker for primary epithelial malignant tumors of the ovary is EpCAM.

[0017] Preferably, the fibroblast marker is α-smooth muscle actin (α-SMA).

[0018] This invention also protects the use of ovarian cancer-associated mesenchymal stem cells in evaluating the efficacy of drugs in inhibiting tumor cell proliferation by supporting the tumor microenvironment matrix.

[0019] This invention also protects the use of ovarian cancer-associated mesenchymal stem cells in evaluating the anti-invasive capabilities of drugs in supporting the tumor microenvironment matrix.

[0020] This invention also protects the use of ovarian cancer-associated mesenchymal stem cells in evaluating the anti-spheroidizing ability of drugs to support the tumor microenvironment matrix.

[0021] This invention also protects the use of ovarian cancer-associated mesenchymal stem cells in evaluating the antitumor efficacy of drugs.

[0022] Preferably, the drug is an active substance, a traditional Chinese medicine monomer, and / or a traditional Chinese medicine compound.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention provides a method for isolating ovarian cancer-related mesenchymal stem cells and their application in anti-tumor therapy. Cells are isolated using a tissue block separation method, purified using immunomagnetic beads, and then screened and confirmed as tumor-derived mesenchymal stem cells through sequential cell morphology identification, surface marker analysis, iterative stemness detection, and tumor and / or fibroblast marker exclusion experiments. Experiments have confirmed that ovarian cancer-associated mesenchymal stem cells can be used to evaluate the preliminary screening, efficacy assessment, drug resistance and toxicology assessment, and pharmacological effects of antitumor drugs. The main benefits are as follows: (1) The co-culture model simulates the structural characteristics of the tumor microenvironment in vitro, providing tumor cells with an attachment and growth environment similar to that in vivo. This helps to more realistically reflect the effect of drugs on tumors in vivo during drug screening; (2) The model better simulates the tumor microenvironment's support for tumor growth and its ability to resist invasion and / or spheroidization of tumor matrix support; (3) In the in vitro and in vivo co-culture models, cells support each other and influence each other through paracrine action, reflecting the influence of secretion on intercellular communication networks. For example, some drugs may exert antitumor effects by blocking growth-promoting signals transmitted from mesenchymal stem cells to tumor cells; (4) It improves the accuracy of drug screening. During the drug screening process, the effects of drugs on tumor cells and mesenchymal stem cell-related targets can be evaluated simultaneously. For example, a drug may not only inhibit the proliferation of tumor cells, but also block the induction of drug resistance in tumor cells by mesenchymal stem cells; (5) reduce the need for animal experiments. By using an in vitro model of the interaction between tumor cells and cancer-associated mesenchymal stem cells, the relationship between the in vivo microenvironment matrix and the tumor can be truly reflected. This can replace some animal experiments to a certain extent, help predict the potential clinical value of drugs, and provide strong data support for new drug development and clinical trials. This not only accelerates the process of new drug launches, but also brings more and better treatment options to cancer patients. It has profound significance for promoting progress in the field of cancer treatment and improving patient survival rates and quality of life. Attached Figure Description

[0025] Figure 1 CA-MSCs of different densities (scale bar: 100 μm).

[0026] Figure 2 This is a graph showing the results of flow cytometry analysis of surface markers.

[0027] Figure 3 The figure shows the results of flow cytometry analysis of surface markers of CA-MSC P3 and P10 stem cells.

[0028] Figure 4 This is a graph showing the results of flow cytometry analysis of CA-MSCs and epithelial tumor cell markers (EpCAM) in tumor tissue.

[0029] Figure 5 The image shows the analysis results of α-SMA expression, a marker of CA-MSC myofibroblasts. The left image shows the change in α-SMA content before and after co-culturing CA-MSCs with tumor cells. The right image shows the α-SMA content of CA-MSCs under a confocal microscope (scale bar: 100 μm). *P<0.05; mouse embryonic fibroblasts 3T3-L1 were used as a positive control.

[0030] Figure 6 This is a graph showing the analysis results of the effects of different concentrations of CA-MSC conditioned medium on tumor cell proliferation in Example 2.2. *P<0.05, **P<0.01.

[0031] Figure 7 The figure shows the results of the analysis of the effects of CA-MSCs on tumor cell invasion and the inhibitory effects of traditional Chinese medicine compound and single Chinese medicine monomer on CA-MSC invasion in Example 3 (scale bar: 200 μm).

[0032] Figure 8 Figure 4 shows the results of the analysis of the effect of CA-MSCs on tumor cell spherogenesis and the inhibition of CA-MSC spherogenesis by traditional Chinese medicine compound and single Chinese medicine monomer (scale bar: 200 μm). P < 0.001.

[0033] Figure 9 The figure shows the analysis results of tumor volume after tissue sampling in each group of mice in Example 5. *P<0.05. Detailed Implementation

[0034] To better understand the present invention, the present invention will be further described below with reference to the embodiments and accompanying drawings. The following embodiments are only illustrative of the present invention and are not intended to limit it.

[0035] Example 1: Isolation and Identification of Ovarian Cancer-Related Mesenchymal Stem Cells

[0036] 1. Extraction and isolation of ovarian cancer-associated mesenchymal stem cells (CA-MSCs)

[0037] Using the direct tissue block separation method, fresh tumor samples from six patients clinically diagnosed with ovarian cancer were washed under sterile conditions with a balanced salt solution containing dual antibodies (300 U / mL penicillin and 300 μg / mL streptomycin, Dalian Meilun Biotechnology Co., Ltd., MA0348). Surrounding blood vessels, fat, and connective tissue were removed using scissors and forceps. The tissue was then cut into 1-5 mm pieces on ice using a blade.3 Small pieces were rinsed three times with a balanced salt solution containing penicillin (300 U / mL) and streptomycin (300 μg / mL). The tissue was then pipetted and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the tissue was further cut into smaller pieces (0.1-1 mm). 3 The tissue, either as a paste or a puree, is evenly seeded into a 10cm culture dish containing 5mL of mesenchymal stem cell culture medium (Suzhou Ekosei Biotechnology Co., Ltd., ME000-N023) and cultured for 24 hours. A coverslip can be placed on top of the tissue to ensure that the tissue block fully contacts the bottom of the culture dish and to prevent the tissue block from floating or moving. After about 48 hours, cells are observed under a microscope to be freed from under the tissue block. The tissue block is then removed, and epithelial cells are removed using the adhesion difference method (which uses the difference in the speed of cell adhesion to purify cells). Mesenchymal stem cells are then extracted and isolated.

[0038] 2. Cell morphology identification

[0039] The isolated mesenchymal stem cells (MSCs) exhibited tripolar or spindle-shaped extensions, with 2-3 cellular processes, relatively large nuclei (approximately 25-30 μm in diameter), and abundant cytoplasm. The cell diameter was larger than that of cancer cells and the ovarian cancer cell line (SKOV3) isolated from the same tissue; CA-MSCs had a diameter of approximately 280-350 μm. After primary culture, low-passage (P<8) CA-MSCs showed spindle-shaped extensions and slow growth (doubling time approximately 50-60 h), while high-passage (P>8) cells showed a significant increase in processes and a faster growth rate (doubling time approximately 35-50 h). Figure 1 The image shows CA-MSCs of different densities under a 10x inverted microscope. Scale bar: 100 μm.

[0040] 3. Surface marker analysis and identification

[0041] Flow cytometry was used to analyze CA-MSC surface markers: CA-MSCs were washed with calcium- and magnesium-free PBS, digested with trypsin, and collected by centrifugation. Cells were resuspended in FACS buffer and adjusted to 5 × 10⁶ cells / mL. 6Cells / mL. CA-MSCs were added with specific fluorescent antibodies (CD105, CD73, CD90, CD44, and PE cocktail (CD45, CD34, CD11b, CD19, HLA-DR) using the Human MSC Analysis Kit (BD 562245). The cells were incubated at room temperature in the dark for 30 minutes. Afterwards, the cells were washed twice with PBS to remove unbound antibodies. The cells were resuspended in 300-500 μl of FACS buffer and analyzed using a flow cytometer (BD, FACS Canto II). The subpopulation of CA-MSCs that showed positive fluorescence signals (CD105, CD73, CD90, CD44) and negative fluorescence signals (PE cocktail (CD45, CD34, CD11b, CD19, HLA-DR)) was defined as follows: Figure 2 As shown.

[0042] 4. Isolation and purification of CA-MSCs using surface markers

[0043] CA-MSCs were isolated and purified using immunomagnetic beads. Cells were washed with PBS and the cell concentration was adjusted to 5 × 10⁶. 7 Cells / mL. Add 20 μl of human CD90 magnetic beads (Miltenyi Biotechnology Co., Ltd., 130-096-253) used for labeling mesenchymal stem cell markers to the cell suspension, mix gently, and incubate at room temperature for 30 minutes. Transfer the cell suspension to a magnetic separator and use magnetic force to separate labeled cells from unlabeled cells. After 3-5 minutes, the magnetic beads will be fully adsorbed. Gently remove the supernatant of unadsorbed cells, retaining the cells adsorbed by the magnetic beads. Wash with 2 mL of culture medium. Release the cells from the magnetic beads with a release reagent. Centrifuge to remove the release reagent, and resuspend the cells in fresh culture medium to obtain purified CA-MSCs.

[0044] 5. Iterative dryness detection

[0045] During continuous passage culture from primary (P1) to the 10th generation (P10), CA-MSCs exhibited good morphology. Flow cytometry analysis of stem cell surface markers revealed that CA-MSCs comprised 76.1% of the total cell population at P3, with positive expression rates of CD105, CD90, and CD73 of 97.7%, 98.9%, and 99.8%, respectively. Further culture to the 10th generation showed that CA-MSCs comprised 80.9% of the total cell population, with positive expression rates of CD105, CD90, and CD73 remaining above 90%. This demonstrates that even after multiple passages, CA-MSCs maintained excellent stem cell characteristics, such as… Figure 3As shown.

[0046] 6. Exclusion of tumor cell markers

[0047] Cell flow cytometry was used to exclude epithelial cells and ephemeral malignant tumor cell marker (EpCAM) positive populations. CA-MSCs were washed with calcium- and magnesium-free PBS, digested with trypsin, and collected by centrifugation. Cells were resuspended in FACS buffer and adjusted to 5 × 10⁶ cells / mL. 6 Cells / mL. Add EpCAM-specific fluorescent antibody (BD, catalog number 324205) and incubate at room temperature in the dark for 30 minutes. Then wash twice with PBS to remove unbound antibody. Resuspend in 300-500 μl FACS buffer and analyze using flow cytometry. Among the analyzed cells, the cell population with a positive EpCAM fluorescence signal represents epithelial cells and primary epithelial malignant tumors of the ovary, while the negative cell population represents CA-MSCs. Figure 4 As shown.

[0048] 6. Exclusion of fibroblast markers

[0049] CA-MSCs have been reported to differentiate into cancer-associated fibroblasts (CAFs) through induction. Alpha smooth muscle actin (α-SMA) is considered a marker of CAF activation. Detection of α-SMA expression in CA-MSCs revealed that, compared to mouse embryonic fibroblasts (3T3-L1), CA-MSCs exhibited significantly lower α-SMA expression levels, approximately 60% of those in 3T3-L1 cells, thus ruling out the possibility of CA-MSCs differentiating into CAFs. After co-culturing CA-MSCs with tumor cells for 24 h and 48 h, respectively, Western blot analysis was performed to detect α-SMA expression (antibody purchased from Wuhan Sanying Biotechnology Co., Ltd., 67735-1-lg) in CA-MSCs. No increase in α-SMA expression was observed compared to before co-culturing, confirming that the obtained ovarian cancer CA-MSCs are a type of mesenchymal stem cell that is not easily induced to differentiate. Figure 5 As shown.

[0050] The ovarian cancer-related mesenchymal stem cells obtained by isolating, screening and identifying through the above steps 1 to 6 were used to construct four ovarian cancer CA-MSC cell lines.

[0051] Example 2: Evaluation of the effect of different concentrations of CA-MSC conditioned medium on tumor cell proliferation using MTT assay.

[0052] 2.1 Preparation of CA-MSC conditioned medium (CM)

[0053] CA-MSC cells were loaded at 1.5 × 10⁻⁶ 6Cells were seeded at a density of 10 cells / dish in 10cm culture dishes. After 24 hours of cell adhesion, the cells were washed 1-2 times with PBS and replaced with fresh serum-free RPMI 1640 medium (Soleb Biotechnology Co., Ltd., 11875). After culturing for 72 hours, the supernatant was collected, filtered through a 0.22μm filter, mixed well, aliquoted, and frozen at -80℃ for later use.

[0054] 2.2 Effect of MTT assay on CA-MSCs on tumor cell proliferation

[0055] Wild-type (WT) and KO cells were seeded at a density of 5000 cells / well in 96-well plates, with blank, control (cells + normal culture medium), and experimental (cells + CA-MSC CM) groups set up. After the cells adhered overnight, different concentrations of CA-MSC CM were added. After culturing for 72 h, 20 μL of MTT solution (5 mg / mL, Lamborghini Biotechnology Co., Ltd., 0793) was added to each well, and the plates were incubated at 37°C for 4 h. Then, the liquid in the wells was discarded, and 150 μL of DMSO was added to each well. The plates were then placed on a 96-well plate shaker and shaken in the dark for 10 min. After the formazan in the wells was completely dissolved, the cells were read using a microplate reader. i3x (Molecular Devices, USA) measured the absorbance (OD) of each well at a wavelength of 570 nm and calculated the cell viability at different concentrations of CM using the following formula:

[0056] Cell viability % = (OD) 实验值 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%

[0057] The results are as follows Figure 6 As shown, CA-MSCs can significantly promote the growth and proliferation of wild-type tumor cells in a concentration-dependent manner.

[0058] Example 3: CA-MSCs can be used to evaluate the anti-invasive ability of traditional Chinese medicine and other active substances to support tumor matrix.

[0059] Tumor cells were cultured in 85% CA-MSC conditioned medium for 24 hours. Using 8 μm pore size Transwell chambers (Corning, catalog number 3422), 60 μL of Matrigel (Corning, catalog number 356234) was added to the upper chamber and incubated at 37°C for 3 hours to allow the Matrigel to solidify. Peritoneal mesothelial cells were cultured at 5 × 10⁻⁶ cells / mL. 5 The prepared tumor cells were seeded at a density of 10 cells / well in the upper chamber of the microplate chamber, and incubated simultaneously in 85% CA-MSC conditioned medium for 24 hours. After incubation, the prepared tumor cells were seeded at a density of 2 × 10⁻⁶ cells / well. 5Mesothelial cells were seeded at a density of cells / well. 200 μL of RPMI 1640 medium containing 5% fetal bovine serum was added to the upper chamber, and 700 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber. A certain concentration of traditional Chinese medicine compound and monomer was also added. The chambers were incubated in an incubator for 72 hours. After incubation, the inner wall of the upper chamber was wiped clean with a cotton swab. The upper chamber was then fixed in 4% paraformaldehyde solution for 15 min, followed by staining with 0.5% crystal violet solution for 15 min. After staining, the excess stain was slowly rinsed off with running water, and the cells were air-dried. The total number of cells passing through the upper chamber was photographed and observed.

[0060] like Figure 7 As shown, CA-MSCs can promote the invasive ability of tumor cells, and traditional Chinese medicine compound and single Chinese medicine substances can effectively inhibit the tumor cell invasive ability of CA-MSCs.

[0061] Example 4: CA-MSCs can be used to evaluate the anti-spheroidizing ability of traditional Chinese medicine and other active substances to support tumor matrix.

[0062] Tumor cells and CA-MSCs were seeded at a density of 3000 cells / well in low-adsorption 96-well U-shaped plates (Corning, catalog number 7007) (tumor cells:CA-MSCs = 1:1). Before seeding, the cells were mixed with a certain amount of matrix gel to prepare a cell suspension, with a final matrix gel concentration of 0.015 mg / well. After the cells formed spheroids overnight, a certain concentration of a traditional Chinese medicine compound and two individual traditional Chinese medicine monomers were added, and the cells were cultured at 37°C. Changes in cell spheroidization were observed periodically. The experiment was terminated on day 8, and 3D scanning images of the cell spheroids in each well were performed using a laser confocal microscope. Simultaneously, the viability of the cell spheroids in each well was detected using the CellTiter-Glo 3D Cell Viability Assay kit (Promega Biotechnology Co., Ltd., catalog number G9682).

[0063] like Figure 8 As shown, by day 8 of culture, the volume of tumor cell spheroids treated with CA-MSCs showed an increasing trend compared to those without CA-MSCs, while no significant increase in spheroid volume was observed in any of the treatment groups; however, the viability of spheroids in the treatment groups was significantly reduced. This indicates that CA-MSCs can promote the spheroidization ability of tumor cells, and that the traditional Chinese medicine compound and its monomeric substances can inhibit the tumor cell spheroidization-promoting ability of CA-MSCs.

[0064] Example 5: CA-MSC can be used to evaluate the antitumor efficacy of traditional Chinese medicine and other active substances.

[0065] 5×10 6 One tumor cell per individual and 1×10 6CA-MSC cells (number per mouse) were mixed with a certain amount of matrix gel and injected subcutaneously into nude mice. One week after cell injection, the mice were randomly divided into four groups: a negative control group (tumor cells only), a control group, a traditional Chinese medicine compound group, and a traditional Chinese medicine single-component group. The traditional Chinese medicine compound group received the medicine once daily by gavage (360 mg / kg), while the traditional Chinese medicine single-component group received the medicine once daily by intraperitoneal injection (20 mg / kg) for 47 days. The negative control group and the control group received the same volume of physiological saline by gavage daily for 47 days. Tumor volume was measured every 3 days. The tumor volume was calculated using the formula: Volume = Tumor long diameter × Tumor short diameter. 2 ×0.5. Tumor tissue was collected on day 52, washed with PBS to remove surface blood, and then weighed.

[0066] like Figure 9 As shown, compared with the negative control group, the tumor volume of mice in the control group was significantly larger, while the tumor volume of mice in the treatment group was significantly smaller than that of the control group. This indicates that the traditional Chinese medicine compound and its monomeric substances can significantly inhibit the tumor-promoting effect of CA-MSCs in vivo.

[0067] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An in vitro antitumor drug screening model based on the synergistic effect of tumor cells and ovarian cancer-associated mesenchymal stem cells, characterized in that: The method for isolating ovarian cancer-related mesenchymal stem cells includes the following steps: (S1) Mesenchymal stem cells were extracted and isolated from fresh tumor samples from ovarian cancer patients using the direct tissue block separation method; (S2) Mesenchymal stem cells were screened and confirmed to be tumor-derived through sequential cell morphology identification, surface marker analysis, iterative stemness detection, and tumor and / or fibroblast marker exclusion experiments; among which, The cell morphology identification is to screen for cells that are tripolar or spindle-shaped, with a diameter of 280-350 μm, have 2-3 cell processes, a nucleus diameter of 25-30 μm, and abundant cytoplasm. The surface marker analysis was performed by screening cells using flow cytometry to identify cells that were positive for CD105, CD73, CD90, and CD44, but negative for CD45, CD34, CD11b, CD19, and HLA-DR. The iterative stemness test is used to screen for CA-MSC cells with good morphology and stable expression levels of stem cell surface markers during continuous passage culture from primary to Nth generation; where N is a natural number ≥10. The exclusion of tumor cell markers refers to the exclusion of individuals who are positive for EpCAM, a cell marker for primary epithelial malignant tumors of the ovary. The exclusion of fibroblast markers refers to the exclusion of ovarian tumor-associated fibroblasts and populations positive for the fibroblast marker α-smooth muscle actin (α-SMA). The drug screening model simultaneously assesses the effects of drugs on tumor cells and mesenchymal stem cell-related targets during the drug screening process, reflecting the relationship between the in vivo microenvironment matrix and the tumor.

2. The drug screening model according to claim 1, characterized in that: The method of extracting and isolating mesenchymal stem cells using direct tissue block separation specifically includes the following steps: Fresh tumor samples from patients clinically diagnosed with ovarian cancer were washed under sterile conditions with a balanced salt solution containing dual antibodies, cut into small pieces, and cultured in a mesenchymal stem cell-specific culture medium for 24 hours. Subsequently, the tissue pieces were removed, and the cells were purified using the adhesion difference method, which utilizes the difference in the speed of cell adhesion to remove epithelial cells, and mesenchymal stem cells were extracted and isolated.

3. The drug screening model according to claim 2, characterized in that: The bispecific antibodies consist of 300 U / mL penicillin and 300 μg / mL streptomycin.

4. The drug screening model according to claim 1, characterized in that: The ovarian cancer-associated mesenchymal stem cells were used to evaluate the efficacy of drugs on the proliferation of tumor cells supported by the tumor microenvironment matrix.

5. The drug screening model according to claim 1, characterized in that: The ovarian cancer-associated mesenchymal stem cells were used to evaluate the anti-invasive ability of drugs to support the tumor microenvironment matrix.

6. The drug screening model according to claim 1, characterized in that: The ovarian cancer-associated mesenchymal stem cells were used to evaluate the anti-globulinization ability of drugs in supporting the tumor microenvironment matrix.

7. The drug screening model according to claim 1, characterized in that: The ovarian cancer-associated mesenchymal stem cells were used to evaluate the antitumor efficacy of drugs.

8. The drug screening model according to claim 1, characterized in that: The drug is an active substance, a single Chinese herbal medicine, and / or a compound Chinese herbal medicine.

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