An ovarian cancer organoid culture kit and a culture method thereof
Patent Information
- Application Number
- CN202611487269.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-23
- Publication Date
- 2026-10-27
AI Technical Summary
[0003]目前，卵巢癌类器官培养普遍面临以下问题：其一，原代培养成功率低且个体差异大，不同患者样本之间类器官形成率波动明显，部分样本难以形成形态均一的类器官结构；其二，长期传代稳定性不足，类器官在传代后容易出现结构崩解、增殖减缓甚至死亡，难以维持稳定的扩增能力；其三，现有培养体系对卵巢癌组织的解离效率不高，组织消化过程耗时较长且容易造成细胞损伤，影响后续培养的细胞活性和类器官形成效率；其四，细胞悬液中细胞分散度不佳，易发生团聚，导致类器官大小不均一
[0032]1）原代类器官形成效率显著提高：在多种卵巢癌样本中，采用本发明的培养基及培养方法，原代培养成功率可达70%以上，且不同样本间的形成率差异明显减小。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of organoid culture technology, specifically to an ovarian cancer organoid culture kit and its culture method. Background Technology
[0002] In recent years, organoid technology has made significant progress in tumor models such as colorectal cancer and gastric cancer, resulting in relatively mature three-dimensional culture systems. However, for ovarian cancer, especially high-grade serous ovarian cancer (HGSOC), primary isolation and in vitro expansion remain challenging due to its complex tissue origins and high heterogeneity. Existing culture systems are mostly based on DMEM / F12 basal medium supplemented with various growth factors, but in some ovarian cancer samples, problems such as low organoid formation success rate, morphological heterogeneity, and easy structural disintegration after passage still exist. Therefore, there is an urgent need for a culture kit and culture method optimized for the characteristics of ovarian cancer to improve the success rate of primary culture and passage stability.
[0003] Currently, ovarian cancer organoid culture generally faces the following problems: First, the success rate of primary culture is low and there are large individual differences. The organoid formation rate fluctuates significantly among different patient samples, and some samples are unable to form organoid structures with uniform morphology. Second, long-term passage stability is insufficient. After passage, organoids are prone to structural disintegration, slowed proliferation, or even death, making it difficult to maintain stable expansion capacity. Third, the existing culture system has low dissociation efficiency for ovarian cancer tissue. The tissue digestion process is time-consuming and easily causes cell damage, affecting the cell activity and organoid formation efficiency of subsequent cultures. Fourth, the cell dispersion in the cell suspension is poor, and it is easy to aggregate, resulting in uneven organoid size.
[0004] Currently, to address the low success rate and significant individual variability in primary culture of ovarian cancer organoids, existing techniques typically aim to improve cell viability by increasing the nutrient levels of the basal culture medium, such as by adding supplements like B27 and N2, or by increasing the concentration of growth factors like EGF and FGF. Additionally, some studies have employed high concentrations of matrix gel to enhance the three-dimensional support structure or added ROCK inhibitors in the early stages of culture to improve cell anti-apoptosis capabilities. However, these methods only improve short-term cell viability to a certain extent and do not fundamentally address the issue of varying culture condition dependencies among different ovarian cancer samples.
[0005] Standardized culture of ovarian cancer organoids faces three major technical bottlenecks: low tissue dissociation efficiency and significant cell damage, large differences in the success rate of primary culture, and insufficient structural stability after long-term passage. Meanwhile, the urgent clinical need for constructing patient-derived tumor models and the broad application prospects of organoid technology in basic research and translational medicine make the research on ovarian cancer organoid culture kits and corresponding culture methods imperative. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the present invention aims to provide an ovarian cancer organoid culture kit and its culture method. By using this culture kit, the efficiency of primary organoid formation, the uniformity of organoid morphology, the long-term passage stability, the cell yield after tissue dissociation, and the reduction of cell aggregation can be achieved.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides an ovarian cancer organoid culture kit, the kit comprising: DMEM / F12 basal medium, penicillin-streptomycin bispecific antibody solution 1×, L-alanyl-L-glutamine solution (Glutamax) 1×, buffer, B27 1×, N2 1×, R-spondin-1, Noggin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), neuroregulatory protein-1 (NRG1), nicotinamide, N-acetylcysteine, and SB203580.
[0009] In this invention, R-spondin-1 and Noggin synergistically promote cell aggregation and three-dimensional structural stability; HGF and NRG1 are added together to enhance cell viability and proliferation; nicotinamide and N-acetylcysteine are added simultaneously to reduce oxidative stress damage; and SB203580 is added to maintain cell activity during passage, significantly improving the initiation rate and structural integrity of organoids.
[0010] Furthermore, the final concentration of R-spondin-1 is 450-550 ng / mL; the final concentration of Noggin is 80-120 ng / mL.
[0011] Furthermore, the final concentration of R-spondin-1 is preferably 500 ng / mL.
[0012] Furthermore, the final concentration of Noggin is preferably 100 ng / mL.
[0013] Furthermore, the R-spondin-1 is His-labeled R-spondin-1 stably expressed and purified in HEK293E cells, with a specific activity ≥1.2×10⁻⁶. 6 U / mg.
[0014] Furthermore, the Noggin is endotoxin-free Noggin expressed by Escherichia coli, with endotoxin <0.1 EU / μg.
[0015] Furthermore, the final concentration of HGF is 8-12 ng / mL; the final concentration of NRG1 is 40-60 ng / mL.
[0016] Furthermore, the final concentration of HGF is preferably 10 ng / mL.
[0017] Furthermore, the final concentration of NRG1 is preferably 50 ng / mL.
[0018] Furthermore, the HGF is a human glycosylated HGF with a purity ≥95%, and it shows a single band on SDS-PAGE.
[0019] Furthermore, the NRG1 is human NRG1-β1 with a monomer content ≥98%.
[0020] Furthermore, the molar ratio of nicotinamide to N-acetylcysteine is 4:1.
[0021] Furthermore, the nicotinamide has a purity of ≥99.5% and a crystal particle size of ≤50 μm, and the N-acetylcysteine is an L-type anhydrous crystal with a purity of ≥99.0%.
[0022] Furthermore, the final concentration of SB203580 is 0.8-1.2 μM.
[0023] Furthermore, the final concentration of SB203580 is preferably 1 μM.
[0024] Furthermore, the SB203580 is prepared as a 10mM stock solution using DMSO as the solvent, stored at -80℃ in the dark, and used only after a single freeze-thaw cycle. The final concentration of DMSO added is ≤0.1%.
[0025] Furthermore, when the buffer solution is HEPES, the final concentration of HEPES is 8~12mM.
[0026] Furthermore, the final concentration of HEPES is preferably 10 mM.
[0027] Furthermore, when the buffer solution is a dual buffer system of HEPES and NaHCO3, the concentration of HEPES is 25 mM, the concentration of NaHCO3 is 5 mM, and the pH of the dual buffer system is 7.35-7.45.
[0028] Secondly, the present invention provides a method for culturing ovarian cancer organoids, which is implemented using the ovarian cancer organoid culture kit of the first aspect, and includes the following steps:
[0029] S1, Mechanically mince fresh ovarian cancer tissue into 1-2mm pieces.3 Tissue blocks were added to a tissue hydrolysate containing 1.5 mg / mL type IV collagenase, and the mixture was shaken at 37°C for 20-25 min. Tissue washing solution was added to stop the digestion. The mixture was filtered through a 100 μm sieve and centrifuged at 300 × g for 5 min to collect the cell pellet.
[0030] S2: Resuspend cells in ovarian cancer organoid complete culture medium containing 10% (v / v) Matrigel, adjust the density to 1×106 cells / mL, seed 20 μL / drop into pre-cooled 24-well plates, incubate at 37°C for 30 min to allow Matrigel to polymerize, add 1 mL of complete culture medium to each well, and incubate at 37°C in a 5% CO2 incubator. Change half of the medium every other day, and change the whole medium every 3-4 days.
[0031] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0032] 1) The efficiency of primary organoid formation is significantly improved: In various ovarian cancer samples, the success rate of primary culture using the culture medium and culture method of the present invention can reach more than 70%, and the difference in formation rate between different samples is significantly reduced.
[0033] 2) Significantly improved organoid morphological uniformity: The coefficient of variation (CV) of the diameter of the organoids cultured by this invention is reduced from more than 25% in the prior art to less than 15%. The organoids are regular spherical or near-spherical with clear edges and dense internal structure, which facilitates subsequent observation, counting and standardized analysis.
[0034] 3) Significantly enhanced long-term passage stability: This invention supports stable passage of ovarian cancer organoids, with the organoid structure remaining intact during passage. In contrast, existing technologies typically result in significant structural disintegration, slowed proliferation, or even cell death after passage. This invention effectively solves the problems of decreased cell viability and structural damage during long-term culture.
[0035] 4) High cell yield and low cell aggregation after tissue dissociation: The present invention uses antioxidant protection in conjunction with tissue dissociation and subsequent processing, which significantly reduces the proportion of cell aggregation in cell suspension, and the size distribution of organoids after plating is more uniform, effectively ensuring the reproducibility of the experiment.
[0036] In summary, this invention significantly outperforms existing technologies in terms of success rate, uniformity, and stability in ovarian cancer organoid culture, providing a reliable in vitro model culture tool for basic research and translational applications of ovarian cancer. Attached Figure Description
[0037] Figure 1Microscopic images of Example 1 and Comparative Example 1 provided for the present invention: (a) Microscopic image of Example 1 after one day of culture; (b) Microscopic image of Example 1 after three days of culture; (c) Microscopic image of Example 1 after seven days of culture; (d) Microscopic image of Comparative Example 1 after one day of culture; (e) Microscopic image of Comparative Example 1 after three days of culture; (f) Microscopic image of Comparative Example 1 after seven days of culture.
[0038] Figure 2 Microscopic images of the fifth generation of ovarian cancer organoids cultured in Example 1 of the present invention: (a) Microscopic image at 4x magnification; (b) Microscopic image at 10x magnification. Detailed Implementation
[0039] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present invention and the specific features in the embodiments are detailed descriptions of the technical solution of the present invention, rather than limitations thereof.
[0040] Example 1
[0041] This embodiment provides an ovarian cancer organoid culture kit and its culture method. The final concentration composition of the ovarian cancer organoid culture kit is as follows: DMEM / F12 basal medium, penicillin / streptomycin 1×, Glutamax 1×, HEPES 10mM, B27 1×, N2 1×, R-spondin-1 500ng / mL, Noggin 100ng / mL, EGF 50ng / mL, HGF 10ng / mL, NRG1 50ng / mL, nicotinamide 5mM, N-acetylcysteine 1.25mM, and SB203580 1μM.
[0042] Among them, R-spondin-1 is His-labeled R-spondin-1 that has been stably expressed and purified in HEK293E cells, with a specific activity ≥1.2×10⁻⁶. 6U / mg; Noggin is endotoxin-free Noggin expressed by E. coli, with endotoxin <0.1 EU / μg; HGF is human glycosylated HGF, with a purity ≥95%, showing a single band on SDS-PAGE; NRG1 is human NRG1-β1, with a monomer content ≥98%; the molar ratio of nicotinamide to N-acetylcysteine is 4:1; nicotinamide purity ≥99.5%, crystal particle size ≤50 μm; N-acetylcysteine is L-type anhydrous crystals with a purity ≥99.0%; SB203580 is a 10 mM stock solution prepared with DMSO as solvent, stored at -80℃ protected from light, used after a single freeze-thaw cycle, and the final concentration of DMSO added is ≤0.1%.
[0043] The cultivation method includes the following steps:
[0044] S1. Take approximately 150 mg of fresh HGSOC tissue, wash three times with tissue cleaning solution to remove bloodstains and necrotic tissue; place the tissue in a culture dish and mechanically cut it to 1.5 mm using sterile scissors. 3 Tissue blocks of various sizes were added to 3 mL of tissue hydrolysate containing 1.5 mg / mL type IV collagenase. The mixture was incubated at 37°C with shaking for 25 min. The tissue dissociation was observed under a microscope during digestion. Once the tissue blocks had largely dispersed into cell clusters, an equal volume of tissue washing solution was added to terminate the digestion. The solution was filtered through a 100 μm sieve, and the filtrate was collected. The filtrate was centrifuged at 300 × g for 5 min, and the supernatant was discarded to obtain the cell pellet.
[0045] S2, resuspend the cell pellet in ovarian cancer organoid complete culture medium containing 10% matrix gel, and adjust the cell density to 1×10⁶ cells / year. 6 Cells / mL; seed 20 μL / drop into pre-chilled 24-well plates, 1 drop per well, and incubate at 37°C for 30 min. After complete polymerization of the matrix gel, add 500 μL of the ovarian cancer organoid culture kit to each well. Incubate at 37°C, 5% CO2. Cell morphology after one day of culture is as follows. Figure 1 As shown in (a); the medium was completely changed every 3 days, and the cell morphology after three days of culture was as follows. Figure 1 As shown in (b); after culturing for 7 days, observe under a microscope (cell morphology as shown). Figure 1 (c) shows the organoid formation rate. The results showed that the organoid formation rate was approximately 80%, and the organoids were regularly spherical with clear edges and an average diameter of (128±17) μm. The morphology of the ovarian cancer organoids at the fifth generation was as follows. Figure 2 (a) and Figure 2 As shown in (b).
[0046] Example 2
[0047] This embodiment provides an ovarian cancer organoid culture kit and its culture method. The final concentration composition of the ovarian cancer organoid culture kit is as follows: DMEM / F12 basal medium, penicillin / streptomycin 1×, Glutamax 1×, HEPES 25mM, NaHCO3 5mM, B27 1×, N2 1×, R-spondin-1 550ng / mL, Noggin 120ng / mL, EGF 50ng / mL, HGF 12ng / mL, NRG1 60ng / mL, nicotinamide 5mM, N-acetylcysteine 1.25mM, and SB203580 1.2μM.
[0048] Among them, R-spondin-1 is His-labeled R-spondin-1 that has been stably expressed and purified in HEK293E cells, with a specific activity ≥1.2×10⁻⁶. 6 U / mg; Noggin is endotoxin-free Noggin expressed by E. coli, with endotoxin <0.1 EU / μg; HGF is human glycosylated HGF, with a purity ≥95%, showing a single band on SDS-PAGE; NRG1 is human NRG1-β1, with a monomer content ≥98%; the molar ratio of nicotinamide to N-acetylcysteine is 4:1; nicotinamide purity ≥99.5%, crystal particle size ≤50 μm; N-acetylcysteine is L-type anhydrous crystals with a purity ≥99.0%; SB203580 is a 10 mM stock solution prepared with DMSO as solvent, stored at -80℃ protected from light, used after a single freeze-thaw cycle, and the final concentration of DMSO added is ≤0.1%; 25 mM HEPES and 5 mM NaHCO3 are used to prepare a buffer solution with a pH of 7.4.
[0049] The cultivation method includes the following steps:
[0050] S1. Take approximately 150 mg of fresh HGSOC tissue and wash it three times with tissue cleaning solution to remove bloodstains and necrotic tissue. Place the tissue in a culture dish and mechanically cut it to 2 mm using sterile scissors. 3 Tissue blocks of various sizes were added to 3 mL of tissue hydrolysate containing 1.5 mg / mL type IV collagenase. The mixture was incubated at 37°C with shaking for 20 min. During digestion, the tissue dissociation was observed under a microscope. Once the tissue blocks had largely dispersed into cell clusters, an equal volume of tissue washing solution was added to terminate the digestion. The solution was filtered through a 100 μm sieve, and the filtrate was collected. The filtrate was centrifuged at 300 × g for 5 min, and the supernatant was discarded to obtain the cell pellet.
[0051] S2, resuspend the cell pellet in ovarian cancer organoid complete culture medium containing 10% matrix gel, and adjust the cell density to 1×10⁶ cells / year. 6 Cells / mL; seed 20 μL / drop into pre-chilled 24-well plates, 1 drop per well, and incubate at 37°C for 30 min until the matrix gel is fully polymerized. Then add 500 μL of the ovarian cancer organoid culture kit to each well. Incubate at 37°C in a 5% CO2 incubator, changing the medium completely every 3 days.
[0052] Example 3
[0053] This embodiment provides an ovarian cancer organoid culture kit and its culture method. The final concentration composition of the ovarian cancer organoid culture kit is as follows: DMEM / F12 basal medium, penicillin / streptomycin 1×, Glutamax 1×, HEPES 12mM, B27 1×, N2 1×, R-spondin-1 450ng / mL, Noggin 80ng / mL, EGF 50ng / mL, HGF 8ng / mL, NRG1 40ng / mL, nicotinamide 5mM, N-acetylcysteine 1.25mM, and SB203580 0.8μM.
[0054] Among them, R-spondin-1 is His-labeled R-spondin-1 that has been stably expressed and purified in HEK293E cells, with a specific activity ≥1.2×10⁻⁶. 6 U / mg; Noggin is endotoxin-free Noggin expressed by Escherichia coli, with endotoxin <0.1 EU / μg; HGF is human glycosylated HGF, with a purity ≥95%, showing a single band on SDS-PAGE; NRG1 is human NRG1-β1, with a monomer content ≥98%; the molar ratio of nicotinamide to N-acetylcysteine is 4:1; nicotinamide purity ≥99.5%, crystal particle size ≤50μm; N-acetylcysteine is L-type anhydrous crystals with a purity ≥99.0%; SB203580 is a 10mM stock solution prepared with DMSO as solvent, stored at -80℃ protected from light, used after a single freeze-thaw cycle, and the final concentration of DMSO added is ≤0.1%.
[0055] The cultivation method includes the following steps:
[0056] S1. Take approximately 150 mg of fresh HGSOC tissue and wash it three times with tissue cleaning solution to remove bloodstains and necrotic tissue; place the tissue in a culture dish and mechanically cut it to 1 mm using sterile scissors. 3Tissue blocks of various sizes were added to 3 mL of tissue hydrolysate containing 1.5 mg / mL type IV collagenase. The mixture was incubated at 37°C with shaking for 22 min. During digestion, the tissue dissociation was observed under a microscope. Once the tissue blocks had largely dispersed into cell clusters, an equal volume of tissue washing solution was added to terminate the digestion. The solution was filtered through a 100 μm sieve, and the filtrate was collected. The filtrate was centrifuged at 300 × g for 5 min, and the supernatant was discarded to obtain the cell pellet.
[0057] S2, resuspend the cell pellet in ovarian cancer organoid complete culture medium containing 10% matrix gel, and adjust the cell density to 1×10⁶ cells / year. 6 Cells / mL; seed 20 μL / drop into pre-chilled 24-well plates, 1 drop per well, and incubate at 37°C for 30 min until the matrix gel is fully polymerized. Then add 500 μL of the ovarian cancer organoid culture kit to each well. Incubate at 37°C in a 5% CO2 incubator, changing the medium completely every 2 days.
[0058] Comparative Example 1
[0059] The operating procedures for this comparative example are basically the same as those in Example 1, except that nicotinamide (5 mM) in the complete culture medium is replaced with an equimolar concentration of nicotinamide mononucleotide (NMN), and N-acetylcysteine (1.25 mM) is replaced with an equimolar concentration of lipoic acid (LA). All other components, concentrations, and culture conditions are consistent with those in Example 1.
[0060] HGSOC from the same source was used for tissue dissociation, cell plating, and culture according to the same method as in Example 1. Microscopic observation was performed on days 1, 3, and 7 of culture. The cell morphology on day 1 is as follows. Figure 1 As shown in (d), the cell morphology on day 3 is as follows. Figure 1 As shown in (e), the cell morphology on day 7 is as follows. Figure 1 As shown in (f), the organoid formation rate was statistically analyzed. The results showed that the organoid formation rate was only 41.6%, the average diameter was (79±22) μm, the coefficient of variation in diameter was 27.8%, the organoid morphology was irregular, and some samples showed fragmentation.
[0061] The culture performance of the ovarian cancer organoid culture kits prepared in Example 1 and Comparative Example 1 was tested, and the specific process is as follows:
[0062] The corresponding ovarian cancer organoid culture kits were prepared according to Example 1 and Comparative Example 1, and the cells were cultured. After one day, three days, and seven days of culture, the corresponding cell morphology was photographed under a microscope, and the organoid formation rate, mean diameter, and coefficient of variation of diameter were statistically analyzed. The test results are as follows: Figure 1 As shown.
[0063] Depend on Figure 1 It is evident that the specific combination of nicotinamide and N-acetylcysteine in this invention exhibits a significant synergistic promoting effect at this concentration, while the tested alternatives (NMN, LA) cannot achieve the same culture support effect.
[0064] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. An ovarian cancer organoid culture kit, characterized in that, The kit consists of DMEM / F12 basal medium, penicillin-streptomycin bispecific antibody solution 1×, L-alanyl-L-glutamine solution 1×, buffer, B27 1×, N2 1×, R-spondin-1, Noggin, 50 ng / mL epidermal growth factor, hepatocyte growth factor, neuromodulatory protein-1, nicotinamide, N-acetylcysteine, and SB203580.
2. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The final concentration of R-spondin-1 is 450-550 ng / mL; the final concentration of Noggin is 80-120 ng / mL.
3. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The R-spondin-1 mentioned is His-labeled R-spondin-1 stably expressed and purified in HEK293E cells, with a specific activity ≥1.2×10⁻⁶. 6 U / mg.
4. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The Noggin in question is endotoxin-free Noggin expressed in Escherichia coli with an endotoxin content of <0.1 EU / μg.
5. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The final concentration of the hepatocyte growth factor is 8-12 ng / mL; the final concentration of the neuroregulatory protein-1 is 40-60 ng / mL.
6. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The hepatocyte growth factor is a human glycosylated hepatocyte growth factor with a purity of ≥95%, and it shows a single band on SDS-PAGE.
7. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The neuroregulatory protein-1 is a human neuroregulatory protein-1 with a monomer content ≥98%.
8. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The molar ratio of nicotinamide to N-acetylcysteine is 4:
1.
9. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The nicotinamide has a purity of ≥99.5% and a crystal particle size of ≤50μm, and the N-acetylcysteine is an L-type anhydrous crystal with a purity of ≥99.0%.
10. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The final concentration of SB203580 is 0.8-1.2 μM.
11. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The SB203580 is prepared as a 10mM stock solution using dimethyl sulfoxide as solvent, stored at -80℃ protected from light, and used only after a single freeze-thaw cycle. The final concentration of dimethyl sulfoxide added is ≤0.1%.
12. The ovarian cancer organoid culture kit according to claim 1, characterized in that, The buffer solution is 4-hydroxyethylpiperazine ethanesulfonic acid or a double buffer system of 4-hydroxyethylpiperazine ethanesulfonic acid and sodium bicarbonate. When the buffer solution is 4-hydroxyethylpiperazine ethanesulfonic acid, the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 8~12mM; when the buffer solution is a double buffer system of 4-hydroxyethylpiperazine ethanesulfonic acid and sodium bicarbonate, the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 25mM, the final concentration of sodium bicarbonate is 5mM, and the pH of the double buffer system is 7.35-7.
45.
13. A method for culturing ovarian cancer organoids, comprising using the ovarian cancer organoid culture kit according to any one of claims 1 to 12, characterized in that, Includes the following steps: S1, Mechanically mince fresh ovarian cancer tissue into 1-2mm pieces. 3 Tissue blocks were added to a tissue hydrolysate containing 1.5 mg / mL type IV collagenase, and the mixture was shaken at 37°C for 20-25 min. Tissue washing solution was added to stop the digestion. The mixture was filtered through a 100 μm sieve and centrifuged at 300 × g for 5 min to collect the cell pellet. S2, cells were resuspended in ovarian cancer organoid complete culture medium containing 10% (v / v) matrix gel and the density was adjusted to 1×10⁻⁶. 6 Inoculate cells / mL at 20 μL / drop into a pre-cooled 24-well plate, incubate at 37°C for 30 min to allow the matrix gel to polymerize, add 500 μL of complete culture medium to each well, and incubate at 37°C in a 5% CO2 incubator. Replace the medium completely every 2-3 days.