Palvian cystic tumor organoid culture medium and application thereof

Abstract

The invention relates to the technical field of organoid construction and culture, and discloses a yolk sac tumor organoid culture medium and application thereof. The culture medium comprises a basic culture medium and specific addition factors, and the specific addition factors comprise L-glutamine, a TGF-BI type receptor inhibitor A83-01, nicotinamide, an N2 supplement, a vitamin A-removed B27 supplement, N-acetylcysteine, beta-estradiol, a P38MAPK inhibitor SB202190, a bone morphogenetic protein antagonist Noggin, a Wnt pathway related protein R-Spondin-1, an epidermal growth factor, an insulin-like growth factor 1, an insulin-like growth factor 2, an insulin-like growth factor 3, an insulin-like growth factor 4, an insulin-like growth factor 5 and an insulin-like growth factor 6. The medicine composition is prepared from a liver cell growth factor, a neuroregulatory protein-1, a ROCK inhibitor Y27632, activin A and interleukin-6. By adopting the culture medium disclosed by the invention, the successful construction of the egg cystic tumor organoid can be realized, and the stable passage of the egg and the stable and rapid growth after passage can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of the construction and culture of organoids, and particularly relates to a yolk sac tumor organoid culture medium and its application. Background Art

[0002] Ovarian yolk sac tumor, also known as ovarian endodermal sinus tumor, belongs to ovarian germ cell malignant tumors, which are more common in children and young women. It has a high degree of malignancy, grows rapidly, is prone to early metastasis, and has a poor prognosis. Yolk sac tumors exhibit various histological patterns. The endodermal sinus or Schiller-Duval body pattern is the most distinctive type. However, solid, alveolar-glandular, multivesicular yolk, myxomatous, papillary, and microcystic patterns are also visible. Constructing an ideal model can provide a technical platform for the basic transformation and clinical trial research of yolk sac tumors, thereby promoting the personalized and precise development of the treatment of yolk sac tumors.

[0003] Currently, the commonly used tumor models are mainly 2D cancer cell lines and patient-derived animal transplantation models, but these two models have many limitations. In recent years, organoid technology has been continuously developed, and organoid models have become a new tool for studying human cancers. Compared with traditional two-dimensional cell culture, organoids have many advantages. For example, organoids can exhibit a cell composition and physiological functions close to the physiological state. Models with these characteristics are particularly suitable for use as a biobank and high-throughput screening. In addition, compared with animal models, organoid models can reduce the complexity of experiments and enable the application of real-time imaging technology. It is difficult for animal models to accurately simulate human development and disease research, but organoid models can. In recent studies, it has been demonstrated that organoids can be developed from a variety of different cancer types, and these tumor-derived organoids maintain type and patient-specific characteristics. Therefore, establishing a yolk sac tumor organoid model is not only beneficial to the basic research of yolk sac tumors, but also helps in the diagnosis and treatment of yolk sac tumors, and improves the overall survival rate of yolk sac tumor patients.

[0004] However, at present, research reports on yolk sac tumor organoids are relatively rare. The possible reason is that the development of yolk sac tumor organoid culture media is relatively difficult because different tumors are in different in vivo environments and there is a large heterogeneity. Therefore, the culture media for different tumor organoids cannot be universal. In addition, the 2D adherent cultured tumor cell lines have been immortalized or laboratory-domesticated. Even if they are all yolk sac tumor cells, the traditional culture media used for constructing and culturing 2D tumor cell lines cannot be used for constructing and culturing yolk sac tumor organoids. Moreover, to achieve the construction and stable and rapid passage of yolk sac tumor organoids, it is necessary to meet various requirements such as maintaining the proliferation activity of yolk sac tumor cells and activating their proliferation pathways. Therefore, it is necessary to reasonably allocate the components and dosages of the yolk sac tumor organoid culture medium. Therefore, there is still an urgent need in the current biomedical technology field for a culture medium that can achieve the construction and stable and rapid passage of yolk sac tumor organoids. Summary of the Invention

[0005] To solve the above technical problem, that is, there is currently no culture medium that can be used for the construction and subculture of yolk sac tumor organoids, the present invention provides a yolk sac tumor organoid culture medium and its application. This culture medium can not only achieve the successful construction of yolk sac tumor organoids from yolk sac tumor cells, but also realize the stable subculture of yolk sac tumor organoids and the stable and rapid growth after subculture.

[0006] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides a yolk sac tumor organoid culture medium, which includes a basal medium and specific additive factors; the specific additive factors include the following components: L-glutamine, TGF-β type I receptor inhibitor A83-01, nicotinamide, N2 supplement, B27 supplement without vitamin A, N-acetylcysteine, β-estradiol, P38 MAPK inhibitor SB202190, bone morphogenetic protein antagonist Noggin, Wnt pathway-related protein R-Spondin-1, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF1), hepatocyte growth factor (HGF), neuregulin-1 (NRG1), ROCK inhibitor Y27632, activin A, interleukin-6.

[0007] After theoretical research and a large number of experiments, the team of the present invention designed the above culture medium for culturing, which can achieve the construction of human-derived yolk sac tumor organoids with a high success rate, and can also achieve the stable subculture of human-derived yolk sac tumor organoids and the rapid and stable growth after subculture. Therefore, it can provide an effective disease model for the study of the pathogenesis of yolk sac tumors, the drug screening of yolk sac tumor patients and the auxiliary clinical treatment, and has broad application prospects in basic research and clinical diagnosis and treatment.

[0008] Preferably, the basal medium is DMEM / F12 medium.

[0009] When DMEM / F12 medium is selected as the basal medium, it can cooperate well with the specific additive factors in the present invention, make the yolk sac tumor organoids have high activity, and can grow and develop rapidly, thus accelerating the construction of yolk sac tumor organoids.

[0010] Preferably, in the yolk sac tumor organoid culture medium, the final concentrations of each component are as follows: L-glutamine, 5-15 mM; TGF-β type I receptor inhibitor A83-01, 0.5-5 μM; nicotinamide, 5-10 mM; N2 supplement, 0.5-1.5% vol; vitamin A-free B27 supplement, 1-3% vol; N-acetylcysteine, 1-2 mM; β-estradiol, 5-15 nM; P38 MAPK inhibitor SB202190, 0.5-5 μM; bone morphogenetic protein antagonist Noggin, 100-500 ng / mL; Wnt pathway-related protein R-Spondin-1, 250-750 ng / mL; epidermal growth factor (EGF), 25-75 ng / mL; insulin-like growth factor 1 (IGF1), 10-30 ng / mL; hepatocyte growth factor (HGF), 5-15 ng / mL; neuregulin-1 (NRG1), 25-75 ng / mL; ROCK inhibitor Y27632, 5-15 μM; activin A, 10-30 ng / mL; interleukin-6, 10-30 ng / mL; the solvent is DMEM / F12 medium.

[0011] Furthermore, in the yolk sac tumor organoid culture medium, the final concentrations of each component are as follows: L-glutamine, 10 mM; TGF-β type I receptor inhibitor A83-01, 0.25 μm; nicotinamide, 5 mM; N2 supplement, 1% vol; vitamin A-free B27 supplement, 2% vol; N-acetylcysteine, 1.25 mM; β-estradiol, 10 nM; P38 MAPK inhibitor SB202190, 0.5 μM; bone morphogenetic protein antagonist Noggin, 100 ng / mL; Wnt pathway-related protein R-Spondin-1, 50 ng / mL; epidermal growth factor (EGF), 50 ng / mL; insulin-like growth factor 1 (IGF1), 20 ng / mL; hepatocyte growth factor (HGF), 10 ng / mL; neuregulin-1 (NRG1), 50 ng / mL; ROCK inhibitor Y27632, 10 μM; activin A, 20 ng / mL; interleukin-6, 20 ng / mL; the solvent is DMEM / F12 medium.

[0012] In a second aspect, the present invention provides the use of the yolk sac tumor organoid culture medium in constructing yolk sac tumor organoids.

[0013] Using the culture medium of the present invention, the successful construction of yolk sac tumor organoids from yolk sac tumor cells can be achieved.

[0014] Preferably, the use includes the following steps: (1) Pretreat and digest the yolk sac tumor tissue to obtain yolk sac tumor cells; (2) After resuspending the yolk sac tumor cells with Matrigel, drop them into a culture container and let it solidify to form a gel droplet containing yolk sac tumor cells; (3) Add the yolk sac tumor organoid medium into the culture container and culture it to obtain yolk sac tumor organoids.

[0015] Preferably, in step (1), the process of pretreatment includes washing and cutting into pieces.

[0016] Preferably, in step (1), the process of digestion specifically includes the following steps: Mix the pretreated yolk sac tumor organoids with digestion solution 1 containing collagenase I for the first digestion, collect the cells after terminating the reaction, mix them with digestion solution 2 containing dispase II for the second digestion, collect the cells, mix them with a buffer solution, filter through a filter screen with a pore size of 95 - 105 μm, and collect the cells from the filtrate to obtain yolk sac tumor cells.

[0017] Furthermore, the digestion solution 1 containing collagenase I is a DMEM / F12 medium containing 1 - 3 mg / mL collagenase I; the digestion solution 2 containing dispase II is a DMEM / F12 medium containing 1 - 3 μg / mL dispase 2.

[0018] Furthermore, before mixing the collected cells with the buffer solution, first mix the collected cells with a red blood cell lysate for red blood cell lysis, and then mix them with the buffer solution.

[0019] Preferably, in step (2), the ratio of yolk sac tumor cells to Matrigel is 1.5×10 5 ~2.5×10 5 cells / 100 μL Matrigel; in step (3), the temperature of the culture is 35 - 37 °C, the CO2 concentration is 4 - 6%, and the medium is changed every 2 - 3 days.

[0020] In a third aspect, the present invention provides the application of the yolk sac tumor organoid medium in the subculture of yolk sac tumor organoids.

[0021] When the medium in the present invention is used for the subculture of yolk sac tumor organoids, stable subculture of this type of tumor organoids can be achieved, and the growth of the organoids after subculture is relatively stable and rapid, thereby realizing the large - scale and rapid propagation of yolk sac tumor organoids, meeting the needs of high - throughput drug screening experiments, helping doctors provide more accurate drugs for patients, and improving the overall survival rate of patients.

[0022] Preferably, the application includes the following steps: S1: Digest the yolk sac tumor organoids; S2: After resuspending the digested yolk sac tumor organoids in step S1 with Matrigel, drop them into a culture container and let it solidify to form a gel droplet; S3: Add the yolk sac tumor organoid medium into the culture container and culture to obtain passage organoids.

[0023] Preferably, in step S1, Tryple digestive juice is used to digest the yolk sac tumor organoids.

[0024] Preferably, in step S3, the temperature of the culture is 35 - 37 °C, the CO2 concentration is 4 - 6%, the medium is changed every 2 - 3 days, and the culture time is 7 - 10 days.

[0025] Compared with the prior art, the present invention has the following advantages: The medium formula provided by the present invention can be used for the construction and passage culture of yolk sac tumor organoids, which can not only successfully construct yolk sac tumor organoids from yolk sac tumor cells, but also achieve the stable passage of yolk sac tumor organoids and their stable and rapid growth after passage. Therefore, it can provide an effective disease model for the study of the pathogenesis of yolk sac tumor, drug screening for yolk sac tumor patients and assisting clinical treatment, and has broad application prospects in basic research and clinical diagnosis and treatment. Description of the Drawings

[0026] Figure 1 It is the growth situation of primary yolk sac tumor organoids in Application Example 1. Among them, Figure 1 A - 1D are representative bright - field images taken at the 0th, 2nd, 6th, and 12th days of culturing primary yolk sac tumor organoids respectively.

[0027] Figure 2 It is the growth situation of passage yolk sac tumor organoids in Application Example 3. Among them, Figure 2 A - 2C are representative bright - field images taken at the 0th, 2nd, and 10th days of culturing passage yolk sac tumor organoids respectively.

[0028] Figure 3 It is the influence of the basal medium on the construction of yolk sac tumor organoids. Among them, Figure 3 A and Figure 3 B are representative bright - field images taken at the 8th day of culturing primary yolk sac tumor organoids in Application Example 1 and Comparative Application Example 1 respectively.

[0029] Figure 4 It is the influence of activin A in the medium on the construction of yolk sac tumor organoids. Among them, Figure 4 A and Figure 4 B are representative bright - field images taken at the 8th day of culturing primary yolk sac tumor organoids in Application Example 1 and Comparative Application Example 2 respectively. Detailed Embodiments

[0030] The present invention will be further described below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection of the present invention is the appended claims and any equivalents thereof.

[0031] General Embodiment A yolk sac tumor organoid culture medium, comprising a basal medium and specific additive factors; the specific additive factors include the following components: L-glutamine, TGF-β type I receptor inhibitor A83-01, nicotinamide, N2 supplement, vitamin A-free B27 supplement, N-acetylcysteine, β-estradiol, P38 MAPK inhibitor SB202190, bone morphogenetic protein antagonist Noggin, Wnt pathway-related protein R-Spondin-1, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF1), hepatocyte growth factor (HGF), neuregulin-1 (NRG1), ROCK inhibitor Y27632, activin A, interleukin-6.

[0032] As a specific embodiment, the basal medium is DMEM / F12 medium.

[0033] As a specific embodiment, in the yolk sac tumor organoid culture medium, the final concentrations of each component are as follows: L-glutamine, 5-15 mM; TGF-β type I receptor inhibitor A83-01, 0.5-5 μM; nicotinamide, 5-10 mM; N2 supplement, 0.5-1.5% vol; vitamin A-free B27 supplement, 1-3% vol; N-acetylcysteine, 1-2 mM; β-estradiol, 5-15 nM; P38 MAPK inhibitor SB202190, 0.5-5 μM; bone morphogenetic protein antagonist Noggin, 100-500 ng / mL; Wnt pathway-related protein R-Spondin-1, 250-750 ng / mL; epidermal growth factor (EGF), 25-75 ng / mL; insulin-like growth factor 1 (IGF1), 10-30 ng / mL; hepatocyte growth factor (HGF), 5-15 ng / mL; neuregulin-1 (NRG1), 25-75 ng / mL; ROCK inhibitor Y27632, 5-15 μM; activin A, 10-30 ng / mL; interleukin-6, 10-30 ng / mL; the solvent is DMEM / F12 medium.

[0034] In a second aspect, the present invention provides the use of the yolk sac tumor organoid culture medium in constructing yolk sac tumor organoids.

[0035] As a specific embodiment, the application includes the following steps: (1) Pretreat and digest the yolk sac tumor tissue to obtain yolk sac tumor cells; (2) After resuspending the yolk sac tumor cells with Matrigel, drop them into a culture container and let it solidify to form a gel droplet containing yolk sac tumor cells; (3) Add the yolk sac tumor organoid medium into the culture container and culture to obtain yolk sac tumor organoids.

[0036] As a specific embodiment, in step (1), the pretreatment process includes washing and cutting into pieces.

[0037] As a specific embodiment, in step (1), the digestion process specifically includes the following steps: Mix the pretreated yolk sac tumor organoids with DMEM / F12 medium containing 1 - 3 mg / mL collagenase I for the first digestion, collect the cells after terminating the reaction, mix them with DMEM / F12 medium containing 1 - 3 μg / mL dispase II for the second digestion, collect the cells, mix them with a buffer solution, filter through a filter with a pore size of 95 - 105 μm, and collect the cells from the filtrate to obtain yolk sac tumor cells. Optionally, in the above process, before mixing the collected cells with the buffer solution, first mix the collected cells with red blood cell lysate for red blood cell lysis, and then mix with the buffer solution.

[0038] As a specific embodiment, in step (2), the ratio of yolk sac tumor cells to Matrigel is 1.5×10 5 ~2.5×10 5 cells / 100 μL Matrigel; in step (3), the culture temperature is 35 - 37 °C, the CO2 concentration is 4 - 6%, and the medium is changed every 2 - 3 days.

[0039] In a third aspect, the present invention provides the application of the yolk sac tumor organoid medium in the subculture of yolk sac tumor organoids.

[0040] Preferably, the application includes the following steps: S1: Digest the yolk sac tumor organoids; S2: After resuspending the digested yolk sac tumor organoids in step S1 with Matrigel, drop them into a culture container and let it solidify to form a gel droplet; S3: Add the yolk sac tumor organoid medium into the culture container and culture to obtain subcultured organoids.

[0041] As a specific embodiment, in step S1, Tryple digestive juice is used to digest the yolk sac tumor organoids.

[0042] As a specific embodiment, in step S3, the temperature of the culture is 35-37 °C, the CO2 concentration is 4-6%, the culture medium is changed every 2-3 days, and the culture time is 7-10 days.

[0043] Preparation Example 1: Preparation of Yolk Sac Tumor Organoid Medium Prepare the yolk sac tumor organoid medium according to the formula shown in Table 1.

[0044] Table 1 Formula of Yolk Sac Tumor Organoid Medium Comparative Preparation Example 1: Preparation of Yolk Sac Tumor Organoid Medium The difference between this comparative preparation example and Preparation Example 1 is only that the DMEM / F12 medium is replaced with RPMI-1640 medium. The other components and their final concentrations are the same as those in Preparation Example 1.

[0045] Comparative Preparation Example 2: Preparation of Yolk Sac Tumor Organoid Medium The difference between this comparative preparation example and Preparation Example 1 is only that activin A is not added. The other components and their final concentrations are the same as those in Preparation Example 1.

[0046] Application Example 1: Construction of Yolk Sac Tumor Organoids Use the yolk sac tumor organoid medium prepared according to the method in Preparation Example 1 to construct yolk sac tumor organoids. The specific steps are as follows: (1) Washing: Obtain human yolk sac tumor tissue by surgery or biopsy, remove connective tissue and necrotic parts, etc., and wash the tissue 5 times with cold PBS buffer containing antibiotics; (2) Mincing: Cut the tissue into minced meat with surgical scissors in turn, which can pass through a 1 mL pipette tip smoothly, evenly loose and non-adherent (operate at low temperature on ice); (3) Digestion: Transfer the tissue to a 15 mL centrifuge tube, add an appropriate amount of Digestive Solution 1 (DMEM / F12 medium containing 1 mg / mL collagenase I), blow and mix well, place it in a 37 °C constant temperature shaker for 30 min, terminate with twice the volume of cold PBS buffer, centrifuge to collect cells, add Digestive Solution 2 (DMEM / F12 medium containing 1 μg / mL dispase II), blow and mix well, and place it in a 37 °C constant temperature shaker for 30 min; (4) Centrifugation: Centrifuge the digested cell suspension at 1500 rpm for 5 min, and discard the supernatant; (5) Hemolysis: When a macroscopic red precipitate appears, add 3 mL of red blood cell lysate, mix well, let it stand on ice for 5 min, add 4 times the volume of cold PBS buffer to terminate the reaction, mix well, filter through a 100 μm filter, centrifuge the filtrate at 1500 rpm for 5 min, and discard the supernatant; (6) Counting: Resuspend with DMEM / F12 medium and then perform counting; after counting, centrifuge at 1500 rpm for 5 min and discard the supernatant; (7) Seeding: Resuspend the cells with Matrigel matrix gel at a ratio of 2×10 5 cells / 100 μL of matrix gel (operate at low temperature on ice), and drop the matrix gel and cell mixture in the center of a 24-well plate at 100 μL / well, and solidify it in an incubator at 37 °C and 5% CO2 for 15 min; (8) Slowly add 700 μL of yolk sac tumor organoid medium (prepared according to the method in Preparation Example 1) along the side wall of the well plate, and culture it in an incubator at 37 °C and 5% CO2. Change the medium every 3 days during the culture period. The growth conditions of the yolk sac tumor organoids at 0, 2, 6, and 12 days of culture are shown in Figure 1 A to Figure 1 D. It can be seen from Figure 1 that by using the method of this application example, yolk sac tumor organoids can be obtained relatively quickly, and the obtained organoids have clear and complete structural morphology and are relatively stable during the growth process.

[0047] Application Example 2: The yolk sac tumor organoids were constructed using the yolk sac tumor organoid medium prepared according to the method in Preparation Example 1. The specific steps for constructing the yolk sac tumor organoids are as follows: (1) Washing: Obtain human yolk sac tumor tissue by surgery or biopsy, remove connective tissue and necrotic parts, etc., and wash the tissue 5 times with cold PBS buffer containing antibiotics; (2) Mincing: Cut the tissue into minced meat with surgical scissors in sequence, and it can pass through a 1 mL pipette tip smoothly, being evenly loose and not sticky (operate at low temperature on ice); (3) Digestion: Transfer the tissue to a 15 mL centrifuge tube, add an appropriate amount of Digestive Solution 1 (DMEM / F12 medium containing 1 mg / mL collagenase I), mix well by pipetting, and place it in a constant temperature shaker at 37 °C for 30 min. After terminating with twice the volume of cold PBS buffer, centrifuge to collect the cells, add Digestive Solution 2 (DMEM / F12 medium containing 1 μg / mL dispase II), mix well by pipetting, and place it in a constant temperature shaker at 37 °C for 30 min; (4) Centrifugation: Centrifuge the digested cell suspension at 1500 rpm for 5 min and discard the supernatant; (5) Filtration: No visible red precipitate was observed, so no red blood cell lysis was performed; add 4 times the volume of cold PBS buffer, mix well, filter through a 100 μm filter mesh, and centrifuge the filtrate at 1500 rpm for 5 min and discard the supernatant; (6) Counting: Resuspend with DMEM / F12 medium and then perform counting; after counting, centrifuge at 1500 rpm for 5 min and discard the supernatant; (7) Seeding: Resuspend the cells with Matrigel matrix gel at a ratio of 2×10 5Resuspend the cells at a ratio of cells / 100 μL Matrigel matrix gel (operate at low temperature on ice), and drop 100 μL of the mixture of Matrigel matrix gel and cells onto the center of a 24-well plate, and solidify it in an incubator at 37 °C and 5% CO2 for 15 min; (8) Slowly add 700 μL of yolk sac tumor organoid medium (prepared according to the method in Preparation Example 1) along the side wall of the well plate, and culture it in an incubator at 37 °C and 5% CO2. Change the medium every 2 - 3 days during the culture period to obtain yolk sac tumor organoids.

[0048] Application Example 3: Subculture of yolk sac tumor organoids Use the yolk sac tumor organoid medium prepared according to the method in Preparation Example 1 to construct yolk sac tumor organoids. The specific steps are as follows: S1: Digestion: Collect the yolk sac tumor organoids obtained after 14 days of culture in Application Example 1, remove the medium in the 24-well plate, and wash it 2 times with PBS buffer. Add 1×Tryple digestive solution at 1 mL / well to the 24-well plate, pipette and mix well, then place it in an incubator at 37 °C and 5% CO2 for digestion for 10 min. Add an equal volume of DMEM / F12 medium to terminate the digestion, then centrifuge at 1500 rpm for 5 min, and discard the supernatant; S2: Plating: Mix Matrigel matrix gel and cell precipitate at a subculture ratio of 1:3, and then drop 100 μL of the obtained mixture onto a new 24-well plate, and place it in an incubator at 37 °C and 5% CO2 to solidify for 15 min; S3: Adding medium: After the matrix gel solidifies, add 700 μL of preheated yolk sac tumor organoid medium (prepared according to the method in Preparation Example 1) to each well, and place it in an incubator at 37 °C and 5% CO2 for incubation. The growth conditions of the organoids at 0, 2, and 10 days are shown in Figure 2 A to Figure 2 C. It can be seen from Figure 2 that using the medium in Preparation Example 1 for the construction and subculture of yolk sac tumor organoids can achieve stable subculture of yolk sac tumor organoids, as well as stable and rapid growth after subculture.

[0049] Comparative Application Example 1: Construction of yolk sac tumor organoids Use the yolk sac tumor organoid medium prepared according to the method in Comparative Preparation Example 1 to construct yolk sac tumor organoids. The specific steps are as follows: (1) Washing: Obtain human yolk sac tumor tissue by surgery or biopsy, remove connective tissue and necrotic parts, etc., and wash the tissue 5 times with cold PBS buffer containing antibiotics; (2) Cutting into pieces: Cut the tissue into minced meat with surgical scissors in sequence, which can pass through a 1 mL pipette tip smoothly, evenly loose and non-adherent (operate at low temperature on ice); (3) Digestion: Transfer the tissue to a 15 mL centrifuge tube, add an appropriate amount of digestion solution 1 (DMEM / F12 medium containing 1 mg / mL collagenase I), pipette and mix well, then place it in a 37 °C constant temperature shaker for 30 min. After termination with twice the volume of cold PBS buffer, centrifuge to collect the cells. Add digestion solution 2 (DMEM / F12 medium containing 1 μg / mL dispase II), pipette and mix well, and then place it in a 37 °C constant temperature shaker for 30 min; (4) Centrifugation: Centrifuge the digested cell suspension at 1500 rpm for 5 min, and discard the supernatant; (5) Red blood cell lysis: When a visible red precipitate appears, add 3 mL of red blood cell lysis buffer, mix well, let it stand on ice for 5 min, add 4 times the volume of cold PBS buffer to terminate the reaction, mix well, filter through a 100 μm filter, and centrifuge the filtrate at 1500 rpm for 5 min, then discard the supernatant; (6) Counting: Resuspend with DMEM / F12 medium and then count; after counting, centrifuge at 1500 rpm for 5 min, and discard the supernatant; (7) Seeding: Resuspend the cells with Matrigel matrix gel at a ratio of 2×10 5 cells / 100 μL of matrix gel (operate at low temperature on ice). Drop the matrix gel and cell mixture at 100 μL / well in the center of a 24-well plate, and let it solidify in a 37 °C, 5% CO2 incubator for 15 min; (8) Slowly add 700 μL of yolk sac tumor organoid medium (prepared according to the method in Comparative Preparation Example 1) along the side wall of the well plate, and culture it in a 37 °C, 5% CO2 incubator. Change the medium every 3 days during the culture period.

[0050] Figure 3 A and Figure 3 B are the organoid growth conditions on the 8th day of culture in Application Example 1 and this Comparative Application Example respectively. It can be seen from Figure 3 that if the DMEM / F12 in the yolk sac tumor organoid medium is replaced with RPMI-1640, it will cause slow growth and poor activity of the yolk sac tumor organoids.

[0051] Comparative Application Example 2: Construction of yolk sac tumor organoids Use the yolk sac tumor organoid medium prepared according to the method in Comparative Preparation Example 2 to construct yolk sac tumor organoids. The specific steps are as follows: (1) Washing: Obtain human yolk sac tumor tissue by surgery or biopsy, remove connective tissue and necrotic parts, etc., and wash the tissue 5 times with cold PBS buffer containing antibiotics; (2) Mincing: Use surgical scissors to cut the tissue into minced meat in sequence, which can smoothly pass through a 1 mL pipette tip, and be evenly loose and non-adherent (operate at low temperature on ice); (3) Digestion: Transfer the tissue to a 15 mL centrifuge tube, add an appropriate amount of digestion solution 1 (DMEM / F12 medium containing 1 mg / mL collagenase I), pipette to mix evenly, place it in a 37 °C constant temperature shaker for 30 min, terminate with twice the volume of cold PBS buffer, centrifuge to collect the cells, add digestion solution 2 (DMEM / F12 medium containing 1 μg / mL dispase II), pipette to mix evenly, and place it in a 37 °C constant temperature shaker for 30 min; (4) Centrifugation: Centrifuge the digested cell suspension at 1500 rpm for 5 min, and discard the supernatant; (5) Red blood cell lysis: When a visible red precipitate appears, add 3 mL of red blood cell lysis buffer, mix evenly, let it stand on ice for 5 min, add 4 times the volume of cold PBS buffer to terminate the reaction, mix evenly, filter through a 100 μm filter, centrifuge the filtrate at 1500 rpm for 5 min, and discard the supernatant; (6) Counting: Resuspend with DMEM / F12 medium and then perform counting; (7) Seeding: Resuspend the cells with Matrigel matrix gel at a ratio of 2×10 5 cells / 100 μL of matrix gel (operate at low temperature on ice), drop the matrix gel and cell mixture at 100 μL / well in the center of a 24-well plate, and let it solidify in a 37 °C, 5% CO2 incubator for 15 min; (8) Slowly add 700 μL of yolk sac tumor organoid medium (prepared according to the method in Comparative Preparation Example 2) along the side wall of the well plate, place it in a 37 °C, 5% CO2 incubator for culture, and change the medium every 3 days during the culture period.

[0052] Figure 4 A and Figure 4 B are respectively the organoid growth conditions on the 8th day of culture in Application Example 1 and this Comparative Application Example. It can be seen from Figure 4 that if activin A is not added to the yolk sac tumor organoid medium, it will cause slow growth and poor activity of the yolk sac tumor organoids.

[0053] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And, the methods used herein, unless otherwise specified, are conventional methods; the reagents, biological materials and equipment used, unless otherwise specified, are common reagents, biological materials and equipment in this field and can be obtained commercially.

[0054] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A yolk sac tumor organoid culture medium, characterized in that, It includes a basal medium and specific additives; the specific additives include the following components: L-glutamine, TGF-β type I receptor inhibitor A83-01, nicotinamide, N2 supplement, B27 supplement without vitamin A, N-acetylcysteine, β-estradiol, P38 MAPK inhibitor SB202190, bone morphogenetic protein antagonist Noggin, Wnt pathway-related protein R-Spondin-1, epidermal growth factor, insulin-like growth factor 1, hepatocyte growth factor, neuregulin-1, ROCK inhibitor Y27632, activin A, interleukin-6.

2. The yolk sac tumor organoid culture medium according to claim 1, wherein, The basal medium is DMEM / F12 medium.

3. The yolk sac tumor organoid culture medium according to claim 2, characterized in that, In the yolk sac tumor organoid medium, the final concentrations of each component are as follows: L-glutamine, 5-15 mM; TGF-β type I receptor inhibitor A83-01, 0.5-5 μM; nicotinamide, 5-10 mM; N2 supplement, 0.5-1.5% vol; B27 supplement without vitamin A, 1-3% vol; N-acetylcysteine, 1-2 mM; β-estradiol, 5-15 nM; P38 MAPK inhibitor SB202190, 0.5-5 μM; bone morphogenetic protein antagonist Noggin, 100-500 ng / mL; Wnt pathway-related protein R-Spondin-1, 250-750 ng / mL; epidermal growth factor, 25-75 ng / mL; insulin-like growth factor 1, 10-30 ng / mL; hepatocyte growth factor, 5-15 ng / mL; neuregulin-1, 25-75 ng / mL; ROCK inhibitor Y27632, 5-15 μM; activin A, 10-30 ng / mL; interleukin-6, 10-30 ng / mL; the solvent is DMEM / F12 medium.

4. Use of the yolk sac tumor organoid medium according to any one of claims 1 to 3 in constructing yolk sac tumor organoids.

5. The application according to claim 4, characterized in that It includes the following steps: (1) Pretreat and digest the yolk sac tumor tissue to obtain yolk sac tumor cells; (2) Resuspend the yolk sac tumor cells with Matrigel, then drop them into a culture container and let it solidify to form a gel droplet containing yolk sac tumor cells; (3) Add the yolk sac tumor organoid medium into the culture container and culture to obtain yolk sac tumor organoids.

6. The application according to claim 5, characterized in that, In step (1), the digestion process specifically includes the following steps: Mix the pretreated yolk sac tumor organoids with digestion solution 1 containing collagenase I for the first digestion, collect the cells after terminating the reaction, mix them with digestion solution 2 containing dispase II for the second digestion, collect the cells, mix them with a buffer solution, filter through a filter screen with a pore size of 95-105 μm, and collect the cells from the filtrate to obtain yolk sac tumor cells.

7. The application according to claim 6, wherein Before mixing with the buffer solution after collecting the cells, first mix the collected cells with red blood cell lysate for red blood cell lysis, and then mix with the buffer solution.

8. The application according to claim 5, wherein In step (2), the ratio of the yolk sac tumor cells to the Matrigel is 1.5×10 5 ~2.5×10 5 cells / 100 μL of Matrigel; in step (3), the temperature of the culture is 35 - 37°C, the CO2 concentration is 4 - 6%, and the medium is changed every 2 - 3 days.

9. Use of the yolk sac tumor organoid culture medium according to any one of claims 1 to 3 in subculture of yolk sac tumor organoids.

10. The application according to claim 9, characterized in that, It includes the following steps: S1: Digest the yolk sac tumor organoids; S2: After resuspending the digested yolk sac tumor organoids in step S1 with Matrigel, drop them into a culture container and let them solidify to form gel droplets; S3: Add the yolk sac tumor organoid culture medium into the culture container and culture to obtain subcultured organoids.

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