Gynecological tumor organoid culture medium, culture method and product

Abstract

The invention provides a gynecological tumor organoid culture medium, a culture method and a product, and belongs to the technical field of biological tissue engineering. The gynecological tumor organoid base comprises a basic culture medium, a cell factor and growth factor composition, a nutritional supplement and a small molecule inhibitor, the small molecule inhibitor comprises Y-27632, A83-01, CHIR99021, SB431542 and DJ001, the culture medium can be used for culture of organoid such as ovarian cancer, cervical cancer and endometrial cancer, the organoid is high in growth speed and culture success rate, and the culture cost is low. And the subculture can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological tissue engineering, and specifically relates to a gynecological tumor organoid culture medium, a culture method and a product. Background Art

[0002] Cervical cancer (CC), endometrial cancer (EC), and ovarian cancer (OC) are known as the three major gynecological malignancies. Inhibiting the occurrence and development of the three major gynecological malignancies is a major challenge facing the current medical field.

[0003] Cell culture and animal models are the experimental cornerstones of studying tissue, organ and body physiology in the medical field. However, their ability to accurately reflect human mechanisms in vivo is limited. Organoids are an emerging in vitro three-dimensional modeling technology that has the characteristics of reproducing the physiological and pathological characteristics of original tissues in vivo. They show good applicability in disease modeling, regeneration mechanisms and precision medicine. Organoids are micro-organs with three-dimensional structures that can highly simulate the morphology and physiological functions of source tissues and produce tissues with stable phenotypes, making them good models for clinical disease research. With the continuous development of research, human-derived models have developed from monolayer cell culture to 3D organoids. Cultivating 3D organoids can be used to explore the mechanisms of disease occurrence and development. Tumor organoids have become the main experimental model for exploring the diagnosis and treatment of tumor diseases. They can be used to analyze genetic or epigenetic basis, reveal drug resistance mechanisms, conduct drug screening, and provide personalized treatment for patients.

[0004] On this basis, it is necessary to further explore the optimal modeling method for gynecological tumor-related organoids, improve the success rate of modeling, and cultivate more types of gynecological tumor-related organoid models to facilitate scientific research and guide clinical treatment. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a gynecological tumor organoid culture medium, culture method and product.

[0006] In one aspect, the present invention provides a gynecological tumor organoid culture medium.

[0007] Specifically, the culture medium includes the following components: a basal culture medium, a cytokine and growth factor composition, a nutrient supplement, and a small molecule inhibitor.

[0008] Specifically, the basic culture medium includes but is not limited to: DMEM / F12.

[0009] Specifically, the cytokine and growth factor composition includes: EGF, IGF1, HGF, Heregulin β-1, Forskolin, Hydrocortisone, β-Estradiol, Noggin, Wnt3a and R-spondin1.

[0010] Specifically, the nutritional supplement includes at least one of: B27, N-2 supplement and / or GlutaMax.

[0011] Specifically, the small molecule inhibitors include: Y-27632, A83-01, CHIR99021, SB431542 and DJ001.

[0012] Further specifically, in the culture medium, the final concentrations of the components of the cytokine and growth factor composition are: EGF: 25-75 ng / mL; IGF1: 10-30 ng / mL; HGF: 5-15 ng / mL; Heregulin β-1: 5-15 ng / mL; Forskolin: 5-15 μM; Hydrocortisone: 0.5-1.5 μM; β-Estradiol: 5-15 nM; Noggin: 50-150 ng / mL; Wnt3a: 50-150 ng / mL; R-spondin 1: 50-150 ng / mL.

[0013] Preferably, in the culture medium, the final concentrations of the components of the cytokine and growth factor composition are: EGF: 25-50 ng / mL; IGF1: 10-20 ng / mL; HGF: 5-10 ng / mL; Heregulin β-1: 5-10 ng / mL; Forskolin: 5-10 μM; Hydrocortisone: 0.5-1 μM; β-Estradiol: 5-10 nM; Noggin: 50-100 ng / mL; Wnt3a: 50-100 ng / mL; R-spondin 1: 50-100 ng / mL.

[0014] Preferably, in the culture medium, the final concentrations of the components of the cytokine and growth factor composition are: EGF: 50-75 ng / mL; IGF1: 20-30 ng / mL; HGF: 10-15 ng / mL; Heregulin β-1: 10-15 ng / mL; Forskolin: 10-15 μM; Hydrocortisone: 1-1.5 μM; β-Estradiol: 10-15 nM; Noggin: 100-150 ng / mL; Wnt3a: 100-150 ng / mL; R-spondin 1: 100-150 ng / mL.

[0015] In certain specific embodiments of the present invention, in the culture medium, the final concentrations of the components of the cytokine and growth factor composition are: EGF: 50 ng / mL; IGF1: 20 ng / mL; HGF: 10 ng / mL; Heregulin β-1: 10 ng / mL; Forskolin: 10 μM; Hydrocortisone: 1 μM; β-Estradiol: 10 nM; Noggin: 100 ng / mL; Wnt3a: 100 ng / mL; R-spondin 1: 100 ng / mL.

[0016] More specifically, the final concentrations of the small molecule inhibitor components are: Y-27632: 5-15 μM; A83-01: 400-600 nM; CHIR99021: 2-4 μM; SB431542: 4-6 μg / mL; DJ001: 4-6 μg / mL.

[0017] Preferably, in the culture medium, the final concentrations of the components of the small molecule inhibitors are: Y-27632: 5-10 μM; A83-01: 400-500 nM; CHIR99021: 2-3 μM; SB431542: 4-5 μg / mL; DJ001: 4-5 μg / mL.

[0018] Preferably, in the culture medium, the final concentrations of the components of the small molecule inhibitors are: Y-27632: 10-15 μM; A83-01: 500-600 nM; CHIR99021: 3-4 μM; SB431542: 5-6 μg / mL; DJ001: 5-6 μg / mL.

[0019] In certain specific embodiments of the present invention, the final concentrations of the components of the small molecule inhibitors in the culture medium are: Y-27632: 10 μM; A83-01: 500 nM; CHIR99021: 3 μM; SB431542: 5 μg / mL; DJ001: 5 μg / mL.

[0020] Specifically, the culture medium also includes antibiotics.

[0021] More specifically, the antibiotics include but are not limited to streptomycin and penicillin.

[0022] Specifically, the culture medium also includes a buffer.

[0023] More specifically, the buffer includes but is not limited to: HEPES, DPBS or HBSS.

[0024] On the other hand, the present invention provides a method for culturing gynecological tumor organoids.

[0025] Specifically, the culture method uses the above-mentioned culture medium.

[0026] More specifically, the culture method comprises the following steps:

[0027] (1) Clean and chop tissue blocks;

[0028] (2) Digestion and filtration;

[0029] (3) Add glue and dot the board;

[0030] (4) Add the above-mentioned gynecological tumor organoid culture medium for cultivation.

[0031] Preferably, in step (2), the enzyme used for digestion includes but is not limited to: at least one of collagenase, trypsin and / or hyaluronidase.

[0032] In another aspect, the present invention provides use of the above-mentioned culture medium and / or culture method in preparing a product for culturing gynecological tumor organoids.

[0033] Specifically, the gynecological tumors include, but are not limited to, ovarian tumors, uterine tumors, cervical tumors, germ cell tumors, sex cord-stromal tumors, vulvar tumors and / or fallopian tube tumors.

[0034] Preferably, the gynecological tumor is an ovarian tumor, a uterine tumor or a cervical tumor.

[0035] In another aspect, the present invention provides a kit,

[0036] Specifically, the kit includes the above-mentioned culture medium.

[0037] More specifically, the kit also includes: a buffer, a digestion solution, a tissue preservation solution and / or an organoid freezing solution.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] The method of the present invention is a general method for culturing gynecological tumor organoids. Using the culture medium formula provided by the present invention can significantly increase the growth rate of organoids. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0041] Table 1 Reagents

[0042]

[0043]

[0044] Example 1 Ovarian cancer organoid culture Organoid culture medium formula:

[0045] Table 2

[0046]

[0047]

[0048] 1. With the informed consent of the patient, the postoperative ovarian cancer tumor tissue of the patient was collected. The patient did not receive chemotherapy before surgery. The tumor tissue was washed with PBS (containing 100U / mL penicillin and 0.1mg / mL streptomycin), and then the PBS was removed. This was repeated 3 times. After removing the PBS buffer, the tissue was cut into pieces of about 0.5mm with sterile scissors. 3 The small pieces were then rinsed three times with PBS (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin).

[0049] 2. Digestion and filtration: Add 10 mL of tissue digestion solution (DMEM / F12 culture medium containing 0.001% v / v DNase, 1 mg / mL collagenase I, 100 U / mL penicillin and 0.1 mg / mL streptomycin), digest at 37°C for 30 min, pass through a 70 μm sieve, centrifuge the filtrate at 4°C, 200 g for 5 min, and collect ovarian cancer cells.

[0050] 3. Organoid plate: Use an automatic cell counter to count the number of cells and use organoid culture medium to adjust the cell density to 5×10 3 cells / μL, and mixed evenly with matrix gel in a ratio of 1:1, and the cell suspension was seeded in a 24-well plate at 20 μL per well. After being placed at 37°C for 30 min, 400 μL of the organoid culture medium of the formula of this example was added to each well.

[0051] 4. Organoid culture: Place the culture plate in a 37°C CO2 incubator and change the culture medium every 2 days. When adding the culture medium, add it slowly with the pipette tip facing the side wall.

[0052] Example 2 Endometrial cancer organoid culture

[0053] With the patient's informed consent, postoperative endometrial cancer tumor tissue was collected from the patient. The patient did not receive chemotherapy before surgery.

[0054] The culture medium formula and organoid culture steps used were the same as in Example 1.

[0055] Example 3 Cervical cancer organoid culture

[0056] With the patient's informed consent, cervical cancer tumor tissue was collected from the patient after surgery. The patient did not receive chemotherapy before surgery.

[0057] The culture medium formula and organoid culture steps used were the same as in Example 1.

[0058] Comparative Example 1

[0059] The difference from Example 1 is that SB431542 is replaced by an equal amount of DJ001.

[0060] Comparative Example 2

[0061] The difference from Example 1 is that DJ001 is replaced by an equal amount of SB431542.

[0062] Comparative Example 3

[0063] The difference from Example 1 is that SB431542 and DJ001 are not added.

[0064] Comparative Example 4

[0065] The difference from Example 1 is that rhabdomyosarcoma organoids are cultured, and the rhabdomyosarcoma tissue is obtained during puncture of the patient with the patient's informed consent, and the patient did not undergo chemotherapy before puncture.

[0066] Experimental Example 1

[0067] There are two main signs of successful organoid formation: 1. Stable maintenance of the in vivo characteristics of the original tumor during large-scale in vitro expansion; 2. Consistent appearance with the source tissue. The number of successfully formed organoids in the 24-well plate was counted, and the success rate was calculated (number of wells with successfully formed organoids / 24×100%). The organoid volume was measured using NoviSight 3D analysis software, and the results are shown in Table 3.

[0068] Table 3

[0069] Group <![CDATA[Tumor organoid volume (mm 3 )]]> Success rate Example 1 58.6±4.02 100％ Example 2 57.4±5.62 96％ Example 3 57.7±3.87 96％ Comparative Example 1 36.5±3.12*** 71％ Comparative Example 2 42.8±1.63*** 63％ Comparative Example 3 30.3±3.02*** 54％ Comparative Example 4 15.3±1.12*** 33％

[0070] Note: Compared with Example 1, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001.

[0071] Experimental Example 2

[0072] Ovarian cancer organoid subculture:

[0073] 1. Select the organoid in the stable state in Example 1, remove the organoid culture medium in the culture plate, add 1 mL of trypsin digestion solution (DMEM / F12 culture medium containing 0.25 g / 100 mL trypsin, 100 U / mL penicillin and 0.1 mg / mL streptomycin, preheated at 37°C for 10 min before use) to each well of the culture plate, repeatedly blow and mix the matrix gel with a pipette, collect it in a 15 mL centrifuge tube, put it in a 37°C, 5% CO2 incubator for digestion for 20 min, after terminating the digestion, centrifuge at 200g, 4°C for 5 min, and discard the supernatant;

[0074] 2. After aspirating all the supernatant, add 10 mL of tissue digestion solution (DMEM / F12 culture medium containing 0.001% v / v DNase, 1 mg / mL collagenase I, 100 U / mL penicillin and 0.1 mg / mL streptomycin), digest at 37°C for 30 min, and centrifuge at 4°C, 200 g for 5 min.

[0075] 3. Plating: Mix an appropriate amount of matrix gel and cell pellet at a subculture ratio of 1:3, seed each well in a 24-well plate at 20 μL, place at 37°C for 30 min, add 400 μL of the organoid culture medium of this example to each well, place the culture plate in a 37°C CO2 incubator, and replace the culture medium every 2 days.

[0076] The culture method can stably propagate ovarian cancer organoids and enable them to grow stably with good growth conditions, and the success rate of subculture can reach 90%.

[0077] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A gynecological tumor organoid culture medium, characterized in that The culture medium comprises the following components: a basal culture medium, a cytokine and growth factor composition, a nutritional supplement and a small molecule inhibitor; The basic culture medium includes: DMEM / F12; The cytokine and growth factor composition includes: EGF, IGF1, HGF, Heregulin β-1, Forskolin, Hydrocortisone, β-Estradiol, Noggin, Wnt3a and R-spondin 1; The nutritional supplement includes: at least one of B27, N-2 Supplement and / or GlutaMax; The small molecule inhibitors include: Y-27632, A83-01, CHIR99021, SB431542 and DJ001.

2. The gynecological tumor organoid culture medium according to claim 1, characterized in that In the culture medium, the final concentration of the cytokine and growth factor composition is: EGF: 25-75ng / mL; IGF1: 10-30ng / mL; HGF: 5-15ng / mL; Heregulinβ-1: 5-15ng / mL; Forskolin: 5-15μM; Hy drocortisone: 0.5-1.5μM; β-Estradiol: 5-15nM; Noggin: 50-150ng / mL; Wnt3a: 50-150ng / mL; R-spondin 1:50-150ng / mL.

3. The gynecological tumor organoid culture medium according to claim 1, characterized in that In the culture medium, the final concentration of each component of the small molecule inhibitor is: Y-27632: 5-15μM; A83-01: 400-600nM; CHIR99021: 2-4μM; SB431542: 4-6μg / mL; DJ001: 4-6μg / mL.

4. A method for culturing gynecological tumor organoids, characterized in that: The culture method uses the gynecological tumor organoid culture medium according to any one of claims 1 to 3.

5. The method for culturing gynecological tumor organoids according to claim 4, characterized in that: The following steps are involved: (1) Clean and chop tissue blocks; (2) Digestion and filtration; (3) Add glue and dot the board; (4) Add gynecological tumor organoid culture medium for culture.

6. The method for culturing gynecological tumor organoids according to claim 5, characterized in that: In step (2), the digestion uses at least one of collagenase, trypsin and / or hyaluronidase.

7. Use of the gynecological tumor organoid culture medium according to any one of claims 1 to 3 and / or the gynecological tumor organoid culture method according to any one of claims 4 to 6 in preparing a product for culturing gynecological tumor organoids, characterized in that: The application is non-disease diagnosis or treatment application.

8. The use according to claim 7, characterized in that: The gynecological tumors include: ovarian tumors, uterine tumors, cervical tumors, vulvar tumors and / or fallopian tube tumors.

9. A kit, characterized in that: The kit comprises the gynecological tumor organoid culture medium according to any one of claims 1 to 3.

10. The kit according to claim 9, characterized in that The kit also includes: a buffer, a digestion solution, a tissue preservation solution and / or an organoid freezing solution.

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