Novel technical method for constructing ovarian cancer organoid

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a culture medium formula for constructing ovarian cancer organoid and a culture method. The culture medium comprises the following components: L-glutamine 100x (L-Glutamine), L-glutamine 100x (L-Glutamine), L-glutamine 100x (L-Glutamine 4-hydroxyethylpiperazine ethanesulfonic acid (1M (hepes)); b-27 is free of a serum additive (B27); the preparation method comprises the following steps of: extracting the raw materials, namely nicotinamide; the preparation method comprises the following steps: adding N, N-acetylcysteine amide (N-Acetylcysteine); a forskolin element (Forskolin); and estradiol (beta-Estradiol), which is used for preparing the pharmaceutical composition. A TGF-[beta] inhibitor (A83-01); a p38 MAPK inhibitor (SB202190), and a p38 MAPK inhibitor, a p160ROCK small molecule inhibitor (Y27632); hepatocyte growth factor (HGF); the invention relates to a recombinant human Hergulin beta-1 protein product (Hergulin beta-1) and a preparation method of the recombinant human Hergulin beta-1 protein product. An epidermal growth factor (EGF); a fibroblast growth factor (FGF10); the invention relates to a method for preparing hydrocortisone. The method comprises the following steps: preparing hydrocortisone; and a primary cell antibiotic (Primocin). The culture solution and the culture method have the advantages that ovarian high-grade serous cancer cells can be effectively cultured and induced to develop into organoid. Noggin is not needed in the scheme, and the method is different from a traditional organoid culture method and is low in cost and simple in manufacturing process. Meanwhile, preparation of a conditioned culture medium is not needed, time can be remarkably saved, the situation that the conditioned culture medium is unstable due to too long time is avoided, and the success rate is high and is about 50%.

Description

[0001] Technical field: The present invention belongs to the field of biomedical technology, and specifically relates to a culture medium formula and culture method for constructing ovarian cancer organoids.

[0002] Background technology: Ovarian cancer is one of the most common malignant tumors in female reproductive organs, and its annual incidence ranks third among female reproductive system tumors. It is often accompanied by poor prognosis and needs to rely on chemotherapy drugs for adjuvant treatment. Ovarian cancer with high invasion and metastasis ability has high resistance to conventional chemotherapy drugs, which brings great challenges to clinical treatment. Organoids are 3D cultures constructed in vitro from primary tissues or pluripotent stem cells that are similar to in vivo tissues and organs. Tumor organoids reproduce the diversity and complexity of their source tumors at a high level in genes, cells and histology, indicating that tumor organoids do simulate various aspects of tumor characteristics to a large extent, becoming a new disease model for drug screening and mechanism research. The cost of ovarian cancer organoids known to date is much higher than that of primary cell culture, and the raw materials such as matrix gel and cytokines used in culture are relatively expensive. In particular, compared with other solid tumors, the culture of ovarian cancer organoids requires the addition of hormones and other specific factors, which makes the cost of ovarian cancer organoids even higher.

[0003] Invention content: In view of the high price of ovarian cancer organoid culture medium and the high cost of organoid culture (or the difficulty of ovarian cancer organoid culture and the low success rate of organoid model construction), the present invention proposes a culture medium formula and culture method for constructing ovarian cancer organoids. The present invention can be implemented through the following technical routes:

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] A culture medium for constructing ovarian cancer organoids, the culture medium is an advanced DMEM / F12 culture medium, and its components include:

[0006] 1x L-Glutamine 100x(L-Glutamine);

[0007] 1x 4-Hydroxyethylpiperazineethanesulfonic acid 1M (hepes);

[0008] 1x B-27 serum-free supplement (B27);

[0009] 5mM Nicotinamide;

[0010] 1.0 mM N-Acetylcysteine;

[0011] 10 μM Forskolin;

[0012] 100nM estradiol (β-Estradiol);

[0013] 0.5 μM TGF-β inhibitor (A83-01);

[0014] 0.5 μM p38 MAPK inhibitor (SB202190);

[0015] 10 μM p160ROCK small molecule inhibitor (Y27632);

[0016] 10 ng / ml hepatocyte growth factor (HGF);

[0017] 37.5ng / ml recombinant human Heregulinβ-1 protein product (Heregulinβ-1);

[0018] 50ng / ml epidermal growth factor (EGF);

[0019] 10 ng / ml fibroblast growth factor (FGF10);

[0020] 0.5 μg / ml hydrocortisone;

[0021] 1x primary cell antibiotic (Primocin).

[0022] A method for constructing ovarian cancer organoids, wherein the method for collecting ovarian cancer tissue samples comprises the following steps:

[0023] S1. Preoperative preparation for collecting ovarian cancer samples: 15ml sterile tubes, tissue preservation solution, sterile gloves, ice and incubator.

[0024] S2. Rapid frozen sections during surgery indicated poorly differentiated ovarian adenocarcinoma, which was further confirmed by postoperative pathology as high-grade serous carcinoma. During surgery, ovarian cancer tissue was cut into 2 cm3 specimens as quickly as possible using sterile instruments under sterile conditions.

[0025] S3. Soak the tissue specimen in tissue preservation solution in a 15 ml centrifuge tube. Place the centrifuge tube in an incubator and transport it on ice at 4°C for organoid culture.

[0026] 4. A method for culturing ovarian cancer organoids according to claim 2, characterized in that the method comprises the following steps of constructing ovarian cancer organoids:

[0027] S4. Place the ovarian cancer tissue sample in a 10 cm culture dish and wash it three times with PBS containing 2% double antibody + 1% amphotericin, each time for 5 minutes.

[0028] S5. Use a scalpel to cut the cleaned ovarian cancer tissue into tissues of about 1mm3 in size, remove necrotic tissue and blood clots, transfer to a 15ml centrifuge tube, add an appropriate amount of digestion solution, and place it in a shaker. After 20 minutes of digestion, the digestion status can be observed under a microscope. Digestion into cell clumps is sufficient, and complete digestion into single cells is likely to damage cell activity. After stopping digestion, blow the sample evenly, stop digestion with serum-containing culture medium, pass through a 100μm cell filter, and obtain a cell clump suspension. Centrifuge at 300g for 5 minutes in a pre-cooled 4℃ centrifuge, discard the supernatant, add 3-5ml of red blood cell lysis solution, keep on ice for 15 minutes, centrifuge at 450g for 5 minutes in a pre-cooled 4℃ centrifuge, and remove the supernatant.

[0029] S6. Resuspend with 1ml advanced DMEM / F12 medium and take 10ul of suspension for counting. Centrifuge the cell suspension at 300g for 5min at 4℃, discard the supernatant, and precool the precipitate on ice for 1min. Resuspend the cells in an appropriate amount of precooled Matrigel using a precooled pipette tip.

[0030] S7. Add 50 μL of Matrigel to each well of a 24-well plate.

[0031] S8. Place the 24-well plate in a carbon dioxide incubator and heat the front side for 10 min and the back side for 10 min to solidify the Matrigel.

[0032] S9. Add 500 μL of pre-warmed HGSOC organoid culture medium to each well.

[0033] S10. Replace the organoid culture medium every 2-3 days. To remove the used culture medium, use a pipette or carefully aspirate it to avoid disturbing Matrigel. Preferably, in step S5, the shaker speed is 100 rpm, the temperature is 37°C, the time is 30-40 minutes, and it is gently shaken every 10 minutes. Compared with the prior art, the beneficial effect of the present invention is that through the culture solution and the culture method, ovarian high-grade serous cancer cells can be effectively cultured and induced to develop into organoids. This scheme does not require Noggin, which is different from the traditional organoid culture method, has low cost and simple production process. At the same time, there is no need to prepare conditioned culture medium, which can significantly save time, and also avoids the instability of the conditioned culture medium for too long, and the success rate is high, about 50%.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS: In order to more clearly illustrate the technical solution of the prior art of the present invention, the following is a brief introduction to the drawings required for the description of the prior art.

[0035] Figure 1-4 The ovarian cancer organoid of the first patient constructed according to the method in Example 1 of the present invention;

[0036] Figure 5 The ovarian cancer organoid of the second patient constructed according to the method in Example 1 of the present invention;

[0037] Figure 6-9 The ovarian cancer organoid of the third patient constructed according to the method in Example 1 of the present invention;

[0038] Fig.10 This is a diagram showing the results of a drug sensitivity test of ovarian high-grade serous carcinoma organoids cultured in the present invention.

[0039] Specific implementation method: The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention.

[0040] It is obvious that the described embodiments are only a part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] A culture medium for constructing ovarian cancer organoids, the culture medium is a DMEM / F12 culture medium, and its components and concentrations include:

[0042] 1x L-Glutamine 100x(L-Glutamine);

[0043] 1x 4-Hydroxyethylpiperazineethanesulfonic acid 1M (hepes);

[0044] 1x B-27 serum-free supplement (B27);

[0045] 5mM Nicotinamide;

[0046] 1.0 mM N-Acetylcysteine;

[0047] 10 μM Forskolin;

[0048] 100nM estradiol (β-Estradiol);

[0049] 0.5 μM TGF-β inhibitor (A83-01);

[0050] 0.5 μM p38 MAPK inhibitor (SB202190);

[0051] 10 μM p160ROCK small molecule inhibitor (Y27632);

[0052] 10 ng / ml hepatocyte growth factor (HGF);

[0053] 37.5ng / ml recombinant human Heregulinβ-1 protein product (Heregulinβ-1);

[0054] 50ng / ml epidermal growth factor (EGF);

[0055] 10 ng / ml fibroblast growth factor (FGF10);

[0056] 0.5 μg / ml hydrocortisone;

[0057] 1x primary cell antibiotic (Primocin).

[0058] Example 1

[0059] 1. Prepare 15 ml sterile tubes, tissue preservation solution, sterile gloves, ice and incubator to collect ovarian cancer samples.

[0060] 2. Rapid frozen sections during surgery indicated poorly differentiated ovarian adenocarcinoma, which was further confirmed by postoperative pathology as high-grade serous carcinoma. During surgery, ovarian cancer tissue was cut into 2 cm3 specimens as quickly as possible using sterile instruments under sterile conditions.

[0061] 3. Soak the tissue specimen in tissue preservation solution in a 15 ml centrifuge tube. Place the centrifuge tube in an incubator and transport it on ice at 4°C for organoid culture.

[0062] 4. Place the ovarian cancer tissue sample in a 10 cm culture dish and wash it three times with PBS containing 2% double antibody + 1% amphotericin, each time for 5 minutes.

[0063] 5. Cut the cleaned ovarian cancer tissue into tissues of about 1mm3 with a scalpel, remove necrotic tissue and blood clots, transfer to a 15ml centrifuge tube and add an appropriate amount of digestion solution, place in a 37℃ shaker, rotate at 100rpm, and shake gently every 10 minutes. After 20 minutes of digestion, the digestion can be observed under a microscope. Digestion into cell clumps is sufficient, and complete digestion into single cells is likely to damage cell activity. After stopping digestion, blow the sample evenly, stop digestion with serum-containing culture medium, pass through a 100μm cell filter, and obtain a cell clump suspension. Centrifuge at 300g for 5 minutes in a pre-cooled 4℃ centrifuge, remove the supernatant, add 3-5ml red blood cell lysis solution, put on ice for 15 minutes, centrifuge at 450g for 5 minutes in a pre-cooled 4℃ centrifuge, and remove the supernatant.

[0064] 6. Resuspend with 1ml advanced DMEM / F12 medium and take 10ul of suspension for counting. Centrifuge the cell suspension at 300g for 5min at 4℃, discard the supernatant, and precool the precipitate on ice for 1min. Resuspend the cells in an appropriate amount of precooled Matrigel using a precooled pipette tip.

[0065] 7. Add 50 μL of Matrigel to each well of a 24-well plate.

[0066] 8. Place the 24-well plate in a carbon dioxide incubator and heat the front side for 10 minutes and the back side for 10 minutes to solidify the Matrigel.

[0067] 9. Add 500 μL of pre-warmed HGSOC Organoid Medium to each well.

[0068] 10. Change the organoid culture medium every 2-3 days. To remove the spent culture medium, use a pipette or carefully aspirate to avoid disturbing the Matrigel.

[0069] Application Example 1

[0070] After culturing the organoids according to the present invention, the organoids are used to perform drug sensitivity testing.

[0071] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture medium for constructing ovarian cancer organoids, characterized in that: The culture medium is DMEM / F12 culture medium, and its components include: 1x L-Glutamine 100x(L-Glutamine); 1x 4-Hydroxyethylpiperazineethanesulfonic acid 1M (hepes); 1x B-27 serum-free supplement (B27); 5mM Nicotinamide; 1.0 mM N-Acetylcysteine; 10 μM Forskolin; 100nM estradiol (β-Estradiol); 0.5 μM TGF-β inhibitor (A83-01); 0.5 μM p38 MAPK inhibitor (SB202190); 10 μM p160ROCK small molecule inhibitor (Y27632); 10 ng / ml hepatocyte growth factor (HGF); 37.5ng / ml recombinant human Heregulinβ-1 protein product (Heregulinβ-1); 50ng / ml epidermal growth factor (EGF); 10 ng / ml fibroblast growth factor (FGF10); 0.5 μg / ml hydrocortisone; 1x primary cell antibiotic (Primocin).

2. A method for culturing ovarian cancer organoids according to claim 1, characterized in that: The following steps are involved in collecting ovarian cancer tissue: S1. Preoperative preparation for collecting ovarian cancer samples: 15ml sterile tubes, tissue preservation solution, sterile gloves, ice and incubator. S2. Rapid frozen sections during surgery indicated poorly differentiated ovarian adenocarcinoma, which was further confirmed by postoperative pathology as high-grade serous carcinoma. During surgery, ovarian cancer tissue was cut into 2 cm3 specimens as quickly as possible using sterile instruments under sterile conditions. S3. Soak the tissue specimen in tissue preservation solution in a 15 ml centrifuge tube. Place the centrifuge tube in an incubator and transport it on ice at 4°C for organoid culture.

3. A method for culturing ovarian cancer organoids according to claim 2, characterized in that: The steps to construct ovarian cancer organoids include: S1. Place the ovarian cancer tissue sample in a 10 cm culture dish and wash it three times with PBS containing 2% double antibody + 1% amphotericin, each time for 5 minutes. S2. Cut the cleaned ovarian cancer tissue into pieces of about 1mm3 with a scalpel, remove necrotic tissue and blood clots, transfer to a 15ml centrifuge tube and add an appropriate amount of digestion solution, place in a 37℃ shaker, rotate at 100rpm, for 30-40 minutes, and shake gently every 10 minutes. After 20 minutes of digestion, the digestion can be observed under a microscope. Digestion into cell clumps is sufficient, and complete digestion into single cells is likely to damage cell activity. After stopping digestion, blow the sample evenly, stop digestion with serum-containing culture medium, pass through a 100μm cell filter, and obtain a cell clump suspension. Centrifuge at 300g for 5 minutes in a pre-cooled 4℃ centrifuge, remove the supernatant, add 3-5ml red blood cell lysis solution, keep on ice for 15 minutes, centrifuge at 450g for 5 minutes in a pre-cooled 4℃ centrifuge, and remove the supernatant. S3. Resuspend with 1ml advancedDMEM / F12 medium and take 10ul of suspension for counting. Centrifuge the cell suspension at 300g for 5min at 4℃, discard the supernatant, and precool the precipitate on ice for 1min. Resuspend the cells in an appropriate amount of precooled Matrigel using a precooled pipette tip. S4. Add 50 μL of Matrigel to each well of a 24-well plate. S5. Place the 24-well plate in a 37°C 5% carbon dioxide incubator and heat the front side for 10 min and the back side for 10 min to solidify the Matrigel. S6. Add 500 μL of pre-warmed HGSOC organoid culture medium to each well. S7. Change the organoid culture medium every 2-3 days. To remove the spent culture medium, use a pipette or carefully aspirate to avoid disturbing the Matrigel.

4. Use of the culture medium as claimed in claim 1 in constructing ovarian cancer organoids.

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