Intrahepatic bile duct cell carcinoma organoid culture medium and culture method

Abstract

The invention provides an intrahepatic cholangiocarcinoma organ culture medium and a culture method. The culture medium is prepared from the following components according to final concentration: 0.5 to 200 mu M of A83-01, 0.1 to 100 mu M of EGF (Epidermal Growth Factor), 10 to 500 ng / ml of FGF10 (Fibroblast Growth Factor), 1 to 200 ng / ml of HGF (Hepatitis Growth Factor), 0.5 to 200 mu M of Forskolin, 0.01 to 20 mu M of Gastrin I, 0.5 to 100 mM of Nicotinamide, 1 to 500 mu M of Y-27632, 0.5 to 100 nM of dexamethasone and 20 to 500 mu g / ml of primary cell antibiotics. All the components are dissolved in a DMEM / F12 culture solution. When the culture medium is used for culturing the intrahepatic cholangiocellular carcinoma organoid, the rapid amplification of the intrahepatic cholangiocellular carcinoma stem cells and the maturation and differentiation of the organoid are facilitated, the passage times are obviously increased, and the same tissue structure and genome characteristics of primary tissues can still be maintained after multiple passages.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to an intrahepatic cholangiocarcinoma organoid culture medium and an application method thereof. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy and the second most common primary liver cancer, accounting for 20% of all liver malignancies and 3% of all gastrointestinal malignancies. The incidence of ICC has increased by more than 140% over the past 40 years. ICC typically develops in the context of chronic inflammation, leading to cholestasis and resulting in bile duct cell damage.

[0003] Organoids are organ-specific cell collections derived from stem cells or precursor cells. Organoids cultured in vitro are highly similar to corresponding organs in terms of cell composition and tissue structure, and have corresponding functional characteristics. Unlike conventional cell culture that cultivates a single cell population in a two-dimensional environment, organoid culture cultivates multiple cell populations contained in specific tissues and organs in a three-dimensional environment, and its culture system is more similar to the in vivo microenvironment. Therefore, it shows great application prospects in basic research on the physiology and pathology of various organs, precision medicine, drug screening and development, gene therapy, regenerative medicine, etc.

[0004] Although a variety of human tumor tissues (such as colorectal cancer, lung cancer, etc.) can be successfully cultured into organoids in vitro using different methods and under different culture conditions, there are currently few studies and reports on the culture methods of intrahepatic cholangiocarcinoma organoids, especially the specific experimental process, operation steps, culture conditions, and culture medium formula.

[0005] Therefore, it is of great significance to provide an intrahepatic cholangiocarcinoma organoid culture medium and an application method thereof. Summary of the invention

[0006] The present invention provides an optimized intrahepatic cholangiocarcinoma organoid culture medium and an application method thereof, aiming to solve the problems of poor stability and easy contamination of existing culture media, which are not conducive to the long-term subculture of intrahepatic cholangiocarcinoma organoids.

[0007] The technical solution of the present invention is: An intrahepatic cholangiocarcinoma organoid culture medium, characterized in that the final concentration composition of the culture medium includes: 0.5 - 200μM A83-01; 0.1 - 100μM EGF; 10 - 500ng / ml FGF10; 1 - 200ng / ml HGF; 0.5 - 200μM Forskolin; 0.01 - 20μM GastrinⅠ; 0.5 - 100mM Nicotinamide; 1 - 500μM Y-27632; 0.5 - 100nM Dexamethasone; 20 - 500ug / ml primary cell antibiotics; all the above ingredients are dissolved in DMEM / F12 culture medium.

[0008] Furthermore, the final concentration composition of the culture medium includes: 1 - 100 μM A83-01; 0.1 - 50 μM EGF; 50 - 500 ng / ml FGF10; 10 - 200ng / ml HGF; 0.5 - 100μM Forskolin; 0.01 - 1μM GastrinⅠ; 0.5 -100mM Nicotinamide; 1 - 200μM Y-27632; 0.5 - 100nM Dexamethasone; 50 - 200ug / ml primary cell antibiotics; all the above ingredients are dissolved in DMEM / F12 culture medium.

[0009] Furthermore, the preparation method is to dissolve the components in DMEM / F12 culture medium, mix them evenly, and form a liquid culture medium.

[0010] Furthermore, FGF10 may be replaced by FGF7.

[0011] Another object of the present invention is to provide an application method of intrahepatic cholangiocarcinoma organoid culture medium.

[0012] The following steps are involved: S1. Pre-treat the obtained intrahepatic cholangiocarcinoma specimen to obtain a cell cluster with a cell number of 3 to 50 cells; S2. Mix the cell mass and Matrigel and plate them. After solidification, invert and fix for 20-30 min. S3. Add the culture medium and culture at 37° C. and 5% CO 2 for 4 to 7 days, during which the culture medium is replaced every 2 to 3 days to obtain intrahepatic cholangiocarcinoma organoids.

[0013] Furthermore, the number of cells in the cell cluster in S1 is 5 - 30 cells.

[0014] Furthermore, the amount of Matrigel used in S2 is 5 - 15 μL.

[0015] Furthermore, the culture time in S3 is 5 days.

[0016] Another object of the present invention is to provide an intrahepatic cholangiocarcinoma organoid.

[0017] An intrahepatic cholangiocarcinoma organoid, characterized in that it is cultured according to any of the above-mentioned intrahepatic cholangiocarcinoma organoid culture methods.

[0018] Another object of the present invention is to provide a use of intrahepatic cholangiocarcinoma organoids.

[0019] The use of the above-mentioned intrahepatic cholangiocarcinoma organoids in screening drugs for preventing or treating intrahepatic cholangiocarcinoma.

[0020] Compared with the prior art, the present invention has significant advantages and beneficial effects: First, the intrahepatic cholangiocarcinoma organoid culture medium provided by the present invention does not rely on bovine serum albumin (FBS), effectively reducing the cytotoxicity and inhibitors caused by FBS, while reducing costs. Secondly, the added specific cytokines promote the rapid expansion of intrahepatic cholangiocarcinoma stem cells and the maturation and differentiation of organoids, thereby improving the culture efficiency. Furthermore, the water-soluble inhibitors used avoid the toxicity of the DMSO solvent, ensuring the safety and effectiveness of the culture medium. Finally, after the component concentration is optimized, the culture medium of the present invention significantly improves the growth rate and passage ability of intrahepatic cholangiocarcinoma organoids, and can still maintain the tissue structure and genomic characteristics of the primary tissue after multiple passages, providing a more stable and reliable in vitro research platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0022] Figure 1 This is an optical microscopic image of the intrahepatic cholangiocarcinoma organoid cultured in Example 1 of the present invention; Figure 2 This is an optical microscopic image of the intrahepatic cholangiocarcinoma organoid cultured in Example 2 of the present invention; Figure 3 This is an optical microscopic image of the intrahepatic cholangiocarcinoma organoid cultured in Example 3 of the present invention; Figure 4 This is an optical microscopic image of the intrahepatic cholangiocarcinoma organoid cultured in Example 4 of the present invention; Figure 5 This is the HE staining image of the intrahepatic cholangiocarcinoma organoid cultured in Example 5 of the present invention; Figure 6 This is an optical microscopic image of the intrahepatic cholangiocarcinoma organoid cultured in Example 6 of the present invention. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0024] Example 1: This example provides an intrahepatic cholangiocarcinoma organoid culture medium and a culture method. The intrahepatic cholangiocarcinoma organoid culture medium is composed of the following final concentrations: 150 μM A83-01, 50 μM EGF, 200 ng / ml FGF10, 100 ng / ml HGF, 100 μM Forskolin, 10 μM Gastrin I, 50 mM Nicotinamide, 200 μM Y-27632, 300 nM Dexamethasone, and 200 ug / ml primary cell antibiotics. The intrahepatic cholangiocarcinoma organoid culture method is: S1. Sample washing: Transfer the obtained intrahepatic cholangiocarcinoma specimens into a centrifuge tube, then wash with sterile saline for 30 seconds, remove the supernatant, and add sterile saline again for washing. Repeat the above method for 3 times to remove impurities on the tissue surface.

[0025] S2. Sample cutting: In a biosafety cabinet, transfer the sample to a 6 cm culture dish and cut the tissue into pieces of 1 - 3 mm in size using sterilized surgical scissors on ice. 3 .

[0026] S3. Sample digestion: Add 5 ml of collagenase type III and shake at 37°C for 30 min. After digestion, add 5 ml of F12 / DMEM medium to terminate digestion.

[0027] S4. Cell collection: Centrifuge the liquid in S3 at 1000 rpm for 5 min, carefully remove the supernatant to obtain a cell pellet with a cell number of 3-50 cells.

[0028] S5. Mix the cell clusters and Matrigel and plate them. The mixing ratio of cell clusters to Matrigel is 1:1. After solidification for 2 min, invert and culture for 20 min.

[0029] S6. Add the above culture medium into the culture dish, and then place it in a constant temperature incubator at 37°C and 5% CO2 concentration for culture.

[0030] S7. The culture medium was replaced every 2 days. After 6 days of culture, intrahepatic cholangiocarcinoma organoids were obtained. The tissue morphology and structure were observed under an ordinary optical microscope. Figure 1 shown.

[0031] Example 2: This example provides an intrahepatic cholangiocarcinoma organoid culture medium and culture method. The intrahepatic cholangiocarcinoma organoid culture medium is composed of the following final concentrations: 100 μM A83-01, 1000 μM EGF, 100 ng / ml FGF10, 80 ng / ml HGF, 70 μM Forskolin, 30 μM GastrinⅠ, 50 mM Nicotinamide, 100 μM Y-27632, 200 nM Dexamethasone, and 100 ug / ml primary cell antibiotic. The intrahepatic cholangiocarcinoma organoid culture method is the same as that of Example 1. After 6 days of culture, intrahepatic cholangiocarcinoma organoids can be obtained, and the tissue morphology and structure can be observed under an ordinary optical microscope. Figure 2 shown.

[0032] Example 3: This example provides an intrahepatic cholangiocarcinoma organoid culture medium and a culture method. The intrahepatic cholangiocarcinoma organoid culture medium is composed of the following final concentrations: 100 μM A83-01, 500 μM EGF, 100 ng / ml FGF10, 50 ng / ml HGF, 50 μM Forskolin, 20 μM Gastrin I, 50 mM Nicotinamide, 100 μM Y-27632, 100 nM dexamethasone, and 100 ug / ml primary cell antibiotics. The intrahepatic cholangiocarcinoma organoid culture method is: S1. Sample washing: Transfer the obtained intrahepatic cholangiocarcinoma specimens into a centrifuge tube, then wash with sterile saline for 30 seconds, remove the supernatant, and add sterile saline again for washing. Repeat the above method for 3 times to remove impurities on the tissue surface.

[0033] S2. Sample cutting: In a biosafety cabinet, transfer the sample to a 6 cm culture dish and cut the tissue into pieces of 1 - 3 mm in size using sterilized surgical scissors on ice. 3 .

[0034] S3. Sample digestion: Add 5 ml of collagenase type III and shake at 37°C for 30 min. After digestion, add 5 ml of F12 / DMEM medium to terminate digestion.

[0035] S4. Cell collection: Centrifuge the liquid in S3 at 1000 rpm for 5 min, carefully remove the supernatant to obtain a cell pellet with a cell number of 3-50 cells.

[0036] S5. Mix the cell clusters and Matrigel and plate them. The mixing ratio of cell clusters to Matrigel is 1:1.3. After solidification for 3 min, invert and culture for 30 min.

[0037] S6. Add the above culture medium into the culture dish, and then place it in a constant temperature incubator at 37°C and 5% CO2 concentration for culture.

[0038] S7. The culture medium was replaced every 2 days. After 6 days of culture, intrahepatic cholangiocarcinoma organoids were obtained. The tissue morphology and structure were observed under an ordinary optical microscope. Figure 3 shown.

[0039] Example 4: This example provides an intrahepatic cholangiocarcinoma organoid culture medium and a culture method. The intrahepatic cholangiocarcinoma organoid culture medium is composed of the following final concentrations: 100 μM A83-01, 500 μM EGF, 100 ng / ml FGF10, 50 ng / ml HGF, 50 μM Forskolin, 20 μM Gastrin I, 50 mM Nicotinamide, 100 μM Y-27632, 100 nM dexamethasone, and 100 ug / ml primary cell antibiotics. The intrahepatic cholangiocarcinoma organoid culture method is: S1. Sample washing: Transfer the obtained intrahepatic cholangiocarcinoma specimens into a centrifuge tube, then wash with sterile saline for 30 seconds, remove the supernatant, and add sterile saline again for washing. Repeat the above method for 3 times to remove impurities on the tissue surface.

[0040] S2. Sample cutting: In a biosafety cabinet, transfer the sample to a 6 cm culture dish and cut the tissue into pieces of 1 - 3 mm in size using sterilized surgical scissors on ice. 3 .

[0041] S3. Sample digestion: Add 5 ml of collagenase type III and shake at 37°C for 30 min. After digestion, add 5 ml of F12 / DMEM medium to terminate digestion.

[0042] S4. Cell collection: Centrifuge the liquid in S3 at 1000 rpm for 5 min, carefully remove the supernatant to obtain a cell pellet with a cell number of 3-50 cells.

[0043] S5. Mix the cell clusters and Matrigel and plate them. The mixing ratio of cell clusters to Matrigel is 1:1.5. After solidification for 3 min, invert and culture for 30 min.

[0044] S6. Add the above culture medium into the culture dish, and then place it in a constant temperature incubator at 37°C and 5% CO2 concentration for culture.

[0045] S7. The culture medium was replaced every 2 days. After 6 days of culture, intrahepatic cholangiocarcinoma organoids were obtained. The tissue morphology and structure were observed under an ordinary optical microscope. Figure 4 shown.

[0046] Example 5: Morphological identification and functional verification of intrahepatic cholangiocarcinoma organoids obtained in Example 4 The intrahepatic cholangiocarcinoma organoids cultured in Example 4 were subjected to HE staining according to the following method: Embedding and sectioning: Embed the organoids in an embedding mold, then cut them into 4 μm slices using a paraffin slicer and mount them on anti-shedding slides.

[0047] Baking: Place the slide on a slide rack and put it in an oven at 65°C for 60 min to dry the moisture on the slide and melt the paraffin.

[0048] Dewaxing: Use xylene to dewax 3 times, 5 min each time; then soak in 100% alcohol 2 times, 5 min each time; soak in 95% alcohol 2 times, 5 min each time; soak in 75% alcohol 2 times, 5 min each time; finally soak in running water for 1 min.

[0049] H&E staining: first stain with hematoxylin for 3 min, then wash with water for 1 min, then differentiate with 1% hydrochloric acid alcohol for 1-2s, then rinse with running water for 10 min, then stain with 1% eosin for 2 min, and finally rinse with running water for 1 min.

[0050] Fixation after staining: Immerse in 95% alcohol and 100% alcohol in sequence, twice in each reagent, 2 min each time.

[0051] Transparent and sealing: Use xylene to transparentize for 2 min, take out and dry, then seal with neutral gum.

[0052] Observe the tissue morphology under an ordinary optical microscope. Figure 5 Shown, showing features of intrahepatic cholangiocarcinoma.

[0053] Example 6: Multiple Passaging of Intrahepatic Cholangiocarcinoma Organoids After obtaining the primary intrahepatic cholangiocarcinoma organoids according to Example 4, the primary intrahepatic cholangiocarcinoma organoids were subcultured. The subculture operation was as follows: Use a pipette to remove the culture medium from the culture dish and take 1-2 ml of TrypLETM Express to digest the organoids. The gel droplets were placed in an incubator and digested at 37°C for 10 min; Add 10 ml of sterile Hank's balanced salt solution and centrifuge at 1000 rpm for 3 min; The supernatant was discarded, 200 μl of the culture medium of Example 4 was added to resuspend the organoids, 250 μl of Matrigel matrix gel was added to mix, and the mixture was dropped into a new 35 mm culture dish, and allowed to stand for 5 min. The dish was moved to an incubator and inverted. After 40 min, 2-4 ml of the culture medium of Example 4 was added and the dish was allowed to stand in an incubator at 37°C.

[0054] After 6 days of subculture, the second generation of intrahepatic cholangiocarcinoma organoids were obtained. After 40 days of continuous subculture according to this method, the sixth generation of intrahepatic cholangiocarcinoma organoids were obtained. The tissue morphology and structure of the organoids were observed under an ordinary optical microscope. Figure 6 As shown, the organoid structure is good, and after 6 generations, the intrahepatic cholangiocarcinoma organoids can still maintain the morphological structure of the original tissue.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An intrahepatic cholangiocarcinoma organoid culture medium, characterized in that The final concentration of the culture medium comprises: 0.5 - 200μM A83-01; 0.1 - 100μM EGF; 10 - 500ng / ml FGF10; 1 - 200ng / ml HGF; 0.5 - 200μM Forskolin; 0.01 - 20μM GastrinⅠ; 0.5 - 100mM Nicotinamide; 1- 500μM Y-27632; 0.5 - 100nM Dexamethasone; 20 - 500ug / ml primary cell antibiotics; all the above ingredients are dissolved in DMEM / F12 culture medium.

2. The intrahepatic cholangiocarcinoma organoid culture medium according to claim 1, characterized in that The final concentration composition of the culture medium includes: 1 - 100 μM A83-01; 0.1 - 50 μM EGF; 50 - 500 ng / ml FGF10; 10 - 200ng / ml HGF; 0.5 - 100μM Forskolin; 0.01 - 1μM GastrinⅠ; 0.5 -100mM Nicotinamide; 1 - 200μM Y-27632; 0.5 - 100nM Dexamethasone; 50 - 200ug / ml primary cell antibiotics; all the above ingredients are dissolved in DMEM / F12 culture medium.

3. An intrahepatic cholangiocarcinoma organoid culture medium according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: dissolving the components in DMEM / F12 culture fluid, mixing the mixture evenly, and forming a liquid culture medium.

4. The intrahepatic cholangiocarcinoma organoid culture medium according to claim 1 or 2, characterized in that FGF10 may be replaced by FGF7.

5. A method for culturing intrahepatic cholangiocarcinoma organoid culture medium, characterized in that: The following steps are involved: S1. Pre-treat the obtained intrahepatic cholangiocarcinoma specimen to obtain a cell cluster with a cell number of 3 to 50 cells; S2. Mix the cell mass and Matrigel and plate them. After solidification, invert and fix for 20-30 min. S3. Add the intrahepatic cholangiocarcinoma organoid culture medium described in any one of claims 1-2, and culture at 37° C. and 5% CO2 for 4-7 days, during which the culture medium is replaced every 2-3 days to obtain intrahepatic cholangiocarcinoma organoids.

6. A method for culturing intrahepatic cholangiocarcinoma organoid culture medium according to claim 5, characterized in that: The number of cells in the cell cluster S1 is 5 - 30 cells.

7. A method for culturing intrahepatic cholangiocarcinoma organoid culture medium according to claim 5, characterized in that: The amount of Matrigel used in S2 is 5 - 15 μL.

8. A method for culturing intrahepatic cholangiocarcinoma organoid culture medium according to claim 5, characterized in that: The culture time in S3 is 5 days.

9. An intrahepatic cholangiocarcinoma organoid, characterized in that The intrahepatic cholangiocarcinoma organoid is obtained by culturing according to the culturing method of any one of claims 5 to 8.

10. Use of the intrahepatic cholangiocarcinoma lung cancer organoid according to claim 9 in screening drugs for preventing or treating intrahepatic cholangiocarcinoma.

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