Construction method of liver tumor organoid with primary vascularization

Abstract

The invention relates to the technical field of medical modeling, in particular to a construction method of liver tumor organoid with primary vascularization, which comprises the following steps: selecting tumor tissue blocks, washing and chopping, adding tissue digestive juice, filtering, centrifuging, and discarding supernatant to obtain liver cancer organoid precipitate; a mixed solution of a basic culture medium and matrigel is used for resuspension counting, incubation is carried out till solidification, a complete culture medium is added for culture, the liver tumor organoid can be obtained, and primary liver tumor organoid vascularization can be achieved through continuous culture. The organoid generated by the technical scheme of the invention has the advantages of short culture period, fast proliferation, spontaneous vascularization and the like, is closer to the real reflection of the human body on the drug effect, and can be applied to the screening of antitumor drugs and the sensitivity test of antitumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of medical modeling, and in particular to a method for constructing liver tumor organoids with primary vascularization. Background Art

[0002] Hepatocellular carcinoma (HCC) is a major type of liver cancer and the third leading cause of cancer death worldwide. It originates from liver cells and is a malignant tumor caused by multiple factors. Hepatocellular carcinoma is the third leading cause of cancer-related death worldwide, second only to lung cancer and prostate cancer. According to the World Health Organization (WHO) and related epidemiological research data, Asia (especially East Asia and Southeast Asia) and Africa are high-incidence areas for hepatocellular carcinoma. The global incidence of hepatocellular carcinoma is about 10-20 cases per 100,000 people per year. And the prognosis of hepatocellular carcinoma is poor, with a 5-year survival rate of only 18%. In China, due to the high rate of hepatitis B virus infection and factors such as drinking, the incidence of hepatocellular carcinoma has been at a high level. In some Western countries, with the increase in obesity and diabetes, the incidence of hepatocellular carcinoma related to non-alcoholic fatty liver disease is also gradually increasing. The incidence of hepatocellular carcinoma is higher in developing countries and regions due to the high incidence of infectious hepatitis (such as hepatitis C virus and viral hepatitis) and other risk factors (such as liver fibrosis and liver cysts). The prognosis has improved with the continuous development and improvement of treatment technology.

[0003] Tumor organoids are three-dimensional cell structures that are cultured in vitro and have the characteristics of tumor cells. Compared with traditional cell culture techniques, organoid culture has great advantages. They can simulate the biological characteristics of tumors to a certain extent, including growth, differentiation and invasion behavior. Tumor organoid vascularization refers to the process of forming a vascular network inside these organoids. This process is of great significance for studying the biological characteristics of tumors, drug responses and developing new treatment strategies. Tumor organoids can maintain their structure and function stably for a long time in in vitro culture. Even after multiple passages, they can maintain certain characteristics of the corresponding organs in the body without obvious genetic or physiological changes. Miniaturized organ analogs can achieve long-term and stable expansion and simulate 3D tissue structure and function.

[0004] Tumor angiogenesis is a highly regulated process involving the proliferation, migration and reconstruction of vascular endothelial cells. During tumor growth, tumor cells release a series of angiogenic factors, such as vascular endothelial growth factor, platelet-derived growth factor, basic fibroblast growth factor, etc. As the tumor grows, its internal environment becomes hypoxic, nutrient-deficient, and acidic. These changes stimulate tumor cells to secrete angiogenic factors to promote the formation of new blood vessels, thereby providing tumor cells with the required oxygen and nutrients. At the same time, vascularization is also an important part of the tumor microenvironment. The realization of vascularization in liver cancer tumor organoids can more realistically simulate the physiological and pathological states of tumors, including angiogenesis, blood supply and nutrient delivery. This helps to study the mechanisms of tumor growth, metastasis and treatment response. It can also be used for drug screening, especially the testing of anti-angiogenic drugs. Through this model, the effects of drugs on tumor blood vessels can be evaluated and the effects of drugs in patients can be predicted, thereby promoting the development of personalized medicine. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing liver tumor organoids with primary vascularization, aiming to construct a vascularized liver cancer organoid model with good simulation, which can stably and spontaneously grow a microvascular network structure, which can be closer to the true reflection of the human body's response to drug effects and can be applied to the screening of anti-tumor drugs and the sensitivity testing of anti-tumor drugs.

[0006] To achieve the above objectives, the present invention provides a method for constructing liver tumor organoids with primary vascularization, comprising the following steps:

[0007] S1: Take fresh biopsy tissue, use scissors and forceps to select tumor tissue blocks and remove non-epithelial tissue, place liver cancer tissue in a 10 cm cell culture dish, rinse it repeatedly with tissue cleaning solution 3-4 times, and then mince the tissue with a sterile scalpel to obtain tumor tissue blocks;

[0008] S2: transferring the tumor tissue block to a centrifuge tube, adding pre-cooled tissue washing solution for washing, waiting for the tumor tissue block to precipitate, discarding the supernatant, and obtaining the washed liver cancer tissue block;

[0009] S3: adding the washed liver cancer tissue block to the tissue digestion solution in an incubator for digestion, and shaking up and down or pipetting to mix the material in the centrifuge tube, and stopping the digestion when there are 50%-100% single cells in the suspension to obtain a tissue block suspension;

[0010] S4: filtering the tissue block suspension with a 100 μm pore size filter membrane, transferring the filtered cell suspension to a centrifuge tube and centrifuging, discarding the supernatant, and obtaining a liver cancer organoid precipitate;

[0011] S5: collecting the liver cancer organoid precipitate, adding red blood cell lysis solution to lyse the red blood cells, then adding tissue washing solution to stop the process, centrifuging and discarding the supernatant to obtain the organoid;

[0012] S6: resuspending and counting the organoids using a hepatocellular carcinoma organoid basal culture medium, dropping the organoid suspension on the bottom of a suspension cell culture plate, and then incubating until the matrix gel is solidified to obtain organoids solidified in the matrix gel;

[0013] S7: adding the organoid solidified in the matrix gel to the complete culture medium of hepatocellular carcinoma organoid for culture.

[0014] Preferably, the culture medium of the hepatocellular carcinoma organoid comprises the following components: DMEM / F12 basal medium, penicillin-streptomycin, human insulin, ascorbic acid, L-glutathione, Noggin, R-spondin-1, N2, B27 serum replacement, GlutaMAX Supplement, HEPES, nicotinamide, N-acetylcysteine, A83-01, recombinant human epidermal growth factor, hepatocyte growth factor, Y27632, mauveine, recombinant human basic fibroblast growth factor, vascular endothelial growth factor, GA-1000, regenerated human R3 insulin-like growth factor-1, hydrocortisone and prostaglandin E2.

[0015] Preferably, in step S1, the tissue washing solution contains DPBS solution and penicillin-streptomycin, wherein the tissue washing solution contains 1% penicillin-streptomycin and 99% DPBS buffer in a volume ratio, and the size of the tissue minced with a sterile scalpel is 0.5-1 mm 3 Small Block

[0016] Preferably, in step S3, the tissue digestion solution includes 97% DPBS buffer, 10 μM ROCK inhibitor, 100 μg / mL antibiotics, 50 ng / mL EGF, 10 mM HEPES, 1% BSA by mass and 1 mg / mL type I collagenase; the digestion time is 30-60 min, and 3-5 times the final volume of the liver cancer tissue block and 2% fetal bovine serum are added to terminate the digestion operation.

[0017] Preferably, in step S5, the volume of the red blood cell lysis solution added is 3-5 mL, the lysis time is 2-4 min, and the volume of the tissue cleaning solution added is 4-6 mL.

[0018] Preferably, in step S6, the hepatocellular carcinoma organoids are inoculated into a culture medium-matrigel mixture, and the number of organoids in every 30-100 μL of the mixed solution is 5000-10000; and the volume ratio of the culture medium of the hepatocellular carcinoma organoids to the matrix gel in the culture medium-matrigel mixture of the hepatocellular carcinoma organoids is 1:1-4.

[0019] Preferably, in step S7, the organoids are replaced with fresh, pre-warmed complete organoid culture medium every 3-5 days and passaged every 6-9 days, with a passage ratio of 1:2-5.

[0020] Preferably, in the culture medium, the content of penicillin-streptomycin is 50-200 μg / mL, the content of human insulin is 1-4 μM, the content of ascorbic acid is 5-20 mg / L, the content of L-glutathione is 10-40 mg / L, the content of Noggin is 10-100 ng / mL, the content of R-spondin-1 is 100-500 ng / mL, the content of N2, B27 serum substitute is 1X, the content of GlutaMAX The content of Supplement is 1X, the content of HEPES is 10mM, the content of nicotinamide is 1-10mM, the content of N-acetylcysteine ​​is 0.1-1mM, the content of A83-01 is 200-500nM, the content of recombinant human epidermal growth factor is 10-100ng / mL, the content of hepatocyte growth factor is 50ng / mL, the content of Y27632 is 1-10μM, the content of maohorn is 1-20μM, the content of recombinant human basic fibroblast growth factor is 100-4000ng / mL, the content of vascular endothelial growth factor is 10-100ng / mL, the content of GA-1000 is 50-200ng / mL, the content of regenerated human R3 insulin-like growth factor-1 is 10-100ng / mL, the content of hydrocortisone is 10-50ng / mL, and the content of prostaglandin E2 is 1-20nM.

[0021] Beneficial technical effects

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The culture medium of the hepatocellular carcinoma organoids used in this method contains unique hepatocellular carcinoma organoid culture components, which can culture organoids derived from multiple types of liver cancer tissues, such as hepatocellular carcinoma, mixed liver cancer and intrahepatic cholangiocarcinoma. Pro-angiogenic cytokines that promote the spontaneous formation of blood vessels in liver cancer organoids are added to the culture medium components, so that the established liver cancer organoids can grow primary vascular networks, maintain the genetic expression and morphological structure of the primary tissue, and better simulate the existence of liver tumor tissue in the human body.

[0024] The culture medium used in this application does not contain fetal bovine serum. The use of serum substitutes can reduce the impact of uncertain components in serum on organoids, thereby promoting the proliferation and structural maintenance of liver cancer organoids, and can effectively reduce the risk of microbial and mycoplasma contamination in liver cancer culture, thereby improving the success rate of liver cancer organoid culture.

[0025] This method is easy to operate, has a short modeling time, a high modeling success rate, and does not require additional genetic engineering technology and microfluidic platforms. It avoids the problems of expensive instruments and cumbersome operating steps and high technical barriers required by other technical platforms. It can be better and more widely applied to basic scientific research, and solves the problem that traditional in vitro cultured hepatocytes lack certain liver-specific genes and specific biological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 A simplified flowchart of the method for constructing primary vascularized liver tumor organoids.

[0028] Figure 2 This is a cell morphology image of the liver tumor organoid under bright field conditions in Example 2 (scale bar: 100 μm).

[0029] Figure 3 This is the result of H&E staining of liver cancer tumor tissue (scale bar: 100 μm).

[0030] Figure 4 This is a picture of the H&E staining results of liver tumor organoids in Example 2 (scale bar: 100 μm).

[0031] Figure 5 This is the result of immunofluorescence staining of liver tumor organoids in Example 2, in which the blood vessels spontaneously generated by the tumor were stained with CD31 antibody, represented by green fluorescence in the figure; tumor cells were stained with HNF-4α antibody, represented by red fluorescence in the figure; DAPI was used to color the cell nucleus, which is blue fluorescence in the figure, indicating all cells, and non-cellular components have no blue fluorescence (scale bar: 100μm).

[0032] Figure 6 This is a cell morphology image of the liver tumor organoid under bright field conditions in Example 3 (scale bar: 100 μm).

[0033] Figure 7 This is a picture of the H&E staining results of liver tumor organoids in Example 3 (scale bar: 100 μm).

[0034] Figure 8 This is the result of immunofluorescence staining of liver tumor organoids in Example 3, in which the blood vessels spontaneously generated by the tumor were stained with CD31 antibody, represented by green fluorescence in the figure; tumor cells were stained with HNF-4α antibody, represented by red fluorescence in the figure; DAPI was used to color the cell nucleus, which is blue fluorescence in the figure, indicating all cells, and non-cellular components have no blue fluorescence (scale bar: 100μm).

[0035] Fig. 9 It is a Venn diagram of the gene expression results of liver tumor organoids and their original liver tumor tissues.

[0036] Fig.10 It is a clustered heat map of gene expression levels of liver tumor organoids and their original liver tumor tissues.

[0037] Fig.11 This is a diagram of the classification results of the genetic metabolic pathways of liver tumor organoids and their original liver tumor tissues. DETAILED DESCRIPTION

[0038] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. The reagents used in this application are commercially available unless otherwise specified. The experimental methods for which specific implementation conditions are not specified in the embodiments are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0039] See also Figures 1 to 11 The present invention provides a method for constructing a liver tumor organoid with primary vascularization, comprising the following steps:

[0040] S1: Take fresh biopsy tissue, use scissors and forceps to select tumor tissue blocks and remove non-epithelial tissue, place liver cancer tissue in a 10 cm cell culture dish, rinse it repeatedly with tissue cleaning solution 3-4 times, and then mince the tissue with a sterile scalpel to obtain tumor tissue blocks;

[0041] Specifically, the tissue washing solution contains DPBS solution and penicillin-streptomycin, and the volume ratio of the tissue washing solution is 1% penicillin / streptomycin and 99% DPBS buffer. The size of the tissue is 0.5-1 mm using a sterile scalpel. 3 .

[0042] S2: transferring the tumor tissue block to a centrifuge tube, adding pre-cooled tissue washing solution for washing, waiting for the tumor tissue block to precipitate, discarding the supernatant, and obtaining the washed liver cancer tissue block;

[0043] S3: adding the washed liver cancer tissue block to the tissue digestion solution in an incubator for digestion, and shaking up and down or pipetting to mix the material in the centrifuge tube, and stopping the digestion when there are 50%-100% single cells in the suspension to obtain a tissue block suspension;

[0044] Specifically, the components of the tissue digestion solution are as follows: 97% DPBS buffer, 10 μM ROCK inhibitor, 100 μg / mL antibiotics, 50 ng / mL EGF, 10 mM HEPES, 1% BSA by mass and 1 mg / mL type I collagenase. The digestion time of the liver cancer tissue block is 30-60 min. To terminate the digestion of the liver cancer tissue block, a basic culture medium with a final volume of 3-5 times that of the liver cancer tissue block and a final concentration of 2% fetal bovine serum need to be added.

[0045] S4: filtering the tissue block suspension with a 100 μm pore size filter membrane, transferring the filtered cell suspension to a centrifuge tube and centrifuging, discarding the supernatant, and obtaining a liver cancer organoid precipitate;

[0046] S5: collecting the liver cancer organoid precipitate, adding red blood cell lysis solution to lyse the red blood cells, then adding tissue washing solution to stop the process, centrifuging and discarding the supernatant to obtain the organoid;

[0047] Specifically, the volume of the red blood cell lysis solution is 3-5 mL, the lysis time is 2-4 min, and the volume of the tissue washing solution is 4-6 mL.

[0048] S6: resuspending and counting the organoids using a hepatocellular carcinoma organoid basal culture medium, dropping the organoid suspension on the bottom of a suspension cell culture plate, and then incubating until the matrix gel is solidified to obtain organoids solidified in the matrix gel;

[0049] Specifically, the organoids are inoculated in a hepatocellular carcinoma organoid basal culture medium, and the number of the organoids in each 30-100 μL matrix gel volume is 5000-10000; the volume ratio of the culture medium of the organoids to the matrix gel in the hepatocellular carcinoma organoid basal culture medium is 1:1-4.

[0050] The culture medium of the hepatocellular carcinoma organoids contains the following components: DMEM / F12 basal medium, penicillin-streptomycin, human insulin, ascorbic acid, L-glutathione, Noggin, R-spondin-1, N2, B27 serum replacement, GlutaMAXSupplement, HEPES, nicotinamide, N-acetylcysteine, A83-01, recombinant human epidermal growth factor, hepatocyte growth factor, Y27632, mauveine, recombinant human basic fibroblast growth factor, vascular endothelial growth factor, GA-1000, regenerated human R3 insulin-like growth factor-1, hydrocortisone and prostaglandin E2.

[0051] The content of penicillin-streptomycin is 50-200 μg / mL, the content of human insulin is 1-4 μM, the content of ascorbic acid is 5-20 mg / L, the content of L-glutathione is 10-40 mg / L, the content of Noggin is 10-100 ng / mL, the content of R-spondin-1 is 100-500 ng / mL, the content of N2, B27 serum substitute is 1X, the content of GlutaMAXSupplement is 1X, the content of HEPES is 10 mM, the content of nicotinamide is 1-10 mM, the content of N-acetylcysteine ​​is 0.1-1 mM, the content of A83-01 is 200 -500nM, the content of recombinant human epidermal growth factor is 10-100ng / mL, the content of hepatocyte growth factor is 50ng / mL, the content of Y27632 is 1-10μM, the content of malocclusion is 1-20μM, the content of recombinant human basic fibroblast growth factor is 100-4000ng / mL, the content of vascular endothelial growth factor is 10-100ng / mL, the content of GA-1000 is 50-200ng / mL, the content of regenerated human R3 insulin-like growth factor-1 is 10-100ng / mL, the content of hydrocortisone is 10-50ng / mL, and the content of prostaglandin E2 is 1-20nM.

[0052] S7: adding the organoid solidified in the matrix gel to the complete culture medium of hepatocellular carcinoma organoid for culture.

[0053] Specifically, the organoids are replaced with fresh, pre-warmed complete organoid culture medium every 3-5 days and passaged every 6-9 days, with a passage ratio of 1:2-5.

[0054] In order to better understand the present technical solution, the following embodiments are provided for further explanation:

[0055] Example 1

[0056] 1. Preparation of Experimental Materials

[0057] Within a short period of time after sampling, liver tumor organoids select areas of tumor tissue with vigorous growth and no necrosis, and take tumor tissue blocks with a mass greater than 300 mg, about the size of 3 soybeans.

[0058] Under a dissecting microscope, remove as much non-epithelial tissue as possible using surgical scissors or a scalpel and forceps.

[0059] 2. Preparation of Culture Medium and Other Reagents

[0060] Tissue washing solution: contains 1% penicillin / streptomycin and 99% DPBS buffer in a volume ratio.

[0061] Tissue digestion solution: 97% DPBS buffer, ROCK inhibitor 10 μM, antibiotic 100 μg / mL, EGF 50 ng / mL, HEPES 10 mM, BSA with a mass fraction of 1%, and type I collagenase with a concentration of 1 mg / mL.

[0062] Stop solution: DMEM / F12 basal medium with a volume 3-5 times that of digestion solution and fetal bovine serum with a final concentration of 2%.

[0063] Organoid Vascularization Induction Medium: Penicillin-Streptomycin, 100 μg / mL; Human Insulin, 1-4 μM; Ascorbic Acid, 5-20 mg / L; L-Glutathione, 10-40 mg / L; Noggin, 10-100 ng / mL; R-spondin-1, 100-500 ng / mL; N2, B27 Serum Replacement, 1X; GlutaMAX Supplement, 1X; HEPES, 10mM; Nicotinamide, 1-10mM; N-acetylcysteine, 0.1-1mM; A83-01, 200-500nM; Recombinant human epidermal growth factor, 10-100ng / mL; Hepatocyte growth factor, 50ng / mL; Y27632, 1-10μM; Mauveine, 1-20μM; Recombinant human basic fibroblast growth factor, 200ng / mL; Vascular endothelial growth factor, 50ng / mL; GA-1000, 100ng / mL; Regenerated human R3 insulin-like growth factor-1, 50ng / mL; Hydrocortisone, 25ng / mL; Prostaglandin E2, 1-20nM.

[0064] Example 2

[0065] An example of a method to construct liver tumor organoids with primary vascularization

[0066] Tissue pretreatment: Take a biopsy sample, soak the tumor tissue in pre-cooled tissue cleaning solution for 10 seconds, gently rinse 4-5 times to rinse the residual blood on the surface of the tissue, remove it with tweezers or hemostatic forceps, and cut the hepatocellular carcinoma tissue into small pieces of 0.5-1mm with scissors. 3 Transfer the chopped tissue chunks to a 15 mL centrifuge tube, add 10 mL of ice-cold tissue washing solution, and gently blow to wash the tissue chunks. Wait for the tissue chunks to settle, discard about 7.5 mL of supernatant, repeat the wash once, and discard the supernatant.

[0067] Tissue digestion: Add the cleaned tissue blocks to 5-10 times the final volume of tissue digestion solution, and digest them in batches in a 37°C incubator; let the tube stand for 10 minutes after each digestion, and collect the turbid upper layer of the digestion solution after the larger tissue blocks naturally settle. This should be done at least three times, during which blowing can be used to help digestion; every 1-3 minutes, mix the contents of the tube by shaking up and down or pipetting. Stop digestion when there are 50%-100% single cells in the suspension, and add 3-5 times the volume of the digestion solution DMEM / F12 basal culture medium and a final concentration of 5-10% fetal bovine serum to terminate digestion.

[0068] Collect organoids: Wash the filter with cleaning solution in advance, and then filter the tissue blocks and cell suspension after digestion with a 100μm pore size filter to remove excess tissue fragments. Add 2mL of red blood cell lysis solution to lyse red blood cells (on ice for 2min), then add 4mL of cleaning solution to stop, centrifuge at 300g for 5min, discard the supernatant, and collect organoids.

[0069] Culture and expansion of organoids: Resuspend the organoids in complete culture medium, count, and resuspend 5,000-10,000 cells in 30-100 μL of HCC organoid culture medium-matrigel mixture, add drops to the culture plate for culture, place in a 37°C incubator for 15-30 minutes to solidify, then add 500-800 μL of HCC organoid culture medium and continue culture.

[0070] Immunofluorescence staining was performed to identify liver cancer markers and generated blood vessels in organoids (Cancer Markers and Vascularization Evaluation).

[0071] The washing solution of the above culture method contains 1% penicillin / streptomycin and 99% DPBS buffer in a volume ratio.

[0072] The tissue digestion solution of the above culture method is 97% DPBS buffer, 10 μM ROCK inhibitor, 100 μg / mL antibiotics, 50 ng / mL EGF, 10 mM HEPES, 1% BSA by mass, and 1 mg / mL type I collagenase.

[0073] The stop solution of the above culture method is DMEM / F12 basic culture medium with a volume of 3-5 times that of the digestion solution and fetal bovine serum with a final concentration of 2%.

[0074] The organoid vascularization induction medium of the above culture method contains: penicillin-streptomycin, 100 μg / mL; human insulin, 1-4 μM; ascorbic acid, 5-20 mg / L; L-glutathione, 10-40 mg / L; Noggin, 10-100 ng / mL; R-spondin-1, 100-500 ng / mL; N2, B27 serum replacement, 1X; GlutaMAX Supplement, 1X; HEPES, 10mM; Nicotinamide, 1-10mM; N-acetylcysteine, 0.1-1mM; A83-01, 200-500nM; Recombinant human epidermal growth factor, 10-100ng / mL; Hepatocyte growth factor, 50ng / mL; Y27632, 1-10μM; Mauveine, 1-20μM; Recombinant human basic fibroblast growth factor, 200ng / mL; Vascular endothelial growth factor, 50ng / mL; GA-1000, 100ng / mL; Regenerated human R3 insulin-like growth factor-1, 50ng / mL; Hydrocortisone, 25ng / mL; Prostaglandin E2, 1-20nM.

[0075] The tumor organoid culture plate of the above-mentioned culture method is a 24-well flat-bottom plate. During the culture process, PBS is used to fill the surrounding blank wells to prevent the culture medium from drying up during the culture process.

[0076] In the culture medium-matrigel mixture of hepatocellular carcinoma organoids in the above-mentioned culture method, the volume ratio of the culture medium of hepatocellular carcinoma organoids to the matrix gel is 1:1-4.

[0077] The culture plate of the above culture method was placed in a humidified incubator at 37°C and 5% CO2, and then replaced with fresh, pre-warmed organoid complete culture medium every 3-5 days. The cells were subcultured every 6-9 days, with a subculture ratio of 1:2-5.

[0078] The matrix gel used in the above culture method is Matrigel.

[0079] To verify whether the tissue structure and marker molecule expression of the constructed liver cancer organoids are consistent with the source liver cancer tissue, the liver cancer organoids and the source cancer tissue were stained with HE and immunofluorescence staining of liver cancer-specific indicators. The tumor organoids were cultured for about 10 days, and the gel droplets containing the organoids were stained with H&E and immunofluorescence staining. The immunofluorescence staining used CD31 and HNF-4α antibodies and DAPI staining solution, and the staining was performed according to the conventional staining steps and photographed under a fluorescence microscope.

[0080] Experimental steps:

[0081] (1) H&E staining

[0082] Use PBS buffer to wash the original liver cancer tissue and organoids for 3 times or more, and add 1 mL of 40% formaldehyde solution to fix for more than 20 minutes. Place the fixed organoids and paired tissue samples and corresponding labels in a dehydration box, put them in a hanging basket, and dehydrate them in a dehydrator with gradient alcohol. Follow the following steps in sequence: 75% alcohol dehydration (4h) - 85% alcohol dehydration (2h) - 90% alcohol dehydration (2h) - 95% alcohol dehydration (1h) - anhydrous ethanol I dehydration (30min) - anhydrous ethanol II dehydration (30min) - phenylethanol dehydration (5-10min) - xylene I dehydration (5-10min) - xylene II dehydration (5-10min) - wax I dehydration (1h) - wax II dehydration (1h) - wax III dehydration (1h). Embed the wax-soaked tissue in an embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool it in a freezing table at -20℃. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block. Place the trimmed wax block on a paraffin slicer and slice it with a thickness of 4μm. The slices float on the 40℃ warm water of the spreader to flatten the tissue, pick up the tissue with a slide, and put it in a 60℃ oven to bake the slices. After the water is dried and the wax is baked, take it out and store it at room temperature for later use. After the paraffin sections are dewaxed, put the sections into xylene I (20min), xylene II (20min), anhydrous ethanol I (10min), anhydrous ethanol II (10min), 95% alcohol (5min), 90% alcohol (5min), 80% alcohol (5min) and 70% alcohol (5min) in turn, and then wash them with distilled water. After the slices were placed in Harris hematoxylin for 3-8 minutes, they were washed with pure water, differentiated with 1% hydrochloric acid alcohol for a few seconds, washed with pure water, blued with 0.6% ammonia water, and rinsed with running water. The slices were then placed in eosin stain for 1-3 minutes. After staining, the slices were dehydrated and sealed. The slices were placed in 95% alcohol I (5 minutes), 95% alcohol II (5 minutes), anhydrous ethanol I (5 minutes), anhydrous ethanol II (5 minutes), xylene I (5 minutes), and xylene II (5 minutes) for dehydration. The slices were taken out of xylene and dried, and sealed with neutral gum. The slices were used for microscopic examination, and images were collected for analysis.

[0083] (2) Immunofluorescence staining of organoids and paired tissues

[0084] After H&E staining and sealing, the slides were processed according to conventional immunofluorescence staining steps to fix the vascularized liver cancer organoids, and then permeabilization, antigen retrieval, primary antibody incubation, secondary antibody incubation and sealing were performed. After sealing, the slides were observed using an inverted fluorescence confocal microscope and photographed for preservation; the functional changes of HNF4α are important parameters reflecting the progression of liver disease, and its expression and loss of function are associated with the progression of chronic liver disease; CD31 is a platelet-endothelial cell adhesion molecule widely present in blood cells and vascular endothelial cells, and is often used to prove the existence of connections between endothelial cells and evaluate angiogenesis; DAPI, or 4',6-diamidino-2-phenylindole, is a fluorescent dye that can strongly bind to DNA and is often used to locate cell nuclei.

[0085] Histomorphology and pathological characteristics

[0086] Figure 2 Under the bright field of the microscope, it was observed that the diameter of liver tumor organoids was between 200 and 400 μm, maintaining good morphology and growth state.

[0087] H&E staining results Figure 3 and Figure 4 Display: The liver cancer organoids have a high nuclear-to-cytoplasmic ratio, the color is consistent with the parental tissue, and have obvious cellular atypia, suggesting that the organoids retain the histopathological characteristics of the parental tumor.

[0088] Immunofluorescence results Figure 5 Display: The expression levels of key markers were detected by immunofluorescence staining. In liver cancer organoids, while the CD31 angiogenesis indicator was highly expressed, there was also a significant expression of the liver cancer marker HNF-4α. The above indicates that liver cancer organoids retain the biomarkers of primary tumor tissues, reproduce the marker expression pattern of the original tumor tissues, and simulate the interaction between human tumors and vascular networks to a certain extent.

[0089] Example 3

[0090] Validation of the effects of tumor organoid culture medium components

[0091] The co-culture medium provided by the present invention consists of DMEM / F12 basic medium, penicillin-streptomycin, human insulin, ascorbic acid, L-glutathione, Noggin, R-spondin-1, N2, B27 serum substitute, GlutaMAX Supplement, HEPES, nicotinamide, N-acetylcysteine, A83-01, recombinant human epidermal growth factor, hepatocyte growth factor, Y27632, mauveine recombinant human basic fibroblast growth factor, vascular endothelial growth factor, GA-1000, regenerated human R3 insulin-like growth factor-1 (R3-IGF-1), hydrocortisone, and prostaglandin E2.

[0092] Vascular endothelial growth factor, regenerated human R3 insulin-like growth factor-1, recombinant human basic fibroblast growth factor and ascorbic acid are angiogenesis-promoting components. This example verifies the effect of adding and removing the angiogenesis-promoting components on the culture results when other formulas remain unchanged.

[0093] Experimental results: Figure 5 and Figure 8 Green represents the generated blood vessels, red represents tumor organoids, and blue represents the cell nucleus, indicating all cells. Non-cellular components have no blue fluorescence. Obviously, under the same culture time and culture conditions, without adding pro-angiogenic components ( Figure 8 ) only small tumor organoids were seen, and there was no obvious sign of angiogenesis; after adding pro-angiogenic components ( Figure 5 ) The volume increased significantly and a large number of vascular network structures were formed.

[0094] Example 4

[0095] Histological consistency between tumor tissue and paired organoids

[0096] Organoids were cultured according to the method for constructing organoids described in Example 2 for 10-15 days and then collected for transcriptome experiments.

[0097] Experimental procedures

[0098] 1. Collect liver tumor organoids and their corresponding tissue specimens.

[0099] 2. Extract organoid and tissue genomic RNA according to the instructions of the cell and tissue RNA extraction kit.

[0100] 3. Send the RNA of organoids and original tissues to an institution qualified for clinical genetic testing for sequencing testing.

[0101] 4. By analyzing the similarity of gene expression levels, the consistency of gene expression levels of organoid sequencing and original tumor tissues and the gene-related metabolic pathway results of sequencing results were compared.

[0102] Transcriptome sequencing results

[0103] Fig. 9 In the Venn diagram, it is shown that the number of common genes expressed between organoids and primary tissues is high, and the consistency of gene expression between organoids and original tissues is above 90%; Fig.10 The gene expression heat map showed that the up- and down-regulation results of gene expression in liver tumor organoids were basically consistent with those in the original tissues, and their similarities were greater than 93%; Fig.11The results of gene enrichment metabolic pathways showed that liver tumor organoids retained most of the genotypes of their parents and could simulate the expression level of liver cancer tissue metabolism in the human body. The experimental results showed that the liver cancer organoids cultured in the culture medium of the liver cancer organoids of the present invention highly restored the genomic characteristics of the original tumor tissue, reflecting the high consistency and genetic stability of tumor organoids and primary tissues.

[0104] The above disclosure is only a preferred embodiment of a method for constructing a liver tumor organoid with primary vascularization of the present invention. Of course, it cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention are still within the scope of the invention.

Claims

1. A method for constructing liver tumor organoids with primary vascularization, characterized in that: The following steps are involved: S1: Take fresh biopsy tissue, use scissors and forceps to select tumor tissue blocks and remove non-epithelial tissue, place liver cancer tissue in a 10 cm cell culture dish, rinse it repeatedly with tissue cleaning solution 3-4 times, and then mince the tissue with a sterile scalpel to obtain tumor tissue blocks; S2: transferring the tumor tissue block to a centrifuge tube, adding pre-cooled tissue washing solution for washing, waiting for the tumor tissue block to precipitate, discarding the supernatant, and obtaining the washed liver cancer tissue block; S3: adding the washed liver cancer tissue block to the tissue digestion solution in an incubator for digestion, and shaking up and down or pipetting to mix the material in the centrifuge tube, and stopping the digestion when there are 50%-100% single cells in the suspension to obtain a tissue block suspension; S4: filtering the tissue block suspension with a 100 μm pore size filter membrane, transferring the filtered cell suspension to a centrifuge tube and centrifuging, discarding the supernatant, and obtaining a liver cancer organoid precipitate; S5: collecting the liver cancer organoid precipitate, adding red blood cell lysis solution to lyse the red blood cells, then adding tissue washing solution to stop the process, centrifuging and discarding the supernatant to obtain the organoid; S6: resuspending and counting the organoids using a hepatocellular carcinoma organoid basal culture medium, dropping the organoid suspension on the bottom of a suspension cell culture plate, and then incubating until the matrix gel is solidified to obtain organoids solidified in the matrix gel; S7: adding the organoid solidified in the matrix gel to the complete culture medium of hepatocellular carcinoma organoid for culture.

2. The method for constructing liver tumor organoids with primary vascularization according to claim 1, characterized in that: The culture medium of the hepatocellular carcinoma organoids contains the following components: DMEM / F12 basal medium, penicillin-streptomycin, human insulin, ascorbic acid, L-glutathione, Noggin, R-spondin-1, N2, B27 serum replacement, GlutaMAXSupplement, HEPES, nicotinamide, N-acetylcysteine, A83-01, recombinant human epidermal growth factor, hepatocyte growth factor, Y27632, mauveine, recombinant human basic fibroblast growth factor, vascular endothelial growth factor, GA-1000, regenerated human R3 insulin-like growth factor-1, hydrocortisone and prostaglandin E2.

3. The method for constructing a liver tumor organoid with primary vascularization according to claim 1, characterized in that: In step S1, the tissue washing solution contains DPBS solution and penicillin-streptomycin, the volume ratio of which is 1% penicillin-streptomycin and 99% DPBS buffer, and the size of the tumor tissue block after treatment is 0.5-1 mm 3 .

4. The method for constructing liver tumor organoids with primary vascularization according to claim 1, characterized in that: In step S3, the components of the tissue digestion solution are as follows: 97% DPBS buffer, ROCK inhibitor 10 μM, antibiotic 100 μg / mL, EGF 50 ng / mL, HEPES 10 mM, BSA with a mass fraction of 1% and type I collagenase with a concentration of 1 mg / mL. The digestion time of the liver cancer tissue block is 30-60 min. To terminate the digestion of the liver cancer tissue block, a basic culture medium with a final volume of 3-5 times that of the liver cancer tissue block and a final concentration of 2% fetal bovine serum need to be added.

5. The method for constructing liver tumor organoids with primary vascularization according to claim 1, characterized in that: In step S5, the volume of the red blood cell lysis solution is 3-5 mL, the lysis time is 2-4 min, and the volume of the tissue washing solution is 4-6 mL.

6. The method for constructing liver tumor organoids with primary vascularization according to claim 1, characterized in that: In step S6, the organoids are inoculated in a hepatocellular carcinoma organoid basal culture medium, and the number of the organoids in each 30-100 μL matrix gel volume is 5000-10000; the volume ratio of the culture medium to the matrix gel in the hepatocellular carcinoma organoid basal culture medium is 1:1-4.

7. The method for constructing liver tumor organoids with primary vascularization according to claim 1, characterized in that: In step S7, the organoids are replaced with fresh, pre-warmed complete organoid culture medium every 3-5 days and passaged every 6-9 days, with a passage ratio of 1:2-5.

8. The method for constructing a liver tumor organoid with primary vascularization according to claim 3, characterized in that: The content of penicillin-streptomycin is 50-200 μg / mL, the content of human insulin is 1-4 μM, the content of ascorbic acid is 5-20 mg / L, the content of L-glutathione is 10-40 mg / L, the content of Noggin is 10-100 ng / mL, the content of R-spondin-1 is 100-500 ng / mL, the content of N2, B27 serum substitute is 1X, the content of GlutaMAXSupplement is 1X, the content of HEPES is 10 mM, the content of nicotinamide is 1-10 mM, the content of N-acetylcysteine ​​is 0.1-1 mM, the content of A83-01 is 200 -500nM, the content of recombinant human epidermal growth factor is 10-100ng / mL, the content of hepatocyte growth factor is 50ng / mL, the content of Y27632 is 1-10μM, the content of malocclusion is 1-20μM, the content of recombinant human basic fibroblast growth factor is 100-4000ng / mL, the content of vascular endothelial growth factor is 10-100ng / mL, the content of GA-1000 is 50-200ng / mL, the content of regenerated human R3 insulin-like growth factor-1 is 10-100ng / mL, the content of hydrocortisone is 10-50ng / mL, and the content of prostaglandin E2 is 1-20nM.

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