Colorectal Cancer Organoid Culture Medium and Method for Constructing Colorectal Cancer or Liver Metastasis Organoids by 3D Printing

Abstract

The present invention discloses a colorectal cancer organoid culture medium and a method for 3D printing to construct colorectal cancer or liver metastasis organoids. The present invention utilizes a modified colorectal cancer organoid culture medium containing various cytokines required for primary tumor cell culture, combines it with the grid-like three-dimensional physical environment created by 3D bioprinting, and regulates the 3D printing conditions, which improves the success rate of model establishment, shortens the model establishment period. The constructed colorectal cancer or liver metastasis organoids can highly retain the heterogeneous characteristics of the parental tumor, and can more truly reflect the real response of in vivo tumor tissues to test drugs, and have broad application prospects in drug screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of tumor organoids, and relates to a colorectal cancer organoid culture medium and a method for constructing colorectal cancer or liver metastasis organoids by 3D printing. Background Art

[0002] Adjuvant chemotherapy after surgery is an important treatment method for patients with colorectal cancer and colorectal cancer liver metastasis. There are various chemotherapy regimens, but different patients have different responses to the same drug. The chemotherapy regimen mainly depends on clinical experience, and the actual response rate of patients is 30-60%. Therefore, how to more accurately predict the patient's response to drugs and develop an individualized optimal chemotherapy regimen is an urgent problem to be solved clinically. At present, preclinical tumor models are mainly used to simulate the occurrence and development of tumors in the human body and predict the drug response. The main preclinical models of colorectal cancer are human tumor-derived cell lines, human-derived tumor tissue xenografts (i.e., PDX models), and patient-derived tumor organoids (PDO models). However, colorectal cancer cells cultured in 2D lose the biological and genetic characteristics of tumor cells in vivo. The PDX tumor model can relatively well reproduce the characteristics related to colorectal cancer and predict the patient's drug response. However, the PDX model has high costs, a long cycle, and low throughput, which limits its wide application. Organoids constructed using Matrigel and primary tumor cells make up for the deficiencies of both. It can highly simulate the physiological structure and function characteristics of in-situ tissues. The latest research reports that PDOs derived from preoperative puncture samples of colorectal cancer patients can accurately predict the effect of neoadjuvant chemotherapy. However, the established methods for colorectal cancer PDOs are all labor-intensive, with poor time and economic cost-effectiveness. More importantly, there is a large variability and poor consistency among colorectal cancer-related organoid samples, lacking reliable methods for consistency, high throughput, repeatability, and standardization, which affects the accuracy of subsequent drug screening results. Therefore, there is an urgent need to develop a better in vitro drug prediction model.

[0003] 3D bioprinting is a technology for printing living tissues using biocompatible materials and living cells, with advantages such as precision, rapidity, and repeatability, and is especially suitable for constructing complex models. Although there have been studies applying 3D bioprinting technology to construct tumor organoids, there is currently no report on a method for rapidly establishing patient-derived colorectal cancer and colorectal cancer liver metastasis organoids with good uniformity and applicable to drug screening based on 3D bioprinting technology. Summary of the Invention

[0004] Aiming at the defects of poor uniformity, poor repeatability, long cycle, complex operation, and unstable prediction results of colorectal cancer organoids constructed by existing methods, the present invention provides a colorectal cancer organoid culture medium and a method for constructing colorectal cancer or liver metastasis organoids by 3D printing.

[0005] The technical solution of the present invention is as follows:

[0006] A colorectal cancer organoid culture medium, the specific components and contents are as follows: Advanced DMEM / F12 basal medium, B27 diluted 5 - 20 times, N2 diluted 2 - 10 times, GlutaMAX diluted 50 - 200 times, HEPES diluted 100 times, Human EGF 50 - 150 ng / ml, N-acetylcysteine 0.5 - 1.5 mM, Prostaglandin E2 5 - 20 ng / ml, A8301 200 - 1000 ng / ml, SB202190 1 - 5 μM, Gastrin I 1 - 20 nM, lipopolysaccharide 0.5 - 2 μg / ml, 1% (mass concentration) Antibiotic - Antimycotic, 0.2% (mass concentration) Primocin.

[0007] For the colorectal cancer organoid culture medium of the present invention, the organoids of colorectal cancer or colorectal cancer liver metastasis can be successfully cultured and formed when the contents of each component are within the above ranges.

[0008] In a specific embodiment of the present invention, the used colorectal cancer organoid culture medium has the specific components and contents as follows: Advanced DMEM / F12 basal medium, B27 diluted 20 times, N2 diluted 10 times, GlutaMAX diluted 100 times, HEPES diluted 100 times, Human EGF 100 ng / ml, N-acetylcysteine 1 mM, Prostaglandin E2 10 ng / ml, A8301 500 ng / ml, SB202190 1 μM, Gastrin I 10 nM, lipopolysaccharide 1 μg / ml, 1% (mass concentration) Antibiotic - Antimycotic, 0.2% (mass concentration) Primocin.

[0009] A method for constructing colorectal cancer or liver metastasis organoids by 3D printing, comprising the following steps:

[0010] (1) Isolation of colorectal cancer cells or colorectal cancer liver metastasis cells: Isolate colorectal cancer cells or colorectal cancer liver metastasis cells from ex vivo colorectal cancer tissue or colorectal cancer liver metastasis cancer tissue to obtain a cell suspension;

[0011] (2) Preparation of bioink: Mix the cell suspension, sodium alginate solution and gelatin solution evenly to prepare bioink with a final cell concentration of 1 - 5×10 6 cells / mL, a final mass concentration of sodium alginate of 1.0 - 1.5%, and a final mass concentration of gelatin of 5 - 7%.

[0012] (3) 3D printing of colorectal cancer or colorectal cancer liver metastasis organoid tissues: Load the bioink into a syringe and assemble the needle. Let it stand at 4°C for 10 - 30 min until the bioink extrudes in a continuous filament. Load the pre-cooled bioink syringe into the 3D printing nozzle, set the nozzle temperature and the printer chamber platform temperature to 15 - 20°C and 5 - 10°C respectively, and perform 3D printing according to the preset model parameters to obtain colorectal cancer or colorectal cancer liver metastasis organoid tissues;

[0013] (4) Cultivation of colorectal cancer or colorectal cancer liver metastasis organoids: Immerse the colorectal cancer or colorectal cancer liver metastasis organoid tissues in a 2% - 4% calcium chloride solution for chemical cross-linking. After cross-linking, wash with HBSS solution, add the colorectal cancer organoid culture medium, and culture in an incubator at 37°C and 5% CO2 until mature to obtain colorectal cancer or colorectal cancer liver metastasis organoids.

[0014] Further, step (1) is specifically: Soak and wash the excised colorectal cancer tissue or colorectal cancer liver metastasis cancer tissue multiple times with PBS containing 5 μM of Y-276325, 2X of Antibiotic - Antimycotic, and 0.2% of Primocin, then cut it into pieces, resuspend and centrifuge with PBS multiple times, digest the resuspended tissue, filter, centrifuge, discard the supernatant, add DMEM / F-12 medium to resuspend and add erythrocyte lysate, centrifuge, and repeat the steps of digestion, filtration, centrifugation, and erythrocyte lysis. Collect the cell pellet, resuspend and wash with PBS, centrifuge, and finally add the medium to resuspend to obtain a cell suspension.

[0015] Further, in step (2), in the bioink, the final cell concentration is 5×10 6 cells / mL, the final mass concentration of sodium alginate is 1.0%, and the final mass concentration of gelatin is 5%.

[0016] Further, in step (3), the nozzle temperature and the printer chamber platform temperature are 15°C and 10°C respectively.

[0017] Further, in step (4), the concentration of the calcium chloride solution is 3%.

[0018] Further, in step (4), the time to culture until mature is 5 - 7 days.

[0019] The present invention also provides the colorectal cancer or colorectal cancer liver metastasis organoids prepared by the above method.

[0020] Further, the present invention provides the application of the above colorectal cancer or colorectal cancer liver metastasis organoids in drug screening.

[0021] Specifically, the application method is as follows: a drug screening experiment is carried out on the 5th to 7th day of culturing the above colorectal cancer or colorectal cancer liver metastasis organoids. As the first day of drug testing, the test drug is added to the fresh culture medium at the set concentration gradient. Fresh culture medium containing the test drug is replaced on the next two days respectively. On the 4th day of drug testing, a cell viability test is carried out to detect the cell survival rate, and the drug sensitivity is analyzed according to the cell survival rate.

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

[0023] (1) By improving the organoid culture medium, the present invention removes the commonly added wnt small molecule substances and wnt agonists, greatly saving costs. At the same time, lipopolysaccharide is added to the culture medium, which can better simulate the colorectal cancer tumor microenvironment. Combining with the grid-like three-dimensional physical environment created by 3D bioprinting, the success rate of model establishment is improved and the model establishment period is shortened.

[0024] (2) In the process of 3D bioprinting, by controlling the concentrations of gelatin and sodium alginate in the bioink and the printing temperature, the uniformity of the constructed organoids is significantly improved, and rapid and batch construction can be achieved.

[0025] (3) The present invention uses the improved colorectal cancer organoid culture medium containing various cytokines required for culturing primary tumor cells, and at the same time combines with the grid-like three-dimensional physical environment created by 3D bioprinting, so that the primary tumor cells can better show their inherent characteristics during the culture process to achieve highly retaining the heterogeneous characteristics of the parental tumor. The colorectal cancer and liver metastasis organoids cultured by the present invention can more reflect the true response of in vivo tumor tissues to the test drug, and have broad application prospects in drug screening. Description of the Drawings

[0026] Figure 1 It is a flowchart of 3D bioprinting colorectal cancer and liver metastasis organoids of the present invention;

[0027] Figure 2 It is a morphological diagram of colorectal cancer organoids formed after culturing for 6 days in Example 1, magnified 200 times;

[0028] Figure 3 It is a cell diameter diagram of colorectal cancer organoids formed after culturing for 6 days in Example 1 and Comparative Example 4;

[0029] Figure 4 It is a cell number diagram of colorectal cancer organoids formed after culturing for 6 days in Example 1 and Comparative Example 4;

[0030] Figure 5Protein expression map of colorectal cancer organoids formed in Example 1 and parental tumors, where 3DP: 3D-printed tumor organoids, Tumor: parental tumor, magnified 400 times;

[0031] Figure 6 Key gene mutation map of colorectal cancer organoids constructed by 3D printing of colorectal cancer tissues from different patients and parental tumors;

[0032] Figure 7 Morphology map of colorectal cancer liver metastasis organoids formed after 6 days of culture in Example 2;

[0033] Figure 8 Protein expression map of colorectal cancer liver metastasis organoids and parental tumors in Example 2, where 3DP: 3D-printed tumor organoids, Tumor: parental tumor, magnified 400 times;

[0034] Figure 9 Genomic mutation feature map of colorectal cancer liver metastasis organoids and parental tumors cultured in Example 2, 3DP: 3D-printed tumor organoids, PDO: tumor organoids, Tumor: parental tumor;

[0035] Figure 10 Response map of 3D-printed colorectal cancer organoids from different cases to 3 chemotherapy drugs in Application Example 1. The abscissa represents the drug type, the ordinate represents the colorectal cancer cases, and different colors represent drug sensitivity;

[0036] Figure 11 Response map of 3D-printed colorectal cancer liver metastasis organoids from different cases to 3 chemotherapy drugs in Application Example 2. The abscissa represents the drug type, the ordinate represents the colorectal cancer cases, and different colors represent drug sensitivity;

[0037] Figure 12 Prediction effect map of 3D-printed tumor organoids in Application Example 2. PR, SD, and PD represent partial control, stable, and progressive of the actual clinical chemotherapy results of patients respectively;

[0038] Figure 13 3D printing effect map of colorectal cancer organoids constructed with different concentration ratios of gelatin and sodium alginate in Comparative Example 1;

[0039] Figure 14 3D printing effect map of colorectal cancer organoids constructed at different printing temperatures in Comparative Example 2;

[0040] Figure 15 Effect map of colorectal cancer organoids formed by culturing in a colorectal cancer organoid medium without adding LPS in Comparative Example 3, magnified 200 times;

[0041] Figure 16Morphological diagram of colorectal cancer organoids constructed by the conventional matrigel embedding method in Comparative Example 4, magnified 400 times. Detailed implementation manners

[0042] The present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings.

[0043] In the following embodiments, unless otherwise specified, the reagents and culture media used are conventional reagents and culture media in the art and can be commercially purchased.

[0044] In the following embodiments, the materials used are as follows:

[0045] DMEM / F12: purchased from GIBCO; Advanced DMEM / F12: purchased from GIBCO; B27: purchased from GIBCO; N-acetylcysteine: purchased from Sigma; human-EGF: purchased from Peprotech; A83-01: purchased from Tocris Bioscience; fibroblast growth factor (FGF10): purchased from Peprotech; Nicotinamide: purchased from Sigma; ROCK-specific pathway blocker Y-27632 dihydrochloride: purchased from Abmole Bioscience; Prostaglandin E2: purchased from Sigma; SB202190: purchased from Selleckchem; Lipopolysaccharide (LPS): purchased from Solarbio; Gelatin: purchased from Sigma; Sodium alginate: purchased from Sigma; Matrigel: purchased from Corning. 3D Cell Viability Assay Kit: purchased from Promega.

[0046] In the following embodiments, the reagents used are as follows:

[0047] 1. Colorectal cancer tissue preservation solution, the component contents are as follows: DMEM / F-12 medium, HEPES 10 mM, GlutaMAX Supplement 1X, Nicotinamide 1 mM, Y-27632 5 μM, Antibiotic-Antimycotic 2X, Primocin 0.2%.

[0048] 2. Colorectal cancer tissue digestion solution, the component contents are as follows: DMEM / F-12 medium, Collagenase II

[0049] 1.5 mg / mL, Neutral protease II 1 mg / mL, Hyaluronidase 20 μg / mL, Y27632 10 μM, SB431542 10 μM, Antibiotic - Antimycotic 2X, Primocin 0.2%.

[0050] 3. Colorectal cancer organoid culture medium, with the components and contents as follows:

[0051] Advanced DMEM / F12 basal medium, diluted 20X with B27, diluted 10X with N2, diluted 100X with GlutaMAX, diluted 100X with HEPES, Human EGF 100 ng / ml, N - acetylcysteine 1 mM, Prostaglandin E2 10 ng / ml, A8301 500 ng / ml, SB202190 1 μM, Gastrin I 10 nM, Lipopolysaccharide 1 μg / ml, 1% (mass concentration) Antibiotic - Antimycotic, 0.2% (mass concentration) Primocin.

[0052] Example 1

[0053] 1. Isolation of colorectal cancer cells or colorectal cancer liver metastasis cells

[0054] (1) Collect colorectal cancer tissue from the patient into the preservation solution and transport it to the laboratory on ice;

[0055] (2) Immerse the tissue in cold PBS containing Y - 27632 5 μM, Antibiotic - Antimycotic 2X, and Primocin 0.2% on ice in the ultra - clean bench 3 times, 5 minutes each time;

[0056] (3) Use sterile ophthalmic scissors to cut the tissue into pieces smaller than 3 mm, transfer it to a 15 - ml centrifuge tube, resuspend it with the above - mentioned PBS, soak it on ice for 3 minutes, and then centrifuge at 100G for 3 minutes in the centrifuge. Repeat this step 3 times;

[0057] (4) Add 5 ml of digestive solution to resuspend the tissue precipitate, then transfer it to a 50 - ml centrifuge tube and digest it on a 37°C shaker for 30 min; after digestion is completed, add DMEM / F - 12 Medium containing Y - 27632 5 μM, Antibiotic - Antimycotic 1X, and Primocin 0.2% to 30 ml to terminate digestion;

[0058] (5) Filter the digested suspension with a 100-μm filter, centrifuge at 300G for 5 minutes, discard the supernatant, resuspend with 5 ml of the above DMEM / F-12 Medium, add 15 ml of red blood cell lysate, place at 4 °C for 10 minutes, and then centrifuge at 300G for 5 minutes; the remaining tissue after filtration is continuously digested in the digestive solution for 20 min, and the steps of digestion, filtration, centrifugation, and red blood cell lysis are repeated; collect the cell pellets from the two digestions, resuspend and wash them with PBS, centrifuge at 300G for 5 minutes, and collect the cell pellets and resuspend them with 3DP-CRC medium for counting.

[0059] 2. Preparation of bioink

[0060] After cell quantification, 4% sodium alginate (SA) and 15% gelatin (Gel) are rapidly added to the mixed cell suspension in a 15-ml centrifuge tube in sequence, and blown and mixed evenly according to a certain volume ratio, so that the final concentration of cells in the bioink is 5×10 6 cells / ml, and the final concentrations of Gel and SA are 5% and 1% respectively.

[0061] 3. 3D printing of colorectal cancer organoids

[0062] (1) Transfer the prepared bioink to a 3-ml screw-cap syringe with a pipette, assemble a 23G flat-tip dispensing needle, place at 4 °C for 20 minutes until the bioink gels and is ready for printing;

[0063] (2) Set the parameters of the 3D bioprinter in a sterile environment for pre-printing. Set the temperatures of the nozzle and the printer chamber platform to 15 °C and 10 °C respectively. The model is designed into a layer-by-layer grid shape by computer, with dimensions of 6 mm × 6 mm × 1.2 mm (layer height is 0.2 mm, line width is 0.8 mm), and the extrusion speed of the printing nozzle is set to 1.5 mm 3 / s; after the above bioink gels, place it in the 3D bioprinter, let it stand for 10 min for temperature equilibration, check and confirm that the extruded bioink is a uniform and smooth gel, and perform the printing process in a 48-well plate.

[0064] 3. Culture of colorectal cancer organoids

[0065] After printing, add 500 μl of sterile 3% calcium chloride solution to each well in the 48-well plate, soak the 3D printed body for 2 minutes for chemical cross-linking, then wash it with HBSS solution, add colorectal cancer organoid medium, and culture it in a 37 °C, 5% CO2 cell incubator; change the medium every 3 days.

[0066] From Figure 2It can be seen that after 6 days of culture, the 3D bioprinted colorectal cancer organoids contain key mature sphere structures. The single 3D-printed organoid was dissociated into individual small organoids, namely 3DP1, 3DP2, and 3DP3, by Figure 3 It can be seen that the cell diameter is 100 μm and the distribution is uniform. CCK8 was added to each printed body and incubated for 4 h, and the OD value was measured. The results are as Figure 4 shown. The OD values among different printed bodies are close, indicating that the quantity uniformity of different 3D-printed organoids is good.

[0067] By Figure 5 immunofluorescence results, it can be seen that the 3D-printed colorectal cancer organoids express parental tumor-specific proteins, such as CK20 and CDX-2.

[0068] Using the method of this example, colorectal cancer organoids were constructed by 3D printing of colorectal cancer tissues from different patients, and the key gene mutation maps of colorectal cancer organoids and parental tumors were drawn by exon sequencing. The results are as Figure 6 shown. It can be seen that the colorectal cancer organoids and parental tumor tissues have similar key mutation gene spectra, indicating that the 3D-printed colorectal cancer organoids highly retain genetic characteristics such as parental tumor heterogeneity.

[0069] Example 2

[0070] This example is basically the same as Example 1, except that the excised tumor tissue used is colorectal cancer liver metastasis tissue.

[0071] By Figure 7 it can be seen that the 3D-printed colorectal cancer liver metastasis organoids can reach a mature morphology in 6 days. By Figure 8 it can be seen that the 3D-printed colorectal cancer liver metastasis organoids also have parental-specific proteins CK20 and CDX-2. By Figure 9 it can be seen that the gene mutation types of 7 patients indicate that the in vitro models 3DP and PDO retain most of the specific gene mutations (gray) of the parental tumor.

[0072] Application Example 1

[0073] (1) Drug sensitivity testing was started on the 6th day of culturing the 3D-printed colorectal cancer organoids constructed in Example 1. Fresh medium containing different chemotherapy drugs (5-fluorouracil (5-Fu), irinotecan (CPT-11), oxaliplatin (Oxaliplatin)) at different concentrations (100 μM, 50 μM, 10 μM, 1 μM, 0 μM) was replaced, and then fresh medium containing different concentrations of different chemotherapy drugs was replaced every 24 h. After 72 h, a 3D cell viability analysis kit (Promega) was used to detect cell viability according to the method described in the instruction manual.

[0074] (2) Preparation of detection reagent: According to the number of printed bodies to be detected, mix DMEM / F-12 medium and the original detection reagent in the kit at a volume ratio of 1:1. Take out the original detection reagent in the kit in advance and thaw it at 4°C, then equilibrate it at room temperature for 20 minutes.

[0075] (3) Discard the drug-containing medium of the sample to be detected, add 300 μl of detection reagent to each well of the 48-well plate, and shake it at room temperature in a thermostatic microplate shaker for 20 minutes to fully lyse the printed body.

[0076] (4) Transfer the liquid in each well to a 1.5 ml EP tube, centrifuge at 12,000 rpm for 3 minutes to avoid interference from cell debris and residual bioink, then transfer the supernatant to a 96-well plate (90 μl per well), and use a multifunctional microplate reader to perform chemiluminescence detection of cell viability and draw a viability curve.

[0077] It can be seen from Figure 10 that colorectal cancer organoids from different patients have different responses to the same drug, and the responses of the same patient to different drugs also vary, indicating that the 3D printed colorectal cancer organoids constructed in the present invention have chemotherapy response heterogeneity, and this model can well predict the chemotherapy response of colorectal cancer patients.

[0078] Application Example 2

[0079] This application example is basically the same as Application Example 1, and the only difference is that the model is 3D bioprinted colorectal cancer liver metastasis organoids.

[0080] It can be seen from Figure 11 that 3D bioprinted colorectal cancer liver metastasis organoids from different patients have heterogeneous drug responses to the same drug, and the responses of the same patient to different drugs also vary, and are consistent with the actual drug responses of the patients before surgery, indicating that the model constructed in the present invention can well predict the chemotherapy response of colorectal cancer liver metastasis patients.

[0081] Comparative Example 1

[0082] This comparative example is basically the same as Example 1, and the only difference is that the final concentrations of gelatin and sodium alginate in the bioink are 3% / 1%, 7% / 1%, 5% / 0.5%, and 5% / 1.5% respectively.

[0083] It can be seen from Figure 13 that the configurations of 3D bioprinted organoids constructed under the conditions of 7% / 1% and 5% / 1.5% are stable, but not smooth enough, with slightly rough silk gels. The 3D bioprinted organoids constructed under the conditions of 3% / 1% and 5% / 0.5% are morphologically incomplete, unstable or non-uniform.

[0084] Comparative Example 2

[0085] This comparative example is basically the same as Example 1, and the only difference is the printing temperature. When printing, the nozzle temperature and the platform temperature are set to 21°C / 10°C, 14°C / 10°C, 15°C / 4°C, and 15°C / 11°C respectively.

[0086] It can be seen from Figure 14 that the 3D bioprinted organoids constructed under the above conditions are incomplete, unstable or non-uniform in morphology.

[0087] Comparative Example 3

[0088] This example is basically the same as Example 1, and the only difference is that LPS is not added to the colorectal cancer organoid culture medium.

[0089] It can be known from Figure 15 that the organoids in the 3D bioprinted colorectal cancer organoid model are smaller and fewer in number on the 6th day, and the maturity of the colorectal cancer organoids is much lower than that of the culture medium added with LPS.

[0090] Comparative Example 4

[0091] The difference between this comparative example and Example 1 lies in the adoption of the traditional organoid construction method. The specific steps are as follows: In the primary tumor cell precipitate before preparing the bioink, Matrigel is used for embedding instead. Each 50 μl contains 10,000 cells. Take 30 μl and drop it in the center of the well of a 24-well plate. Place the culture dish in a 37°C, 5% CO2 incubator for 15 min to solidify the gel solution; Add 500 μl of the culture medium of Example 1 to each well and culture it in a 37°C, 5% CO2 cell incubator. The constructed colorectal cancer organoids are named PDO.

[0092] Table 1

[0093]

[0094] It can be seen from Table 1, Figure 2 and 3 that the construction success rate, speed, and uniformity among different organoid models of the 3D bioprinted colorectal cancer organoid model of the present invention are higher than those of the colorectal cancer organoids constructed by the traditional Matrigel embedding method.

Claims

1. Colorectal cancer organoid culture medium, characterized in that, The specific components and contents are as follows: Advanced DMEM / F12 basal medium, B27 diluted 5 - 20 times, N2 diluted 2 - 10 times, GlutaMAX diluted 50 - 200 times, HEPES diluted 100 times, Human EGF 50 - 150 ng / ml, N-acetylcysteine 0.5 - 1.5 mM, Prostaglandin E2 5 - 20 ng / ml, A8301 200 - 1000 ng / ml, SB202190 1 - 5 μM, Gastrin I 1 - 20 nM, lipopolysaccharide 0.5 - 2 μg / ml, 1% (mass concentration) Antibiotic-Antimycotic, 0.2% (mass concentration) Primocin.

2. The colorectal cancer organoid culture medium according to claim 1, wherein The specific components and contents are as follows: Advanced DMEM / F12 basal medium, B27 diluted 20 times, N2 diluted 10 times, GlutaMAX diluted 100 times, HEPES diluted 100 times, Human EGF 100 ng / ml, N-acetylcysteine 1 mM, Prostaglandin E2 10 ng / ml, A8301 500 ng / ml, SB202190 1 μM, Gastrin I 10 nM, lipopolysaccharide 1 μg / ml, 1% (mass concentration) Antibiotic-Antimycotic, 0.2% (mass concentration) Primocin.

3. A method for 3D printing to construct colorectal cancer or liver metastasis organoids, characterized in that, It includes the following steps: (1) Isolation of colorectal cancer cells or colorectal cancer liver metastasis cells: Isolate colorectal cancer cells or colorectal cancer liver metastasis cells from ex vivo colorectal cancer tissues or colorectal cancer liver metastasis cancer tissues to obtain a cell suspension; (2) Preparation of bioink: Mix the cell suspension, sodium alginate solution and gelatin solution evenly to prepare a bioink with a final cell concentration of 1-5×10 6 cells / mL, a final mass concentration of sodium alginate of 1-1.5%, and a final mass concentration of gelatin of 5-7%. (3) 3D printing of colorectal cancer or colorectal cancer liver metastasis organoid tissues: Load the bioink into a syringe and assemble a needle, let it stand at 4°C for 10 - 30 min until the bioink extrudes in a continuous filament, load the pre-cooled bioink syringe into the 3D printing nozzle, set the nozzle temperature and the printer chamber platform temperature to 15 - 20°C and 5 - 10°C respectively, and perform 3D printing according to the preset model parameters to obtain colorectal cancer or colorectal cancer liver metastasis organoid tissues; (4) Cultivation of colorectal cancer or colorectal cancer liver metastasis organoids: Immerse the colorectal cancer or colorectal cancer liver metastasis organoid tissues in a 2% - 4% calcium chloride solution for chemical cross-linking. After the cross-linking is completed, wash with HBSS solution, add the colorectal cancer organoid culture medium described in Claim 1 or 2, and culture in an incubator at 37°C and 5% CO2 until mature to obtain colorectal cancer or colorectal cancer liver metastasis organoids.

4. The method according to claim 3, characterized in that, Step (1) is specifically as follows: The excised colorectal cancer tissue or colorectal cancer liver metastasis tissue is soaked and washed multiple times with PBS containing 5 μM of Y-27632, 2X of Antibiotic-Antimycotic, and 0.2% of Primocin, then minced, resuspended and centrifuged multiple times with PBS. The resuspended tissue is digested, filtered, centrifuged, the supernatant is removed, and it is resuspended with DMEM / F-12 medium and red blood cell lysate is added, then centrifuged. The steps of digestion, filtration, centrifugation, and red blood cell lysis are repeated, the cell pellet is collected, resuspended and washed with PBS, centrifuged, and finally resuspended with medium to obtain a cell suspension.

5. The method according to claim 3, characterized in that In step (2), in the bioink, the final cell concentration is 5×10 6 cells / mL, the final mass concentration of sodium alginate is 1.0%, and the final mass concentration of gelatin is 5%.

6. The method according to claim 3, wherein In step (3), the nozzle temperature and the printer chamber platform temperature are 15 °C and 10 °C respectively.

7. The method according to claim 3, wherein In step (4), the concentration of the calcium chloride solution is 3%, and the culture time to maturity is 5 - 7 days.

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