New 3D microtissue model based on tissue microenvironment reduction

Abstract

Provided in the present invention is a new 3D microtissue model based on tissue microenvironment reduction. Specifically, provided in the present invention is the use of a cytokine combination in the promotion of the growth of a tumor 3D micromicrotissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF and IGF-1. The combination of cytokines can promote the proliferation, differentiation and tissue formation of a tumor microtissue model to obtain more microtissue models with larger volumes, so that the growth microenvironment of a tumor can be reproduced in a more realistic manner, and a more comprehensive tool is provided for the subsequent research of disease mechanisms and the screening and development of drugs.

Description

A new 3D microtissue model based on tissue microenvironment restoration Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to the field of tumor microenvironment simulation technology, and in particular to a method for constructing a new 3D microtissue model based on the tumor microenvironment and its application in the research of colorectal cancer, bile duct cancer and lung adenocarcinoma. Background Art

[0002] The tumor microenvironment (TME) is a highly structured ecosystem composed of multiple factors, including tumor cells, surrounding stromal cells, immune cells, blood vessels, and cytokines. Interactions and signaling mechanisms within the microenvironment directly influence tumor cell proliferation, invasion, angiogenesis, and metastasis, playing a crucial role in tumor initiation and progression. Therefore, a deep understanding and simulation of the TME is crucial for cancer research, drug screening, and the development of personalized therapies.

[0003] Traditional 2D cell culture models limit the study of the tumor microenvironment because they cannot reproduce the complex structures and interactions found in three-dimensional organisms. In contrast, the development of 3D microtissue models provides new possibilities for more realistically simulating the tumor microenvironment. These microtissue models can provide cell-cell and cell-matrix interactions that are closer to in vivo conditions and better reflect the behavior of tumor cells in complex microenvironments. However, some current 3D microtissue models still have some problems, such as the lack of controllable microenvironment construction methods and the difficulty in accurately simulating multiple aspects of the tumor microenvironment, which limits their application in cancer research and treatment.

[0004] Therefore, it is necessary to develop a new 3D microtissue model that can more accurately simulate the tumor microenvironment in order to more deeply explore the mechanisms of tumor occurrence and development and provide more effective strategies for personalized treatment.

[0005] Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] A 3D tumor microtissue model is a laboratory model used to study and simulate the tumor tissue microenvironment. It is designed to better understand tumor development and treatment response, as well as to evaluate the effectiveness of new anticancer drugs and therapeutic strategies. 3D tumor microtissue models are typically cultured in a three-dimensional extracellular matrix hydrogel to mimic the structure and function of real tumor tissue. This model can provide conditions closer to the actual tumor growth environment and better reflect the complexity of tumors compared to traditional two-dimensional cell culture models. Here, the inventors selected factors associated with tumor growth and differentiation in the tumor microenvironment, based on factors that are highly abundant or highly expressed in the tumor microenvironment for different tumors. These factors or factor combinations were added to the 3D tumor microtissue models during the culture process. The inventors found that the presence of these factors or factor combinations resulted in a greater number of cultured 3D microtissue models, a larger volume, and a more realistic simulation of the in vivo tumor microenvironment, providing a more comprehensive tool for subsequent research on disease mechanisms, drug screening, evaluation, and development.

[0008] Therefore, the present invention proposes a highly controllable 3D microtissue model based on the reduction culture of the tissue microenvironment. By precisely combining relevant factors in the tumor microenvironment in vitro, it achieves accurate simulation of different tumor microenvironments. This model can more realistically reproduce the in vivo tumor microenvironment and is a valuable tool for studying tumor progression and metastasis, with broad potential applications in drug development and personalized medicine.

[0009] In a first aspect of the present invention, the present invention proposes the use of a cytokine combination in promoting the growth of a 3D tumor microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF and IGF-1.

[0010] It should be noted that the "IL-6" mentioned in the present invention is the immune cell-related cytokine interleukin-6; "Gremlin-1" is expressed by fibroblasts; "IGF-1" is a growth factor highly expressed in common tumor tissues, namely insulin growth factor; "FGF2" is fibroblast growth factor; and "HGF" is hepatocyte growth factor.

[0011] According to an embodiment of the present invention, the cytokine combination is selected from at least two of IL-6, Gremlin-1, HGF and IGF-1.

[0012] After extensive experiments, the inventors discovered that by adding a specific combination of cytokines during the culture process, they could significantly increase the volume and number of cultured 3D tumor microtissue models. This cytokine combination provides key signals and regulatory factors during the culture process, promoting cell proliferation, differentiation, and tissue formation, resulting in the formation of larger and richer 3D microtissue models. This provides an effective means for biomedical research and tissue engineering, enabling the generation of larger and more complex tissue structures in vitro, facilitating the study and treatment of a variety of diseases.

[0013] In the second aspect of the present invention, the present invention proposes the use of a cytokine combination in preparing a reagent for promoting the growth of a tumor 3D microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF or IGF-1.

[0014] As previously mentioned, the addition of a specific cytokine combination significantly increases the volume and number of cultured 3D tumor microtissue models. Therefore, the reagents described above have the same efficacy. Using these reagents to culture 3D microtissue models can increase the number and size of cultured 3D microtissue models, enabling a more realistic simulation of the tumor microenvironment and providing a more comprehensive tool for subsequent research on disease mechanisms and drug screening and development.

[0015] In the third aspect of the present invention, the present invention proposes the use of a cytokine combination in preparing a kit for culturing a tumor 3D microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF or IGF-1.

[0016] As previously mentioned, the addition of a specific cytokine combination can significantly increase the volume and number of cultured 3D tumor microtissue models. Therefore, the kit has the same efficacy. Using this kit to culture 3D microtissue models can increase the number and size of cultured 3D microtissue models, enabling a more realistic simulation of the tumor microenvironment, providing a more comprehensive tool for subsequent research on disease mechanisms and drug screening and development.

[0017] According to an embodiment of the present invention, the use described in the first aspect, the second aspect, or the third aspect further includes at least one of the following additional technical features:

[0018] According to an embodiment of the present invention, the tumor is selected from colorectal cancer, bile duct cancer or lung adenocarcinoma.

[0019] According to an embodiment of the present invention, the tumor is selected from colorectal cancer, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1, FGF2 and HGF.

[0020] According to an embodiment of the present invention, the tumor is selected from colorectal cancer, and the cytokine combination is selected from IL-6 and Gremlin-1. After extensive experiments and in-depth research on the characteristics of colorectal cancer tumors and the effects of cytokines, the inventors discovered that the combination of IL-6 and Gremlin-1 can interact with each other to synergistically promote cell proliferation, differentiation, and tissue formation, achieving optimal results when culturing 3D tumor microtissue models, resulting in a larger number and volume of cultured 3D microtissue models. This discovery helps further understand the developmental mechanisms of colorectal cancer and provides important experimental models and methods for related research and treatment.

[0021] According to an embodiment of the present invention, the tumor is selected from bile duct carcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1 and FGF2.

[0022] According to an embodiment of the present invention, the tumor is selected from cholangiocarcinoma, and the cytokine combination is selected from IGF-1 and Gremlin-1. After extensive experiments and in-depth research on the characteristics of cholangiocarcinoma tumors and the effects of cytokines, the inventors discovered that the combination of IGF-1 and Gremlin-1 can interact with each other to synergistically promote cell proliferation, differentiation, and tissue formation, achieving optimal results when culturing 3D tumor microtissue models, resulting in a larger number and volume of cultured 3D microtissue models. This discovery helps further understand the developmental mechanisms of cholangiocarcinoma and provides important experimental models and methods for related research and treatment.

[0023] According to an embodiment of the present invention, the tumor is selected from lung adenocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1 and HGF.

[0024] According to an embodiment of the present invention, the tumor is selected from lung adenocarcinoma, and the cytokine combination is selected from IL-6, Gremlin-1, and HGF. After extensive experiments and in-depth research into the characteristics of lung adenocarcinoma tumors and the effects of cytokines, the inventors discovered that the combination of IL-6, Gremlin-1, and HGF can interact with each other to synergistically promote cell proliferation, differentiation, and tissue formation, achieving optimal results when culturing 3D tumor microtissue models, resulting in a larger number and volume of cultured 3D microtissue models. This discovery helps further understand the developmental mechanisms of lung adenocarcinoma and provides an important experimental model and method for related research and treatment.

[0025] In a fourth aspect, the present invention provides a method for culturing a 3D tumor microtissue model. According to an embodiment of the present invention, the method comprises culturing tumor cells in a culture medium containing a cytokine combination to obtain the 3D tumor microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF, and IGF-1.

[0026] As mentioned above, the addition of a specific cytokine combination can significantly increase the volume and number of cultured tumor 3D microtissue models. Therefore, culturing the tumor cells in a culture medium containing a specific cytokine combination can significantly increase the number and volume of cultured tumor 3D microtissue models by promoting cell proliferation, differentiation and tissue formation processes. The addition of this specific cytokine combination provides key signals and regulatory factors during the culture process, which has a positive effect on the proliferation, differentiation and tissue formation of tumor cells, thereby leading to the formation of larger and richer tumor 3D microtissue models. The method provides an effective tool for tumor biology research and anti-tumor therapy, enabling researchers to better simulate and study the growth and development of tumors, and provide new ideas and strategies for the treatment of related diseases.

[0027] According to an embodiment of the present invention, the tumor cells are selected from colorectal cancer, bile duct cancer or lung adenocarcinoma.

[0028] According to an embodiment of the present invention, the tumor cells are provided in the form of cell clusters.

[0029] According to an embodiment of the present invention, the final concentration of each single factor in the cytokine combination in the culture medium is 10 to 250 ng / mL.

[0030] According to an embodiment of the present invention, the final concentration of each single factor in the cytokine combination in the culture medium is 30-100 ng / mL.

[0031] According to an embodiment of the present invention, the final concentration of each single factor in the cytokine combination in the culture medium is 40-60 ng / mL, for example, 40 ng / mL, 42 ng / mL, 44 ng / mL, 46 ng / mL, 48 ng / mL, 50 ng / mL, 52 ng / mL, 54 ng / mL, 56 ng / mL, 58 ng / mL, 60 ng / mL, etc.

[0032] According to an embodiment of the present invention, the final concentration of each single factor in the cytokine combination in the culture medium is 50 ng / mL. For example, the cytokine combination is selected from IL-6 and Gremlin-1, the final concentration of IL-6 in the culture medium is 50 ng / mL, and the final concentration of Gremlin-1 in the culture medium is also 50 ng / mL.

[0033] According to an embodiment of the present invention, the tumor cells are selected from colorectal cancer, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1, FGF2 and HGF.

[0034] According to an embodiment of the present invention, the tumor cells are selected from colorectal cancer, and the cytokine combination is selected from IL-6 and Gremlin-1. After extensive experiments and in-depth research on the characteristics of colorectal cancer tumors and the effects of cytokines, the inventors discovered that when culturing colorectal cancer cells, adding a combination of IL-6 and Gremlin-1 to the culture medium can promote cell proliferation, differentiation, and tissue formation, resulting in a larger number and volume of cultured 3D microtissue models of colorectal cancer. This discovery helps further understand the development mechanism of colorectal cancer and provides an important experimental model and method for related research and treatment.

[0035] According to an embodiment of the present invention, the tumor cells are selected from cholangiocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1 and FGF2.

[0036] According to an embodiment of the present invention, the tumor cells are selected from cholangiocarcinoma, and the cytokine combination is selected from IGF-1 and Gremlin-1. After extensive experiments and in-depth research on the characteristics of cholangiocarcinoma tumors and the effects of cytokines, the inventors discovered that adding a combination of IGF-1 and Gremlin-1 to the culture medium during culturing cholangiocarcinoma cells can promote cell proliferation, differentiation, and tissue formation, resulting in a greater number and larger volume of 3D microtissue models of cholangiocarcinoma. This discovery helps further understand the developmental mechanisms of cholangiocarcinoma and provides an important experimental model and method for related research and treatment.

[0037] According to an embodiment of the present invention, the tumor cells are selected from lung adenocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1 or HGF.

[0038] According to an embodiment of the present invention, the tumor cells are selected from lung adenocarcinoma, and the cytokine combination is selected from IL-6, Gremlin-1, and HGF. After extensive experiments and in-depth research into the characteristics of lung adenocarcinoma tumors and the effects of cytokines, the inventors discovered that adding a combination of IL-6, Gremlin-1, and HGF to the culture medium of lung adenocarcinoma cells can promote cell proliferation, differentiation, and tissue formation, resulting in a larger number and volume of 3D lung adenocarcinoma microtissue models. This discovery helps further understand the developmental mechanisms of lung adenocarcinoma and provides an important experimental model and method for related research and treatment.

[0039] According to an embodiment of the present invention, the culture medium includes a basal culture medium, sterile water and specific factors.

[0040] According to an embodiment of the present invention, the basal culture medium is selected from Advanced DMEM / F12.

[0041] According to an embodiment of the present invention, the specific factor is selected from at least one of A8301, B27, HEPES, L-glutamine, penicillin, and streptomycin. Adding the specific factor can promote cell survival and proliferation, stabilize the acid-base balance of the culture medium, maintain cell growth and function in a suitable environment, and prevent and inhibit bacterial contamination, thereby maintaining the purity and sterility of the cell culture.

[0042] According to an embodiment of the present invention, the mass volume ratio of the basic culture medium to sterile water is (96-103):1, for example, 96:1, 97:1, 98:1, 99:1, 100:1, 101:1, 102:1, 103:1, etc.

[0043] According to an embodiment of the present invention, the final concentration of A8301 in the culture medium is 100-1000 nM.

[0044] According to an embodiment of the present invention, the final concentration of A8301 in the culture medium is 450-550nM, for example, 450nM, 455nM, 460nM, 465nM, 470nM, 475nM, 480nM, 485nM, 490nM, 495nM, 500nM, 505nM, 510nM, 515nM, 520nM, 525nM, 530nM, 535nM, 540nM, 545nM, 550nM, etc.

[0045] According to an embodiment of the present invention, the final concentration of B27 in the culture medium is 0.5× to 1.5×, for example, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, etc.

[0046] According to an embodiment of the present invention, the final concentration of HEPES in the culture medium is 5-15 mM, for example, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, etc.

[0047] According to an embodiment of the present invention, the final concentration of L-glutamine in the culture medium is 1-3 mM, for example, 1 mM, 2 mM, 3 mM, etc.

[0048] According to an embodiment of the present invention, the final concentration of penicillin in the culture medium is 50-200 U / mL.

[0049] According to an embodiment of the present invention, the final concentration of penicillin in the culture medium is 70-130 U / mL, for example, 70 U / mL, 75 U / mL, 80 U / mL, 85 U / mL, 90 U / mL, 95 U / mL, 100 U / mL, 105 U / mL, 110 U / mL, 115 U / mL, 120 U / mL, 125 U / mL, 130 U / mL, etc.

[0050] According to an embodiment of the present invention, the final concentration of streptomycin in the culture medium is 50-200 μg / mL.

[0051] According to an embodiment of the present invention, the final concentration of streptomycin in the culture medium is 70-130 μg / mL, for example, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, 120 μg / mL, 125 μg / mL, 130 μg / mL, etc. Beneficial effects

[0052] The method of culturing a 3D tumor microtissue model of the present invention has the following beneficial effects:

[0053] ① Improved biological relevance: By utilizing endogenous factors and real nutritional components, the 3D microtissue model of the present invention provides excellent biological relevance, producing more reliable and clinically meaningful results.

[0054] ② Enhanced drug screening: Using a physiologically accurate tumor microenvironment can better enable drug screening, thereby identifying more effective and targeted treatments.

[0055] ③ Reduce animal testing: The invention of the 3D microtissue model provides a valuable alternative to animal testing, contributing to more ethical and cost-effective research practices.

[0056] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0058] FIG1 is an optical microscope diagram of a single-factor test of a colorectal cancer microtissue model according to an embodiment of the present invention; CRC stands for colorectal cancer, and #3 and #4 refer to the numbers of human tissue samples;

[0059] FIG2 is a diagram of a single-factor cell viability analysis of a colorectal cancer microtissue model according to an embodiment of the present invention; wherein CRC stands for colorectal cancer, and 3 and 4 refer to the numbers of human tissue samples;

[0060] FIG3 is an optical microscopic image of a dual-factor combined colorectal cancer microtissue model according to an embodiment of the present invention; wherein CRC stands for colorectal cancer, #6, #7, and #8 refer to the numbers of human tissue samples, and P1-day7 refers to the first passage after primary construction, and the photograph taken on the seventh day;

[0061] FIG4 is an optical microscope image and a cell viability analysis image of a single factor test of a bile duct cancer microtissue model according to an embodiment of the present invention;

[0062] FIG5 is an optical microscopic image of a two-factor combined cholangiocarcinoma microtissue model according to an embodiment of the present invention; CHOL stands for cholangiocarcinoma, #1, #2, and #3 refer to the numbers of the human tissue samples, and P1-day9 refers to the first passage after primary establishment, taken on the ninth day;

[0063] 6 is an optical microscope image and cell viability analysis image of a single factor test of a lung adenocarcinoma microtissue model according to an embodiment of the present invention;

[0064] Figure 7 is an optical microscopic image of a multifactorial test of a lung adenocarcinoma microtissue model according to an embodiment of the present invention; LUAD represents lung adenocarcinoma, #35, #37, and #39 refer to the numbers of human tissue samples, and P1-day6 refers to the first passage after primary construction, and the photograph was taken on the sixth day. DETAILED DESCRIPTION

[0065] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0066] The reagents involved in this embodiment are shown in Table 1:

[0067] Table 1

[0068] Complete medium preparation: Prepare a mixture of Advanced DMEM and sterile water at a mass ratio of 99:1, and add HEPES to a final concentration of 10 mM, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 500 nM A8301, and 1× B27. The concentrations of each component of the above-mentioned supplements are based on their concentrations in the mixture of basal medium and sterile water.

[0069] Example 1 Construction of a 3D microtissue model of colorectal cancer

[0070] In this example, the construction and culture of the colorectal cancer 3D microtissue model were performed according to the following experimental steps:

[0071] 1) Discarded or purchased fresh colorectal cancer surgical resection tissue samples were placed in the prepared Advanced DMEM / F12 culture medium and sent to the laboratory for pretreatment within 12 hours.

[0072] 2) Sample Washing: Transfer the tissue sample to a 15 mL centrifuge tube and wash with 5 mL of Advanced DMEM / F12 medium for 30 seconds. Discard the supernatant and add 5 mL of Advanced DMEM / F12 medium for another wash. Repeat this washing method three times to remove impurities on the tissue sample surface.

[0073] 3) Sample shearing: In a biosafety cabinet, transfer the washed tissue sample to a 6 cm culture dish and cut the tissue into pieces of 1-3 mm using sterilized surgical scissors on ice. 3 The shearing process should not exceed 10 min to avoid cell damage.

[0074] 4) Tissue Digestion: Transfer the minced tissue sample to a 15 mL centrifuge tube, add 10 mL of tissue digestion buffer (purchased from Bozhen Biotechnology (Suzhou) Co., Ltd.), and incubate at 37°C with shaking for 60 min. After digestion, add FBS to a final concentration of 2% to the tissue digestion buffer to terminate digestion. Centrifuge at 300 g for 3 min, and retain the pellet and discard the supernatant.

[0075] 5) Cell filtration: Filter the precipitate from step 4) through a 100 μm filter to remove large undigested tissue fragments. Centrifuge the filtered cell solution at 300 g for 3 minutes and carefully remove the supernatant to obtain the cell pellet.

[0076] 6) Red blood cell lysis: Add 1 mL of red blood cell lysis buffer (purchased from Bozhen Biotechnology (Suzhou) Co., Ltd.) to the cell pellet from step 5), gently pipette to resuspend the cell pellet, and lyse at room temperature for 2-3 minutes.

[0077] 7) Cell collection: Centrifuge the liquid from step 6) at 300 g for 3 min, carefully remove the supernatant and obtain a cell pellet for later use.

[0078] 8) Take an appropriate amount of extracellular matrix hydrogel to resuspend the cell pellet, and then use a pipette to drop the extracellular matrix hydrogel mixed with cells into a 24-well plate, with approximately 25 μL per well.

[0079] 9) Place the 24-well plate inoculated with the extracellular matrix hydrogel droplets in a CO2 incubator and let it stand for 15-20 minutes to allow it to fully solidify.

[0080] 10) From the microenvironment factor library, five factors related to the colorectal cancer microenvironment were selected: IL-6, Gremlin-1, IGF-1, FGF2, and HGF. After adding each factor to a final concentration of 50 ng / mL in complete culture medium, the cells were added to the 24-well plate containing the cell clusters in step 9) for culture, and single-factor testing experiments were performed. The experimental results are shown in Figures 1 and 2, showing that single factors IL-6 and Gremlin-1 significantly promoted the growth of tumor cells.

[0081] Next, after adding single factors and dual factors with a final concentration of 50 ng / mL to the complete culture medium (the final concentration of each single factor in the dual factor was also 50 ng / mL), single factor and dual factor combination tests were performed on tumor tissue samples from three colorectal cancer patients. It was found that only the dual factor combination of IL-6 + Gremlin-1 could increase the number of cells and the size of the cells in the colorectal cancer 3D microtissue model. The morphological structure of the microtissue model observed under an inverted optical microscope is shown in Figure 3. The cell number and volume of the colorectal cancer 3D microtissue model obtained by culturing IL-6 and Gremlin-1 with the dual factor combination of other cytokines were much lower than those in the IL-6 + Gremlin-1 group.

[0082] Example 2 Construction of a 3D microtissue model of cholangiocarcinoma

[0083] In this example, the construction and culture of the cholangiocarcinoma 3D microtissue model were performed according to the following experimental steps:

[0084] 1) Discarded or purchased fresh bile duct cancer surgical resection tissue samples were placed in the prepared Advanced DMEM / F12 culture medium for storage.

[0085] 2) Assess whether the tissue sample obtained is composed of pure epithelium. If fat or muscle tissue is present, remove as much of this non-epithelial component as possible using surgical scissors or a scalpel and forceps under a dissecting microscope.

[0086] 3) Use surgical scissors to place the tissue sample into a cell culture dish to make a 1-3 mm 3 Incubate the fragments with tissue digestion solution at 37°C for 30 minutes to 1 hour, and monitor the digestion process.

[0087] 4) After digestion, add FBS to a final concentration of 2% to the tissue digestion solution to terminate digestion. Filter through a 100 μm filter and collect the filtered cells (check the pellet to see if red blood cell lysis is necessary). Centrifuge at 300 g for 3 minutes and carefully remove the supernatant to obtain a cell pellet for later use.

[0088] 5) Take an appropriate amount of extracellular matrix hydrogel to resuspend the cell pellet, and then use a pipette to drop the extracellular matrix hydrogel mixed with cells into a 24-well plate, with approximately 25 μL per well.

[0089] 6) Place the 24-well plate seeded with the extracellular matrix hydrogel droplets in a CO2 incubator and let it stand for 15-20 minutes to allow it to fully solidify.

[0090] From the microenvironmental factor library, four factors related to the cholangiocarcinoma microenvironment were selected: IL-6, Gremlin-1, IGF-1, and FGF2. After adding a single factor at a final concentration of 50 ng / mL to the complete culture medium, the cells were added to the 24-well plate containing the cell clusters in step 6) for culture, and a single factor test experiment was performed. The experimental results are shown in Figure 4, and it was found that the single factors Gremlin-1 and IGF-1 had a significant promoting effect on the growth of tumor cells.

[0091] Next, after adding single factors and dual factors with a final concentration of 50 ng / mL to the complete culture medium (the final concentration of each single factor in the dual factor was also 50 ng / mL), single factor and dual factor combination tests were performed on tumor tissue samples from three cholangiocarcinoma patients. It was found that the dual factor combination of Gremlin1+IGF-1 could make the cholangiocarcinoma 3D microtissue model larger in volume and have more cells. The morphological structure of the microtissue model observed under an inverted optical microscope is shown in Figure 5. The cell number and volume of the cholangiocarcinoma 3D microtissue model obtained by culturing Gremlin-1 and IGF-1 with other cytokines were much lower than those in the Gremlin-1+IGF-1 group.

[0092] Example 3 Construction of a 3D microtissue model of lung adenocarcinoma

[0093] In this example, the construction and culture of the lung adenocarcinoma 3D microtissue model were performed according to the following experimental steps:

[0094] 1) Discarded or purchased fresh lung adenocarcinoma surgical resection tissue samples were placed in the prepared Advanced DMEM medium for storage.

[0095] 2) Use surgical scissors to place the tissue sample into a cell culture dish and make a 1-3 mm 3 Incubate the fragments with tissue digestion solution at 37°C for 30 minutes to 1 hour, and monitor the digestion process.

[0096] 3) After digestion, add FBS to a final concentration of 2% to the tissue digestion solution to terminate digestion, filter through a 100 μm filter, collect the filtered cells, and centrifuge at 300 g for 3 minutes. Carefully remove the supernatant to obtain a cell pellet for later use.

[0097] 4) Take an appropriate amount of extracellular matrix hydrogel to resuspend the cell pellet, and then use a pipette to drop the extracellular matrix hydrogel mixed with cells into a 24-well plate, with approximately 25 μL per well.

[0098] 5) Place the culture plate inoculated with the extracellular matrix hydrogel droplets in a CO2 incubator and let it stand for 15-20 minutes to allow it to fully solidify.

[0099] 6) From the microenvironmental factor library, three factors related to the lung adenocarcinoma microenvironment were selected: IL-6, Gremlin-1, and HGF. Each factor was added to complete culture medium at a final concentration of 50 ng / mL and then added to the culture plate containing the cell clusters from step 5) for culture. Single-factor testing was also performed. The experimental results, shown in Figure 6 , showed that IL-6, Gremlin-1, HGF, and IL-6 + Gremlin-1 all promoted the growth of lung adenocarcinoma, with IL-6 + Gremlin-1 and HGF having the most significant effects.

[0100] Next, after adding a single factor, a dual factor (the final concentration of each single factor in the dual factor was also 50 ng / mL), and a triple factor (the final concentration of each single factor in the triple factor was also 50 ng / mL) with a final concentration of 50 ng / mL to the complete culture medium, tumor tissue samples from three patients with lung adenocarcinoma were tested for single factor, dual factor combination, and triple factor combination. It was found that compared with the single factor and dual factor combination, the triple factor combination of IL-6+Gremlin-1+HGF could increase the number of cells and the size of the lung adenocarcinoma 3D microtissue model. The morphological structure of the microtissue model observed under an inverted optical microscope is shown in Figure 7.

[0101] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0102] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of a cytokine combination in promoting the growth of a tumor 3D microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF, and IGF-1.

2. Use of a cytokine combination in the preparation of a reagent for promoting the growth of a tumor 3D microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF, or IGF-1.

3. Use of a cytokine combination in the preparation of a kit for culturing a tumor 3D microtissue model, wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF, or IGF-1.

4. The use according to any one of claims 1 to 3, characterized in that The tumor is selected from colorectal cancer, cholangiocarcinoma, or lung adenocarcinoma.

5. The use according to claim 4, wherein The tumor is selected from colorectal cancer, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1, FGF2, and HGF; preferably, the cytokine combination is selected from IL-6 and Gremlin-1; Optionally, the tumor is selected from cholangiocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1, and FGF2; preferably, the cytokine combination is selected from IGF-1 and Gremlin-1; Optionally, the tumor is selected from lung adenocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, and HGF; preferably, the cytokine combination is selected from IL-6, Gremlin-1, and HGF.

6. A method for culturing a 3D tumor microtissue model, characterized in that, Comprising: Culturing tumor cells in a culture medium containing a cytokine combination to obtain the tumor 3D microtissue model; wherein the cytokine combination is selected from at least two of IL-6, Gremlin-1, FGF2, HGF, and IGF-1.

7. The method according to claim 6, wherein The tumor cells are selected from colorectal cancer, cholangiocarcinoma, or lung adenocarcinoma; Optionally, the tumor cells are provided in the form of cell clusters; Optionally, the final concentration of each single factor in the cytokine combination in the culture medium is 10 - 250 ng / mL.

8. The method according to claim 7, wherein The tumor cells are selected from colorectal cancer, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1, FGF2, and HGF; preferably, the cytokine combination is selected from IL-6 and Gremlin-1; Optionally, the tumor cells are selected from cholangiocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, IGF-1, and FGF2; preferably, the cytokine combination is selected from IGF-1 and Gremlin-1; Optionally, the tumor cells are selected from lung adenocarcinoma, and the cytokine combination is selected from at least two of IL-6, Gremlin-1, or HGF; preferably, the cytokine combination is selected from IL-6, Gremlin-1, and HGF.

9. The method according to claim 6, wherein The culture medium comprises a basal medium, sterile water, and a specific factor.

10. The method according to claim 9, characterized in that, The basal medium is selected from Advanced DMEM / F12; Optionally, the specific factor is selected from at least one of A8301, B27, HEPES, L-glutamine, penicillin, and streptomycin; Optionally, the mass-volume ratio of the basal medium to sterile water is (96-103):1; Optionally, the final concentration of A8301 in the medium is 100-1000 nM; Optionally, the final concentration of B27 in the medium is 0.5×-1.5×; Optionally, the final concentration of HEPES in the medium is 5-15 mM; Optionally, the final concentration of L-glutamine in the medium is 1-3 mM; Optionally, the final concentration of penicillin in the medium is 50-200 U / mL; Optionally, the final concentration of streptomycin in the medium is 50-200 μg / mL.

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