Chip co-culture method, high-content imaging analysis method based on chip co-culture method and application of chip co-culture method in anti-tumor drug screening

Abstract

The invention particularly relates to a chip co-culture method, a high-content imaging analysis method based on the chip co-culture method and application of the chip co-culture method in anti-tumor drug screening. The tumor-related fibroblasts and the tumor organoid cells are respectively subjected to resuspension counting with a PBS buffer solution, the two kinds of cells are fully and uniformly mixed according to the quantity ratio of 1: (2-3) to form co-culture cell suspension, and the co-culture cell suspension is inoculated into a chip according to the volume dose of 1.5-2 mL per row; placing the culture chip in a 5% CO2 incubator at 37 DEG C for first culture, after incubation for 20-30 minutes, slightly and completely sucking PBS in the chip by using a pipette, adding 1.5-2 mL of an organoid culture medium into each hole of the chip, and placing the chip in the 5% CO2 incubator at 37 DEG C for second culture to obtain the co-culture cell system. Rapid and high-quality detection of a large amount of samples can be realized, and an important technical support is provided for deeply understanding a tumor microenvironment and developing a new treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug screening, and specifically relates to a chip co-culture method and a high-content imaging analysis method based on the method, and its application in anti-tumor drug screening. Background Art

[0002] In oncology research, the interaction between cancer-associated fibroblasts (CAFs) and tumor cells plays a crucial role in tumor development and metastasis. While traditional two-dimensional (2D) cell culture methods can provide a basic cell growth environment, they often fail to simulate the complex interactions between cells and microenvironmental conditions. This makes it impossible to observe the interaction between clinical drugs and tumor cells in the tumor microenvironment, and thus, it is impossible to efficiently and accurately analyze the anti-tumor effects of drugs, limiting the effective screening of anti-tumor clinical drugs. Summary of the Invention

[0003] In response to the above technical problems, the present invention proposes a chip co-culture method, which specifically comprises the following steps:

[0004] S1 obtains tumor organoid cells through three-dimensional organoid culture;

[0005] S2: resuspending the tumor-associated fibroblasts and the tumor organoid cells in PBS buffer and counting them respectively, mixing the two cells at a ratio of 1:2-3 to form a co-culture cell suspension, and inoculating the co-culture cell suspension into the chip at a volume of 1.5-2 mL per row;

[0006] S3: Place the chip in a 37°C, 5% CO2 incubator for the first culture. After incubation for 20-30 minutes, gently use a pipette to completely remove the PBS solution, add 1.5-2 mL of organoid culture medium to each well of the chip, and place it in a 37°C, 5% CO2 incubator for the second culture to obtain a co-culture cell system.

[0007] Furthermore, the step of obtaining the tumor organoid cells through three-dimensional organoid culture specifically includes:

[0008] Take the tumor cell suspension, add 5-20% Matrigel to it, mix thoroughly and inoculate into a low-absorption 24-well culture plate, inoculating 200-300 μl per well. Note that the culture medium must be pre-chilled in advance.

[0009] The inoculated 24-well low-absorption culture plate was placed in a 37°C, 5% CO2 incubator for incubation for 20-30 minutes, 200-300 μl of organoid culture medium was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for organoid culture to obtain tumor organoids;

[0010] The tumor organoids are enzymatically digested into tumor organoid cells.

[0011] Furthermore, the S3 tumor-associated fibroblasts are prepared by the following steps:

[0012] Take fresh tissue samples, wash them with PBS buffer, add type IV collagenase tissue dissociation solution with a concentration of 0.1-5 mg / ml, and mince the tissue samples to obtain uniform tissue fragments;

[0013] Place the tissue fragments in a 5-10 times volume of type IV collagenase tissue digestion solution with a concentration of 0.1-5 mg / ml and incubate at 37°C for 40-60 minutes for digestion;

[0014] After digestion, the cells are released from the matrix of the tissue fragments and filtered through a 70 μm mesh to remove undigested tissue clusters to obtain a cell suspension containing single cells or cell clusters.

[0015] Dilute the digestive enzyme with 2 times the volume of DMEM medium of the cell suspension and continue digestion, then centrifuge at 200-300g for 3-5 minutes to collect the cell pellet;

[0016] The cell pellet collected by centrifugation was treated with red blood cell lysis, and then added with liver cancer organoid culture medium containing 5-10% serum, inoculated into a cell culture plate, and placed in a 37°C, 5% CO2 incubator for two-dimensional adherent culture.

[0017] In a second aspect, the present invention proposes a high-content imaging analysis method, which uses a high-content imaging system to evaluate the effects of drugs on tumor cells in a co-culture cell system obtained by a chip co-culture method.

[0018] In a third aspect, the present invention also proposes the application of the above-mentioned high-content imaging analysis method in anti-tumor drug screening.

[0019] In a fourth aspect, the present invention also proposes the application of the above-mentioned high-content imaging analysis method in clinical drug screening for the treatment of liver cancer.

[0020] The beneficial technical effects of the present invention are embodied in the following aspects:

[0021] First, the chip-based co-culture method can ensure the homogeneity of the co-culture samples, which is crucial for the accuracy and reproducibility of experimental results;

[0022] Second, the analysis of co-cultured cells using a high-content imaging system improved the quality of experimental data and analysis efficiency;

[0023] Third, drug screening that is directly applied to clinical drug sensitivity improves the accuracy of drug screening and the success rate of clinical application, and can better simulate in vivo conditions, thereby improving the clinical relevance of drug screening.

[0024] In summary, the technical solution of the present invention provides a more precise and efficient experimental platform compared to traditional culture methods. Through the application of high-content imaging systems, rapid and high-quality detection of large sample volumes can be achieved, providing important technical support for a deeper understanding of the tumor microenvironment and the development of new treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : A flow chart of an embodiment of a chip co-culture method;

[0026] Figure 2 : A flow chart of an embodiment of a chip co-culture method implementation method. DETAILED DESCRIPTION

[0027] First of all, it should be noted that the following terms in the technical field related to the present invention should be understood as follows:

[0028] Co-culture involves culturing two or more different cell types simultaneously within the same culture system to simulate the in vivo microenvironment. This allows for the study of the biological processes of cell-cell interactions, providing a more realistic model of cell interaction and contributing to a deeper understanding of key biological characteristics such as intercellular communication, signal transduction, cell growth, and differentiation. 3D culture, in particular, provides cells with a three-dimensional environment, better simulating the cellular structure and function found in tissues, enabling the study of the growth and interactions of different cells.

[0029] The High-Content Imaging (HCI) system comprises three components: fully automated high-speed microscopy, image analysis, and data management. It can generate a large number of images and extract extensive data from them in a short period of time. Multiple fluorescent probes can be used simultaneously in a single experiment. Simultaneous analysis of large numbers of cells across multiple parameters and types enables a deeper understanding of complex cellular mechanisms and interactions.

[0030] DMEM is a medium containing various amino acids and glucose, including formulations with higher glucose concentrations and with or without sodium pyruvate.

[0031] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are within the scope of the present invention.

[0032] The chip co-culture method of some embodiments includes: Figure 1The steps specifically include the following steps:

[0033] S1 obtains tumor organoid cells through three-dimensional organoid culture;

[0034] S2: resuspending the tumor-associated fibroblasts and the tumor organoid cells in PBS buffer and counting them respectively, mixing the two cells at a ratio of 1:2-3 to form a co-culture cell suspension, and inoculating the co-culture cell suspension into the chip at a volume of 1.5-2 mL per row;

[0035] S3: Place the chip in a 37°C, 5% CO2 incubator for the first culture. After incubation for 20-30 minutes, gently use a pipette to completely remove the PBS solution, add 1.5-2 mL of organoid culture medium to each well of the chip, and place it in a 37°C, 5% CO2 incubator for the second culture to obtain a co-culture cell system.

[0036] It should be noted that the "chip" in the above embodiments includes a cell culture microarray, such as the one disclosed in the prior art provided by Black Jade Star Rock International Science and Technology (Beijing) Co., Ltd., 2015106642904, a method for preparing a microarray chip and its products, and products with equivalent technical effects.

[0037] In some more specific embodiments of the above embodiments, the step of obtaining the tumor organoid cells through three-dimensional organoid culture specifically includes:

[0038] Take the tumor cell suspension, add 5-20% Matrigel to it, mix thoroughly and inoculate into a low-absorption 24-well culture plate, inoculating 200-300 μl per well. The culture medium should be pre-chilled in advance.

[0039] The inoculated 24-well low-absorption culture plate was placed in a 37°C, 5% CO2 incubator for incubation for 20-30 minutes, 200-300 μl of organoid culture medium was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for organoid culture to obtain tumor organoids;

[0040] The tumor organoids are enzymatically digested into tumor organoid cells.

[0041] It should be noted that the “matrigel” may specifically be Matrigel produced by Corning Incorporated in the United States.

[0042] In the above protocol, more specifically, the S3 tumor-associated fibroblasts are prepared by the following steps:

[0043] Take fresh tissue samples, wash them with PBS buffer, add type IV collagenase tissue dissociation solution with a concentration of 0.1-5 mg / ml, and mince the tissue samples to obtain uniform tissue fragments;

[0044] Place the tissue fragments in a 5-10 times volume of type IV collagenase tissue digestion solution with a concentration of 0.1-5 mg / ml and incubate at 37°C for 40-60 minutes for digestion;

[0045] After digestion, cells were released from the matrix of tissue fragments and filtered through a 70 μm mesh to remove undigested tissue clusters to obtain a cell suspension containing single cells or cell clusters. The cell suspension was diluted with 2 times the volume of DMEM medium (Wuhan Saiweier Biotechnology Co., Ltd., G4511-500ML)

[0046] ) After continued digestion with diluted digestive enzyme, the cell pellet was collected by centrifugation at 200-300 g for 3-5 min;

[0047] The cell pellet collected by centrifugation was treated with red blood cell lysis, and then added with liver cancer organoid culture medium containing 5-10% serum, inoculated into a cell culture plate, and placed in a 37°C, 5% CO2 incubator for two-dimensional adherent culture.

[0048] The co-culture system organoid culture medium used in the above examples contains 2% matrigel and 4% FBS buffer.

[0049] In the above embodiment, the drug sensitivity results of the co-culture system are subjected to a cell live and dead fluorescence labeling step, so that the drug sensitivity results are presented by fluorescence intensity, including Calcein AM labeling live cells to emit green fluorescence and PI labeling dead cells to emit red fluorescence.

[0050] Example 1 Application of high-content imaging analysis based on chip co-culture method in drug screening for the treatment of liver cancer

[0051] like Figure 2 As shown, this embodiment is achieved through the following steps: I) Two-dimensional culture of liver cancer-related tumor-associated fibroblasts: single cells are obtained by separating tumor tissue cells and directly inoculated into a six-well plate to obtain tumor-associated fibroblasts. II) Three-dimensional culture of liver cancer organoids: liver cancer enzymatically hydrolyzed single cells are combined with matrix gel for three-dimensional culture to obtain organoids. III) Chip co-culture: cancer-related tumor-associated fibroblasts and liver cancer organoids are inoculated on a microarray cell culture chip in a certain proportion to obtain liver cancer organoids containing a fibroblast microenvironment. IV) Improvement of clinical drug sensitivity screening: based on the above equipment principles, a uniform microenvironment is obtained to improve the accuracy and importance of clinical drug sensitivity screening.

[0052] Step I: Two-dimensional culture of liver cancer-related tumor-associated fibroblasts. Liver cancer tissue is digested to obtain a single cell suspension, and the cells are inoculated on a two-dimensional surface for culture to obtain tumor-associated fibroblasts.

[0053] The cancer-associated tumor-associated fibroblasts described above are obtained as follows: Fresh liver tissue or liver cancer tissue is washed with PBS to remove blood. A small amount of type IV collagenase at a concentration of 0.1-5 mg / ml is added, with the volume of the solution sufficient to completely cover the tissue sample. The tissue sample is then minced to obtain uniform fragments. The tissue fragments are placed in type IV collagenase at a concentration of 0.1-5 mg / ml, at a volume 5-10 times that of the tissue sample, and incubated at 37°C for 40-60 minutes. After digestion, the cells are gently pipetted several times with a pipette or pipette tip to release the cells from the loosely encapsulated matrix. Undigested tissue clumps are filtered through a 70 μm mesh to obtain a cell suspension of single cells or a few cell clumps. The digestive enzyme is diluted with 2 times the volume of DMEM medium as the cell suspension to ease further digestion. The cell pellet is collected by centrifugation at 200-300 g for 3-5 minutes. The cell pellet collected by centrifugation was treated with red blood cell lysis, and then added with liver cancer organoid culture medium containing 5-10% serum, inoculated into a cell culture plate, and placed in a 37°C, 5% CO2 incubator for two-dimensional adherent culture.

[0054] Step II: Liver cancer three-dimensional organoid culture: Liver cancer tissue is digested to obtain a single cell suspension, and the cells are combined with matrix gel for three-dimensional culture to obtain organoids.

[0055] Proceed as follows: Add 5-20% Matrigel to the prepared liver cancer cell suspension, mix thoroughly, and seed 200-300 μL of the medium into each well of a low-absorption 24-well culture plate. Perform the entire process on ice. Incubate the plate in a 37°C, 5% CO2 incubator for 20-30 minutes. Then, add 200-300 μL of organoid culture medium to each well and incubate the plate in a 37°C, 5% CO2 incubator for organoid culture.

[0056] Step III: Co-culture of liver cancer and fibroblasts on the chip: Digest tumor-associated fibroblasts and liver cancer organoids to obtain single-cell suspensions, which are then inoculated on the chip at a certain ratio to achieve co-culture of fibroblasts and liver cancer organoids.

[0057] The following steps were performed: Centrifuge the tumor-associated fibroblast and liver cancer organoid cell suspensions prepared above, discard the supernatant, and resuspend each in 1 mL of PBS for counting. The ratio of tumor-associated fibroblasts to liver cancer organoids was 1:2-3. Resuspend the fibroblasts and liver cancer cells in PBS according to a specific ratio, mix thoroughly, and then inoculate them onto the chip, using a seeding volume of 1.5-2 mL per row at a constant speed. Incubate the culture chip in a 37°C, 5% CO2 incubator for 20-30 minutes. After that, completely aspirate the PBS from the chip and add 2% Matrigel and 4% FBS organoid culture medium. Add 1.5-2 mL of organoid culture medium to each well and incubate at 37°C, 5% CO2.

[0058] Step IV: Clinical drug sensitivity screening: Tumor cells and tumor-associated fibroblasts are cultured in a cell culture microarray and clinical drugs are added for testing.

[0059] Example 2 Application of a high-content imaging analysis method based on a chip co-culture method in drug screening for the treatment of breast cancer

[0060] The difference from Example 1 is that the tumor tissue is triple-negative breast cancer tissue.

[0061] Test Example 1 Preclinical efficacy study of anti-liver cancer drugs

[0062] At least two liver cancer cell lines derived from mouse liver cancer tissue and human liver cancer tissue are taken, and before the co-culture step as in Example 1, the liver cancer cells are fluorescently labeled.

[0063] Through high-content imaging equipment analysis technology and multi-well plate scanning (such as 96 / 384-well plates), thousands of single-cell images are obtained to observe the effects of drugs on liver cancer cells, including changes in growth, apoptosis, cell morphology, etc.

[0064] Specifically, F-actin was labeled by phalloidin staining, and cell morphology, pseudopodia number and length were analyzed.

[0065] The integrity of the microtubule network can be assessed by fluorescent labeling with tubulin antibodies (e.g., β-tubulin).

[0066] The invasiveness and spatial morphological changes of tumor cells in the test cases and the negative and positive control cases after drug administration were analyzed.

[0067] Use software (such as MetaXpress, Harmony, CellProfiler) to extract parameters (fluorescence intensity, morphological indicators, colocalization, etc.) to evaluate the efficacy and toxicity of drugs on liver cancer cells.

[0068] Record changes in tumor cells under drug action, such as apoptosis / necrosis / survival cells, count changes at the single cell or population level, and perform data processing and analysis to compare the inhibitory effects of different drugs on liver cancer cells to determine the best drug candidate or combination treatment plan.

[0069] Table 1. Results of 4 drug screening tests based on liver cancer cell source 1 (after administration)

[0070]

[0071] Table 2. Results of drug screening test based on liver cancer cell source 2 (after administration)

[0072]

[0073] Based on the above experiments, it can be concluded that among the above four test drugs, the relative anti-tumor effect of Test Example 2 is outstanding in that it has a good tumor growth inhibition rate and can be combined with pembrolizumab and lenvatinib in clinical practice for the synergistic treatment of liver cancer.

[0074] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A chip co-culture method, characterized in that: The method specifically comprises the following steps: S1 obtains tumor organoid cells through three-dimensional organoid culture; S2: resuspending the tumor-associated fibroblasts and the tumor organoid cells in PBS buffer and counting them respectively, mixing the two cells at a ratio of 1:2-3 to form a co-culture cell suspension, and inoculating the co-culture cell suspension into the chip at a volume of 1.5-2 mL per row; S3: Place the chip in a 37°C, 5% CO2 incubator for the first culture. After the chip is incubated for 20-30 minutes, observe the sedimentation of the cells in the chip under a microscope, gently remove the PBS solution with a pipette, add 1.5-2 mL of organoid culture medium to each well of the chip, and place it in a 37°C, 5% CO2 incubator for the second culture to obtain a co-culture cell system.

2. The chip co-culture method according to claim 1, characterized in that The step of obtaining the tumor organoid cells by three-dimensional organoid culture specifically includes: Take the tumor cell suspension, add 5-20% Matrigel to it, mix thoroughly and inoculate into a low-absorption 24-well culture plate, inoculating 200-300 μl per well. Note that the culture medium must be pre-chilled in advance. The inoculated 24-well low-absorption culture plate was placed in a 37°C, 5% CO2 incubator for incubation for 20-30 minutes, 200-300 μl of organoid culture medium was added to each well, and the plate was placed in a 37°C, 5% CO2 incubator for organoid culture to obtain tumor organoids; The tumor organoids are enzymatically digested into tumor organoid cells.

3. The chip co-culture method according to claim 2, characterized in that The S3 tumor-associated fibroblasts were prepared by the following steps: Take fresh tissue samples, wash them with PBS buffer, add type IV collagenase tissue dissociation solution with a concentration of 0.1-5 mg / ml, and mince the tissue samples to obtain uniform tissue fragments; Place the tissue fragments in a 5-10 times volume of type IV collagenase tissue digestion solution with a concentration of 0.1-5 mg / ml and incubate at 37°C for 40-60 minutes for digestion; After digestion, the cells are released from the matrix of the tissue fragments and filtered through a 70 μm mesh to remove undigested tissue clusters to obtain a cell suspension containing single cells or cell clusters. Dilute the digestive enzyme with 2 times the volume of DMEM medium of the cell suspension and continue digestion, then centrifuge at 200-300g for 3-5 minutes to collect the cell pellet; The cell pellet collected by centrifugation was treated with red blood cell lysis, and then added with liver cancer organoid culture medium containing 5-10% serum, inoculated into a cell culture plate, and placed in a 37°C, 5% CO2 incubator for two-dimensional adherent culture.

4. The chip co-culture method according to any one of claims 1 to 3, characterized in that: The organoid culture medium contains 2% Matrigel and 4% FBS buffer.

5. The chip co-culture method according to any one of claims 1 to 3, characterized in that: The drug sensitivity results of the co-culture system are subjected to a cell live and dead fluorescence labeling step, so that the drug sensitivity results are presented through fluorescence intensity, including Calcein AM labeling live cells to emit green fluorescence and PI labeling dead cells to emit red fluorescence.

6. A high-content imaging analysis method, characterized in that: The effects of drugs on tumor cells in the co-culture cell system obtained by any one of the chip co-culture methods according to claims 1-3 are evaluated by a high-content imaging system.

7. The high-content imaging analysis method according to claim 5, characterized in that: The high-content imaging analysis method uses high-content imaging equipment to observe the effects of drugs on tumor cells, including changes in growth, apoptosis, cell morphology, etc.

8. The high-content imaging analysis method according to claim 7, characterized in that: The high-content imaging analysis method also includes: recording changes in tumor cells under the action of drugs, and performing data processing and analysis to compare the inhibitory effects of different drugs on tumor cells to determine the best drug candidate or combination treatment plan.

9. Use of any one of the high-content imaging analysis methods according to claims 1-8 in anti-tumor drug screening.

10. Use of any one of the high-content imaging analysis methods according to claims 1-8 in clinical drug screening for the treatment of liver cancer.

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