Tumor organoid culture medium and culture method

Abstract

The invention discloses a tumor organoid culture medium and a culture method, and the tumor organoid culture medium comprises the following components: 20-50% v / v of a culture medium basic solution selected from DMEM / F12 or RPMI-1640; 5-10% v / v of fetal calf serum (FBS); 1-2% v / v of low-molecular peptide genome factors (such as EGF, bFGF and the like); 0.1-0.5% v / v of synthetic matrix factors, including but not limited to Matrigel, sodium alginate, hyaluronic acid and the like; 0.5-1% v / v of tumor microenvironment simulation factors, including tumor-related fibroblast factors (such as TGF-beta and IL-6) and immune cell factors (such as IL-2 and GM-CSF), are used for regulating the immune microenvironment of the organoid. The components of the culture medium used in the method are standardized, and the culture medium can be repeatedly used in different laboratories and researches, so that the comparability and repeatability of experimental results are improved, and the experimental cost can be reduced by optimizing the components of the culture medium and simplifying the culture method. Different tumor types can be cultured in the same culture medium, so that a high-cost special culture medium is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a tumor organoid culture medium and a culture method. Background Art

[0002] Tumor organoid culture medium is a culture medium specifically designed to simulate the tumor microenvironment and is used to culture and study tumor organoids in vitro. It typically contains a variety of growth factors, hormones, sugars, amino acids, and vitamins to support the growth, proliferation, differentiation, and tissue structure formation of tumor cells. Tumor organoids are three-dimensional (3D) cell culture models that can more realistically reproduce the biological characteristics of tumor tissue, such as tumor heterogeneity, cell-cell interactions, and drug response.

[0003] To cultivate tumor organoids, tumor tissue or cells must first be isolated from a patient or mouse model. These cells are then mixed with a suitable matrix (such as Matrigel) and placed in a culture dish containing tumor organoid culture medium. Under appropriate conditions, tumor cells will form tumor-like organoids within the three-dimensional matrix. During the culture process, the culture medium is regularly replaced to maintain the cell's nutrient supply and growth factor concentration, ensuring that the organoids maintain their growth and differentiation properties.

[0004] This culture method is widely used in tumor biology research, drug screening, tumor immunity research and other fields, and can provide an important experimental platform for precise treatment of tumors and personalized medicine.

[0005] Although existing technologies have made progress in many aspects of tumor organoid culture media and methods, they still have some shortcomings and challenges. The main issues include:

[0006] Lack of standardization: There is no unified standard for tumor organoid culture medium composition and culture methods. Different laboratories and researchers may adjust the composition of the culture medium according to their research objectives, which may lead to poor reproducibility of results and difficulty in cross-laboratory comparison or data sharing.

[0007] High cost: Existing tumor organoid culture media and related cell culture materials (such as Matrigel) are expensive and require regular purchase and replacement. This increases the cost burden of experiments for researchers, especially laboratories with limited resources.

[0008] Complex operational procedures: Cultivating tumor organoids requires high technical requirements and tedious operational steps, including tumor tissue isolation, cell seeding, 3D matrix configuration, and adjustment of culture conditions. These procedures are difficult and are highly sensitive to culture conditions (such as oxygen, pH, and nutrients), which can easily lead to experimental failure or unstable results.

[0009] Insufficient cellular heterogeneity: Although tumor organoids can reflect tumor heterogeneity to a certain extent, because certain types of tumor cells are usually selected during the culture process, the cultured organoids may lack a comprehensive representation of different cell subpopulations. This limits their application in the study of tumor heterogeneity, microenvironment, and immune response.

[0010] Inadequate reproduction of the tumor microenvironment: Tumor organoid culture media and culture methods often lack accurate simulation of multiple key elements in the tumor microenvironment, such as cancer-associated fibroblasts (CAFs), immune cells, and vascular endothelial cells. Existing culture systems struggle to fully simulate the complex interactions between tumors and their microenvironment, resulting in a gap between organoids and real tumors.

[0011] Tumor organoids have limited growth and expansion: Although they can closely mimic the structure and function of tumor tissue, their growth rate and duration in vitro are often limited. Certain types of tumor organoids may stop growing or degenerate within a few weeks, limiting their use as long-term drug screening platforms.

[0012] Lack of clinical relevance: Most current tumor organoids are derived from cell lines or mouse tumors, while culturing organoids from patient clinical samples remains a significant challenge. Patient-derived tumor organoids are difficult to culture and may require specific, personalized culture conditions, which has become a bottleneck in clinical translational research.

[0013] Limitations of Immune Cell Response Research: Tumor organoids typically focus on studying tumor cells themselves, with insufficient attention paid to the involvement of the immune system and the roles of immune cells. In vitro, organoids cannot fully mimic the tumor immune microenvironment, limiting their application in immunotherapy and research on tumor immune escape mechanisms.

[0014] To this end, we propose a tumor organoid culture medium and culture method. Summary of the Invention

[0015] To achieve the above objectives, the present invention provides the following technical solution: a tumor organoid culture medium comprising the following components:

[0016] 20-50% v / v culture medium basal solution, selected from DMEM / F12 or RPMI-1640;

[0017] 5-10% v / v fetal bovine serum (FBS);

[0018] 1-2% v / v low molecular weight peptide genomic factors (such as EGF, bFGF, etc.);

[0019] 0.1-0.5% v / v synthetic matrix factors, including but not limited to Matrigel, sodium alginate, or hyaluronic acid;

[0020] 0.5-1% v / v of tumor microenvironment mimicking factors, including tumor-associated fibroblast factors (such as TGF-β, IL-6) and immune cytokines (such as IL-2, GM-CSF, etc.), to regulate the immune microenvironment of organoids;

[0021] Other common media components such as vitamins, minerals, electrolytes and essential amino acids.

[0022] Preferably, the tumor microenvironment mimicking factor is obtained by co-culturing with specific immune cells and fibroblasts and optimizing cytokines. This factor can be personalized according to the immune microenvironment requirements of different tumor types.

[0023] A method for culturing tumor organoids, comprising the following steps:

[0024] S1 Obtain tumor tissue or tumor cells from patients or mouse models;

[0025] S2: mixing tumor cells or tumor tissue with a matrix material, wherein the matrix material is Matrigel, hyaluronic acid or sodium alginate;

[0026] S3 seeding the mixture in a culture dish containing the tumor organoid culture medium according to claim 1;

[0027] S4 was cultured at 37°C and 5% CO2, with the culture medium regularly changed to observe the growth and differentiation of tumor organoids;

[0028] S5 can adjust the concentrations of immune factors and cytokines in the culture medium according to the culture requirements and the microenvironment of different tumor types during the culture process to further enhance the clinical relevance of organoids.

[0029] Preferably, in step S5, the immune factors include but are not limited to IL-2, IL-6, GM-CSF, TNF-α, etc., and the fibroblast factors include TGF-β, CXCL12, etc., which are used to regulate the immune response of the tumor and the tumor microenvironment.

[0030] Preferably, the maintenance time of the tumor organoids can be adjusted according to the tumor type, usually 3 to 8 weeks, and the organoid morphology, function and immune response during the culture process can more truly reflect the characteristics of the original tumor tissue.

[0031] Compared with the prior art, the present invention provides a tumor organoid culture medium and culture method, which has the following beneficial effects:

[0032] 1. The tumor organoid culture medium and culture method used in this invention have standardized culture medium components and can be reused across different laboratories and research studies, thereby improving the comparability and reproducibility of experimental results. By optimizing culture medium components and simplifying culture methods, experimental costs can be reduced. Different tumor types can be cultured in the same culture medium, avoiding the high cost of specialized culture media.

[0033] 2. The tumor organoid culture medium and culture method, by adding tumor microenvironment simulation factors to the culture medium, can better simulate the immune microenvironment and extracellular matrix of the tumor, thereby enhancing the clinical relevance of the organoids. The culture method of the present invention reduces the operation steps and special treatment of cells, making the tumor organoid culture process simpler and more efficient.

[0034] 3. This tumor organoid culture medium and culture method, by using patient-derived tumor cells or tissues, can achieve personalized culture, providing a more reliable model for clinical translational research such as tumor drug screening and treatment response prediction. It is not only suitable for common tumor types (such as breast cancer, lung cancer, etc.), but also can adjust the culture medium and culture conditions according to the characteristics of different tumor types, thereby being widely used in the study of various tumor types. DETAILED DESCRIPTION

[0035] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0036] Example

[0037] An embodiment of a tumor organoid culture medium and culture method

[0038] A tumor organoid culture medium comprising the following components:

[0039] 20-50% v / v culture medium basal solution, selected from DMEM / F12 or RPMI-1640;

[0040] 5-10% v / v fetal bovine serum (FBS);

[0041] 1-2% v / v low molecular weight peptide genomic factors (such as EGF, bFGF, etc.);

[0042] 0.1-0.5% v / v synthetic matrix factors, including but not limited to Matrigel, sodium alginate, or hyaluronic acid;

[0043] 0.5-1% v / v of tumor microenvironment mimicking factors, including tumor-associated fibroblast factors (such as TGF-β, IL-6) and immune cytokines (such as IL-2, GM-CSF, etc.), to regulate the immune microenvironment of organoids;

[0044] Other common media components such as vitamins, minerals, electrolytes and essential amino acids.

[0045] Specifically, the tumor microenvironment mimicking factor is obtained by co-culturing with specific immune cells and fibroblasts and optimizing cytokines. This factor can be personalized according to the immune microenvironment requirements of different tumor types.

[0046] A method for culturing tumor organoids, comprising the following steps:

[0047] S1 Obtain tumor tissue or tumor cells from patients or mouse models;

[0048] S2: mixing tumor cells or tumor tissue with a matrix material, wherein the matrix material is Matrigel, hyaluronic acid or sodium alginate;

[0049] S3 seeding the mixture in a culture dish containing the tumor organoid culture medium according to claim 1;

[0050] S4 was cultured at 37°C and 5% CO2, with the culture medium regularly changed to observe the growth and differentiation of tumor organoids;

[0051] S5 can adjust the concentrations of immune factors and cytokines in the culture medium according to the culture requirements and the microenvironment of different tumor types during the culture process to further enhance the clinical relevance of organoids.

[0052] Specifically, in step S5, the immune factors include but are not limited to IL-2, IL-6, GM-CSF, TNF-α, etc., and fibroblast factors include TGF-β, CXCL12, etc., which are used to regulate the immune response of the tumor and the tumor microenvironment.

[0053] Specifically, the maintenance time of tumor organoids can be adjusted according to the type of tumor, usually 3 to 8 weeks, and the morphology, function and immune response of the organoids during the culture process can more truly reflect the characteristics of the original tumor tissue.

[0054] Through the above technical solution, in the present invention, the components of the culture medium used in the present invention are standardized and can be reused in different laboratories and studies, thereby improving the comparability and repeatability of the experimental results. By optimizing the components of the culture medium and simplifying the culture method, the cost of the experiment can be reduced. Different tumor types can be cultured under the same culture medium, avoiding the high cost of special culture medium. By adding tumor microenvironment simulation factors to the culture medium, the immune microenvironment and extracellular matrix of the tumor can be better simulated, and the clinical relevance of the organoid can be enhanced. The culture method of the present invention reduces the operating steps and special treatment of cells, making the culture process of tumor organoids simpler and more efficient. By using patient-derived tumor cells or tissues, personalized culture can be achieved, providing a more reliable model for clinical translational research such as tumor drug screening and treatment response prediction. It is not only suitable for common tumor types (such as breast cancer, lung cancer, etc.), but also can adjust the culture medium and culture conditions according to the characteristics of different tumor types, so that it is widely used in the study of various tumor types.

[0055] Composition ratio of culture medium:

[0056] 45% DMEM / F12 culture medium

[0057] 7% fetal bovine serum (FBS)

[0058] 1% bFGF, EGF

[0059] 0.2% Matrigel (for three-dimensional support)

[0060] 0.5% TGF-β, IL-6, GM-CSF and other tumor microenvironment mimicking factors

[0061] 0.5% fibronectin, laminin and other extracellular matrix molecules

[0062] Other standard culture ingredients (such as amino acids, vitamins, etc.)

[0063] Cultivation method:

[0064] Tumor tissue was removed from breast cancer patients and tumor cells were isolated using enzymatic digestion.

[0065] Tumor cells are mixed with Matrigel to form a three-dimensional culture structure.

[0066] The mixture was inoculated into a culture dish containing the above culture medium, ensuring that the cells were evenly distributed.

[0067] The cells were cultured at 37°C in an atmosphere of 5% CO2, and the culture medium was replaced every three days.

[0068] Organoids were observed weekly for morphological changes and their growth status and drug response were assessed.

[0069] Patient-derived small cell lung cancer (SCLC) cells were cultured using the aforementioned medium supplemented with immune microenvironmental factors such as VEGF and IL-2. Organoids were able to grow stably and respond to immune checkpoint inhibitors.

[0070] Through these embodiments, tumor organoids can be maintained for 3 to 6 weeks with stable morphology and good function, effectively simulating the immune escape mechanisms and drug responses of patient tumors. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A tumor organoid culture medium, characterized in that: Contains the following ingredients: 20-50% v / v culture medium basal solution, selected from DMEM / F12 or RPMI-1640; 5-10% v / v fetal bovine serum (FBS); 1-2% v / v low molecular weight peptide genomic factors (such as EGF, bFGF, etc.); 0.1-0.5% v / v synthetic matrix factors, including but not limited to Matrigel, sodium alginate, or hyaluronic acid; 0.5-1% v / v of tumor microenvironment mimicking factors, including tumor-associated fibroblast factors (such as TGF-β, IL-6) and immune cytokines (such as IL-2, GM-CSF, etc.), to regulate the immune microenvironment of organoids; Other common media components such as vitamins, minerals, electrolytes and essential amino acids.

2. A tumor organoid culture medium and culture method according to claim 1, characterized in that: The tumor microenvironment mimicking factor is obtained by co-culturing with specific immune cells and fibroblasts and optimizing cytokines. This factor can be personalized according to the immune microenvironment requirements of different tumor types.

3. A method for culturing tumor organoids, characterized in that: The following steps are involved: S1 Obtain tumor tissue or tumor cells from patients or mouse models; S2: mixing tumor cells or tumor tissue with a matrix material, wherein the matrix material is Matrigel, hyaluronic acid or sodium alginate; S3 seeding the mixture in a culture dish containing the tumor organoid culture medium according to claim 1; S4 was cultured at 37°C and 5% CO2, with the culture medium regularly changed to observe the growth and differentiation of tumor organoids; S5 can adjust the concentrations of immune factors and cytokines in the culture medium according to the culture requirements and the microenvironment of different tumor types during the culture process to further enhance the clinical relevance of organoids.

4. The method for culturing tumor organoids according to claim 3, wherein: In step S5, the immune factors include but are not limited to IL-2, IL-6, GM-CSF, TNF-α, etc., and the fibroblast factors include TGF-β, CXCL12, etc., which are used to regulate the immune response of the tumor and the tumor microenvironment.

5. The method for culturing tumor organoids according to claim 3, wherein: The maintenance time of the tumor organoids can be adjusted according to the tumor type, usually 3 to 8 weeks, and the organoid morphology, function and immune response during the culture process can more truly reflect the characteristics of the original tumor tissue.