Breast cancer organoid culture reagent combination and culture method thereof

Abstract

The invention discloses a breast cancer organoid culture reagent combination and a culture method thereof, and the formula is prepared according to the following raw materials: a basic culture medium: a DMEM / F12 culture medium, calf serum, Penicillin-Streptomycin, L-glutamine and HEPES buffer solution growth factors: EGF, IGF and HGF, and other additives: an N2 supplement, glucose, hydrocortisone, a trypsin inhibitor, an antifungal agent and an inflammation regulator. According to the breast cancer organoid culture reagent combination and the culture method thereof, the growth factor and the polymer nanoparticles are combined through an adsorption method, so that the stability of the growth factor and the controllable release of a carrier are ensured, the stability of the growth factor is improved, the release rate is controlled, and the stability of the growth factor is improved. According to the present invention, the cell culture medium has characteristics of simple operation, accurate delivery to the specific cell or tissue so as to promote the cell growth and development, and the inflammation regulator arranged in the culture medium can inhibit the inflammation in the multi-generation cell culture so as to maintain the stability and the health state of the tissue.

Description

Technical Field

[0001] The invention relates to a breast cancer organoid culture reagent combination and a culture method thereof, and belongs to the technical field of bioengineering. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide, with complex genetic and pathological characteristics. The application of early screening, early diagnosis, targeted drugs and immunotherapy has significantly improved the survival rate of breast cancer patients, but recurrence and metastasis still threaten the survival of breast cancer patients, and individualized treatment of patients is still a difficult point in clinical diagnosis and treatment. Breast cancer organoids use three-dimensional culture technology to simulate the tumor microenvironment, culture breast cancer tumor cells in vitro, and form tissue structures similar to in vivo tumors. They have the advantages of simple operation, short cycle and high throughput, and are gradually being widely used in drug sensitivity screening, personalized medication and breast cancer biology research.

[0003] It is difficult to maintain the structural and functional stability of breast organoids in long-term or multi-generation cultures, which may affect the continuity and accuracy of the research, and growth factors cannot be accurately delivered to the cells. Therefore, a breast cancer organoid culture reagent combination and a culture method thereof are urgently needed. Summary of the invention

[0004] The object of the present invention is to provide a breast cancer organoid culture reagent combination and a culture method thereof to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a breast cancer organoid culture reagent combination and a culture method thereof, wherein the breast cancer organoid culture reagent combination formula is prepared according to the following raw materials:

[0006] Basal medium:

[0007] DMEM / F12 medium 500 mL

[0008]

[0009] Growth Factors:

[0010] EGF 10-30 ng / mL

[0011] IGF 30-70 ng / mL

[0012] HGF 10-50 ng / mL

[0013] Other additives:

[0014]

[0015] wherein the growth factors are delivered to breast cancer cells via a method of nanoparticle delivery of growth factors;

[0016] The specific steps are as follows:

[0017] Step 1: Select polymer nanoparticles as carriers of growth factors;

[0018] Step 2: Combine the growth factor and polymer nanoparticles by adsorption to ensure the stability of the growth factor and the controlled release of the carrier;

[0019] Step 3: Add the growth factors delivered by the nanoparticles to the breast cancer organoid cell culture medium.

[0020] As a preferred technical solution of the present invention, the breast cancer organoid culture reagent combination formula is prepared according to the following raw materials:

[0021] Basal medium:

[0022]

[0023]

[0024] Growth Factors:

[0025] EGF 10ng / mL

[0026] IGF 30ng / mL

[0027] HGF 10 ng / mL

[0028] Other additives:

[0029]

[0030] As a preferred technical solution of the present invention, the breast cancer organoid culture reagent combination formula is prepared according to the following raw materials:

[0031] Basal medium:

[0032]

[0033] Growth Factors:

[0034] EGF 20ng / mL

[0035] IGF 50 ng / mL

[0036] HGF 30ng / mL

[0037] Other additives:

[0038]

[0039] As a preferred technical solution of the present invention, the breast cancer organoid culture reagent combination formula is prepared according to the following raw materials:

[0040] Basal medium:

[0041]

[0042] Growth Factors:

[0043] EGF 30ng / mL

[0044] IGF 70ng / mL

[0045] HGF 50ng / mL

[0046] Other additives:

[0047]

[0048]

[0049] As a preferred technical solution of the present invention, the trypsin inhibitor includes one or more of aprotinin, sulfamozone, soybean trypsin inhibitor or benzamide.

[0050] As a preferred technical solution of the present invention, the inflammatory regulator includes one or more of flavonols and anthocyanidins natural plant compounds or cyclooxygenase.

[0051] As a preferred technical solution of the present invention, the method for culturing breast cancer organoids comprises the following steps:

[0052] It mainly includes the following contents:

[0053] Step 1: Cell source and culture medium preparation: Select breast cancer cells, place the breast cancer organoid culture reagent combination formula in the culture medium, and culture the breast cancer cells therein;

[0054] Step 2: Cell passaging and expansion: Passage the cells into culture dishes to adapt them to the culture environment and expand them, and replace the culture medium regularly;

[0055] Step 3: 3D cell culture: In order to better simulate the growth environment of breast cancer tissue, breast cancer organoids are cultured using scaffolds, gels or porous materials;

[0056] Step 4: Cell morphology observation: Regularly observe the morphology and status of cells, and check the cell attachment, proliferation status and possible abnormal changes.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows: a breast cancer organoid culture reagent combination and a culture method thereof of the present invention combine growth factors and polymer nanoparticles by an adsorption method to ensure the stability of the growth factors and the controllable release of the carrier, so as to improve the stability of the growth factors, control the release rate, and accurately deliver them to specific cells or tissues, thereby promoting cell growth and development. Moreover, by setting an inflammatory regulator in the culture medium, the occurrence of inflammation can be inhibited in multi-generation cell culture, thereby maintaining the stability and health of the tissue. DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] The present invention provides a breast cancer organoid culture reagent combination and a culture method thereof

[0060] Example 1: The breast cancer organoid culture reagent combination formula is prepared according to the following raw materials: Basic culture medium:

[0061]

[0062] Growth Factors:

[0063] EGF 10ng / mL

[0064] IGF 30ng / mL

[0065] HGF 10 ng / mL

[0066] Other additives:

[0067]

[0068] Example 2: The breast cancer organoid culture reagent combination formula is prepared according to the following raw materials: Basic culture medium:

[0069]

[0070] Growth Factors:

[0071] EGF 20ng / mL

[0072] IGF 50 ng / mL

[0073] HGF 30ng / mL

[0074] Other additives:

[0075]

[0076] Example 3: The breast cancer organoid culture reagent combination formula is prepared according to the following raw materials: Basic culture medium:

[0077]

[0078] Growth Factors:

[0079] EGF 30ng / mL

[0080] IGF 70ng / mL

[0081] HGF 50ng / mL

[0082] Other additives:

[0083]

[0084] wherein the growth factors are delivered to breast cancer cells via a method of nanoparticle delivery of growth factors;

[0085] The specific steps are as follows:

[0086] Step 1: Select polymer nanoparticles as carriers of growth factors;

[0087] Step 2: Combine the growth factor and polymer nanoparticles by adsorption to ensure the stability of the growth factor and the controlled release of the carrier;

[0088] Step 3: Adding Nanoparticle-Delivered Growth Factors to Breast Cancer Organoid Cell Culture Medium

[0089] Wherein, the trypsin inhibitor includes one or more of aprotinin, sulfasulfazone, soybean trypsin inhibitor or benzamide.

[0090] Furthermore, the inflammatory regulator includes one or more of flavonols and anthocyanidins natural plant compounds or cyclooxygenase.

[0091] Furthermore, the method for culturing breast cancer organoids comprises the following steps:

[0092] It mainly includes the following contents:

[0093] Step 1: Cell source and culture medium preparation: Select breast cancer cells, place the breast cancer organoid culture reagent combination formula in the culture medium, and culture the breast cancer cells therein;

[0094] Step 2: Cell passaging and expansion: Passage the cells into culture dishes to adapt them to the culture environment and expand them, and replace the culture medium regularly;

[0095] Step 3: 3D cell culture: In order to better simulate the growth environment of breast cancer tissue, breast cancer organoids are cultured using scaffolds, gels or porous materials;

[0096] Step 4: Cell morphology observation: Regularly observe the morphology and status of cells, and check the cell attachment, proliferation status and possible abnormal changes.

[0097] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A breast cancer organoid culture reagent combination and a culture method thereof, characterized in that: The breast cancer organoid culture reagent combination formula is prepared according to the following raw materials: Basal medium: Growth Factors: EGF 10-30 ng / mL IGF 30-70 ng / mL HGF 10-50ng / mLOther additives: wherein the growth factors are delivered to breast cancer cells via a method of nanoparticle delivery of growth factors; The specific steps are as follows: Step 1: Select polymer nanoparticles as carriers of growth factors; Step 2: Combine the growth factor and polymer nanoparticles by adsorption to ensure the stability of the growth factor and the controlled release of the carrier; Step 3: Add the growth factors delivered by the nanoparticles to the breast cancer organoid cell culture medium.

2. A breast cancer organoid culture reagent combination and culture method according to claim 1, characterized in that: The breast cancer organoid culture reagent combination formula is prepared according to the following raw materials: basic culture medium: Growth Factors: EGF 10ng / mL IGF 30ng / mL HGF 10 ng / mL Other additives:

3. A breast cancer organoid culture reagent combination and culture method according to claim 1, characterized in that: The breast cancer organoid culture reagent combination formula is prepared by mixing the following raw materials into the basal culture medium: Growth Factors: EGF 20ng / mL IGF 50ng / mL HGF 30ng / mL Other additives:

4. A breast cancer organoid culture reagent combination and culture method according to claim 1, characterized in that: The breast cancer organoid culture reagent combination formula is prepared according to the following raw materials: basic culture medium: Growth Factors: EGF 30ng / mL IGF 70ng / mL HGF 50ng / mL Other additives:

5. A breast cancer organoid culture reagent combination and culture method according to claim 1, characterized in that: The trypsin inhibitor includes one or more of aprotinin, sulfasulfazone, soybean trypsin inhibitor or benzamide.

6. A breast cancer organoid culture reagent combination and culture method according to claim 1, characterized in that: The inflammatory regulators include one or more of flavonols and anthocyanidins natural plant compounds or cyclooxygenase.

7. A breast cancer organoid culture reagent combination and culture method according to claim 1, characterized in that: The method for culturing breast cancer organoids comprises the following steps: It mainly includes the following contents: Step 1: Cell source and culture medium preparation: Select breast cancer cells, place the breast cancer organoid culture reagent combination formula in the culture medium, and culture the breast cancer cells therein; Step 2: Cell passaging and expansion: Passage the cells into culture dishes to adapt them to the culture environment and expand them, and replace the culture medium regularly; Step 3: 3D cell culture: In order to better simulate the growth environment of breast cancer tissue, breast cancer organoids are cultured using scaffolds, gels or porous materials; Step 4: Cell morphology observation: Regularly observe the morphology and status of cells, and check the cell attachment, proliferation status and possible abnormal changes.

Images