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A special culture medium and 3D culture method for breast cancer organoids

A culture medium, breast cancer technology, applied in the field of cell culture, can solve the problems of genotype change, long time required for establishment, deviation, etc., and achieve the effect of less impact of personnel operation, simple operation and high stability

Active Publication Date: 2022-04-12
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the two models have made great contributions to the research of breast cancer, they both have certain limitations, such as the possibility of cross-contamination in the CCL model, changes in the genotype, and difficulty in permanent proliferation. The tumor transplantation of the PDTX model Low success rate, long time required for establishment, lack of immune ability of receptors, and inability to perform high-throughput drug screening, etc. These shortcomings may lead to deviations between experimental results and the actual situation of tumors in humans, so there is an urgent need for a better one. The model appears

Method used

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  • A special culture medium and 3D culture method for breast cancer organoids
  • A special culture medium and 3D culture method for breast cancer organoids
  • A special culture medium and 3D culture method for breast cancer organoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] This embodiment provides a mammary organized organ specific medium and 3D culture method including a base medium and a specific addition factor; the specific additive factor includes the following final concentrations: GSK429286A: 10μm; DoraMapimod : 8 μm; A83-01: 0.8 μm; EGF: 150 ng / ml; β-Estradiol: 5 nm; cholera Toxin: 12 ng / ml; FGF2: 2 μm; hepes: 20mm; B27: 1X; Glutamax: 2X; ITS: 0.5X; Gentamicin: 25 μg / ml; Neuregulin-1: 6 nm; BSA: 4mg / ml; TN-C recombinant protein: 70 ng / ml. The culture method includes: after cleaning the removed breast cancer, cutting 10 ml of trypsin resuscitation, transferred to 37 ° C, transferred to 37 ° C, 220 rpm shaker for 10 minutes, add 5 ml of DMEM / F12 after digestion Terminate digestion. The cell suspension was filtered through a 70 μm cell mesh, and 5 minutes were centrifuged, 5 ml of red blood cell cleavage was resuspended. After 5 mL of DMEM / F12 terminate cleaves, centrifuge, remove the supernatant, cell count, mix Matrigel, ...

Embodiment 2

[0041]This example provides a special medium for breast cancer organs and 3D culture methods including base medium and specific additive factors; the specific additive factor includes the following final concentrations: GSK429286A: 15μm; DoraMapimod : 6 μm; A83-01: 0.7 μm; EGF: 200 ng / ml; β-estradiol: 4 nm; cholera Toxin: 15 ng / ml; FGF2: 5μm; HEPES: 15mm; B27: 5X; Glutamax: 1x; ITS: 1X; Gentamicin : 30 μg / ml; Neuregulin-1: 5 nm; BSA: 5 mg / mL; TN-C recombinant protein: 80 ng / ml. The culture method is in Example 1. Cultivate 7 days of type organ topography Image 6 As shown, the formation of the tumor type of tumor organ cells, and the diameter can exceed 200 μm, and the activity is better.

Embodiment 3

[0043] This embodiment provides a special medium for breast cancer organs and 3D culture methods including a base medium and a specific addition factor; the specific additive factor includes the following final concentrations: GSK429286A: 5μm; Doramapimod : 15 μm; A83-01: 0.5 μm; EGF: 500 ng / ml; β-estradiol: 2 nm; cholera Toxin: 25 ng / ml; FGF2: 3μm; HEPES: 10mm; B27: 3X; Glutamax: 1x; ITS: 0.1X; Gentamicin: 10 μg / ml; Neuregulin-1: 2 nm; BSA: 10 mg / ml; TN-C recombinant protein: 100 ng / ml. The culture method is in Example 1. Cultivate a variety of organ planning Figure 7 As shown, the formation of tumor types of tumor organ cells, and large diameter, better activity.

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Abstract

The present invention provides a special culture medium and 3D culture method for breast cancer organoids. The culture medium includes basal medium and specific additive factors; the specific additive factors include GSK429286A; Doramapimod; A83-01; EGF; β-estradiol ; Cholera Toxin; FGF2; HEPES; B27; GlutaMAX; ITS; Gentamicin; Neuregulin‑1; BSA. The culture method includes: digesting and filtering the isolated breast cancer tissue to obtain cell pellets; lysing the cell pellets, then mixing with matrigel gel and inoculating, adding the above medium after the mixed gel is solidified, and incubating , 5%CO 2 Cultivate for 7-14 days under the concentration. The culture medium of the present invention can realize the rapid growth of breast cancer organoids, and can be stably cultured for a long time, and the formed tumor organoid cells are spherical in shape and relatively uniform in size, and can well retain the heterogeneity of tumor tissues of patients in vitro .

Description

Technical field [0001] The present invention belongs to the field of cell culture, and is specifically involved in a special medium for breast cancer organs and 3D culture methods. Background technique [0002] Breast cancer is one of the most common malignant tumors in women in global women, but also the second big hand that leads to death in patients with female cancer. In recent years, with the development of my country and the changes in people's lifestyle, the incidence of breast cancer in my country has increased year by year, and the patient has a low-age trend. At present, scholars mainly use traditional tumor cells model (CCL) and patient sources of tumor xenograft model (PDTX) to study the development of breast cancer. Although the two models have made great contributions for breast cancer, there is a certain limitations. If the CCL model may have hypocrosis, genotype will change and difficult to permanently proliferation, etc., tumor transplantation of PDTX model The s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0631C12N2513/00C12N2501/724C12N2501/11C12N2501/115C12N2501/392C12N2501/13C12N2501/405C12N2501/15C12N2501/01C12N2501/998C12N2509/00C12N2533/90C12N2500/90
Inventor 于言朱宇陈泽新黄敏
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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