Primary mammary epithelial cell culture medium, culture method and application

A breast epithelial cell and primary cell technology, applied in the field of cell efficacy evaluation and screening of drugs, can solve the problems of high cost, necrosis, impact on response results, etc., and achieve a high success rate, high efficiency, and controllable culture cost. Effect

Active Publication Date: 2021-05-11
PRECEDO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, both techniques have certain limitations
Cell reprogramming is a technique in which the patient's own primary epithelial cells are co-cultured with murine feeder cells, however, the presence of these murine cells interferes with the patient's primary cells when performing drug susceptibility testing The drug sensitivity test results of autologous primary cells; however, if the mouse-derived feeder cells are withdrawn, the patient's autologous primary cells will be out of the reprogramming environment, and the cell proliferation rate and intracellular signaling pathways will be significantly changed (Liu et al., Am J Pathol, 183(6):1862-1870, 2013; Liu et al., Cell Death Dis., 9(7):750, 2018), so that the response of the patient's own primary cells to the drug is greatly affected
Organoid technology is a technology in which the patient's own primary epithelial cells are embedded in the extracellular matrix for three-dimensional culture in vitro. This technology does not

Method used

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  • Primary mammary epithelial cell culture medium, culture method and application
  • Primary mammary epithelial cell culture medium, culture method and application
  • Primary mammary epithelial cell culture medium, culture method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Isolation of human primary mammary epithelial cells and optimization of primary mammary epithelial cell culture medium

[0060] (1) Isolation of human primary mammary epithelial cells

[0061]Breast cancer tissue samples were obtained from five breast cancer patients who had been explained and consented to surgically resected cancer tissue samples, which were HMFL-XN1, HMFL-XN3, HMFL-XN4, HMFL-XN6, and HMFL-XN8. One of the samples (HMFL-XN1) will be described below. The above tissue samples were collected within half an hour of the patient's surgical resection or biopsy. More specifically, in a sterile environment, tissue samples from non-necrotic sites were excised, with a volume of 0.5 cm 3 Above, it was placed in pre-cooled 20mL DMEM / F12 medium (manufactured by Corning Company), and the medium was contained in a 50mL plastic sterile centrifuge tube with a cover, and transported to the laboratory on ice; among them, the DMEM / F12 medium Contains 100U / mL penicillin a...

Embodiment 2

[0075] Culture of Human Primary Mammary Epithelial Cells

[0076] (1) Preparation of primary breast epithelial cell culture medium

[0077] First, prepare the basal medium according to the same method as step (2) of Example 1. Human Amphiregulin (manufactured by R&D Systems) was added to the basal medium at a final concentration of 20 ng / ml, EGF (manufactured by Peprotech) was added at a final concentration of 10 ng / ml, and B27 was added at a 1:50 dilution (Thermo Fisher SCIENTIFIC), human neurotonin 1 (manufactured by Peprotech) at a final concentration of 10 nM, FGF7 (manufactured by R&D Systems) at a final concentration of 10 ng / ml, and TGFβ1 inhibitor at a final concentration of 500 nM A8301 (manufactured by MCE), and P38 / MAPK inhibitor SB202190 (manufactured by MCE) were added at a final concentration of 500 nM to prepare a culture medium for primary mammary gland epithelial cells.

[0078] (2) Culture of primary breast cancer tumor cells derived from breast cancer tiss...

Embodiment 3

[0088] Effect of amphiregulin on the proliferation of primary breast cancer cells derived from breast cancer tissue

[0089] (1) The primary mammary gland epithelial cell culture medium was prepared by the same method as in step (1) of Example 2. In addition, amphiregulin was removed from the formula of primary mammary epithelial cell culture medium, and another primary medium without amphiregulin was prepared.

[0090] (2) Use the same method as step (1) of Example 1 to obtain primary breast cancer tumor cells (HMFL-XN2, HMFL-XN12) from two different cases of breast cancer patients and a case of breast cancer Primary breast cancer tumor cells (HMFL-XN13) from a patient biopsy.

[0091] Primary breast cancer tumor cells (HMFL-XN2) derived from surgically resected cancer tissue were inoculated at the same density (5×10 4 per well) were inoculated into 12-well plates coated with Matrigel (registered trademark) (manufactured by BD Biotechnology Co., Ltd.). The primary mammary ...

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PUM

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Abstract

The invention provides a primary cell culture medium for culturing primary mammary epithelial cells and containing amphiregulin, and a culture method using the primary cell culture medium. Through the culture method, the primary cell culture medium is used for culturing primary cells on a culture vessel coated with extracellular matrigel, the primary cells grow on the culture vessel coated with the extracellular matrigel, and the primary cells are rapidly proliferated under the combined action of nutritional factors contained in the primary cell culture medium and extracellular matrigel. A cell model obtained by the primary cell culture medium and the primary cell culture method can be used for curative effect evaluating and screening of medicines.

Description

technical field [0001] The invention belongs to the technical field of medicine, in particular, it relates to a culture medium and a culture method for culturing or expanding primary mammary gland epithelial cells in vitro, and also relates to a method and application of the cultured cells in evaluating and screening the curative effect of drugs . Background technique [0002] Breast cancer is one of the most important malignant tumors affecting women's health. The latest statistical results show that globally, the incidence and mortality of breast cancer rank first and second in the incidence and mortality of female malignant tumors, respectively. In recent years, although people have made a lot of progress in the research on the molecular typing and pathogenesis of breast cancer, the current standard drug treatment methods for breast cancer are still based on hormones and cytotoxic drugs, lacking personalized precise drug guidance . Because of the diversity and complexi...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09C12Q1/02
CPCC12N5/0631C12N5/0693G01N33/5044C12N2501/999C12N2501/11C12N2501/33C12N2501/405C12N2501/195C12N2501/117C12N2501/15C12N2533/90G01N2500/10C12N2501/12C12N2501/115C12N2501/105C12N2501/235C12N2501/375C12N2501/119C12N2501/727
Inventor 刘青松刘飞扬梅沪生王文超赵明蒋宗儒任涛王黎
Owner PRECEDO PHARMA CO LTD
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