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Culture medium and culture method for mammary epithelial stem cells

A technology of mammary epithelium and culture medium, which is applied in the direction of cell culture active agent, non-embryonic pluripotent stem cells, epidermal cells/skin cells, etc., can solve the problem of high cost of organoid culture and detection, difficult to control the size of organoids, and easy operation. The problems of poor reproducibility and repeatability can be achieved to achieve long-term maintenance of differentiation ability, controllable culture cost, and high efficiency.

Active Publication Date: 2021-10-22
PRECEDO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology does not require feeder cells, so there is no interference with mouse-derived feeder cells. However, a variety of specific factors need to be added to the medium of organoid technology, especially the essential components of the medium, Wnt protein and R-spondin family proteins, The cost of organoid culture and detection is expensive, and it is not suitable for popularization in clinic for large-scale application
In addition, this technology needs to embed organoids in Matrigel during the whole process of culture and detection, and the size of the formed organoids is not easy to control, resulting in poor operability and reproducibility of this technology, which limits The large-scale application of this technology in clinical in vitro drug efficacy and evaluation, high-throughput drug screening and toxicity testing (Nick Barker et al., Nat Cell Biol, 18(3):246-54, 2016; Huch M. et al., Development, 144, 938-941, 2017)

Method used

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  • Culture medium and culture method for mammary epithelial stem cells
  • Culture medium and culture method for mammary epithelial stem cells
  • Culture medium and culture method for mammary epithelial stem cells

Examples

Experimental program
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Embodiment approach 1

[0137] The culture method according to Embodiment 1 of the present invention is a culture method for culturing epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any of these cells derived from normal breast tissue or diseased breast tissue .

[0138] Wherein, described method comprises the following steps:

[0139] (1) preparing extracellular matrix;

[0140] (2) adhering epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues comprising at least any of these cells to an extracellular matrix, adding to the extracellular matrix or embedding in the extracellular matrix;

[0141] (3) using the medium of the present invention to culture the epithelial stem cells, epithelial cells, epithelial tumor cells, or tissues containing at least any one of these cells, to obtain expanded, corresponding epithelial stem cells, epithelial cells, Epithelial tumor cell progeny or organoid progeny.

[0142] If the culture method ...

Embodiment 1

[0174] 1. Preparation of MST1 / 2 Kinase Inhibitor Compound 1

[0175] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0176]

[0177] 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.

[0178] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Add 2-amino- After adding acetone (30 ml) and potassium carbonate (2.2 g) to 2-(2,6-difluorophenyl) methyl acetate (2 g), the system was cooled to -10 ° C with an ice-salt bath, and then slowly added 2,4-Dichloro-5-nitropyrimidine (3.1...

Embodiment 2

[0204] 1. Prepare basal medium in the same manner as in Example 1.

[0205] 2. Obtain a freshly isolated epithelial tumor cell sample (HMFL-XN40) according to the steps in Embodiment 1. Then, inoculate epithelial tumor cells onto Matrigel-coated TM (manufactured by BD Biosciences) 6-well plate. The basal medium was added to the wells inoculated with the above-mentioned epithelial tumor cells, and cultured at 37°C under an oxygen concentration of 20%. Digestion, subculture, cultivation and counting were carried out according to the steps of 4 in Example 1, and the cultivation was continued for 3 generations. When inoculating the 4th passage tumor cells, the epithelial tumor cells were divided into 3×10 4 Cells / well were evenly seeded to the cells coated with Matrigel TM (manufactured by BD Biosciences) in each well of a 24-well plate. For inoculation, add 1 mL of the medium containing basal medium + DMSO and the medium containing basal medium + compound 1 to each well, and...

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Abstract

The invention provides a culture medium for culturing mammary epithelial stem cells. The culture medium contains a TGF-beta inhibitor, B27 and / or N2, insulin, a receptor tyrosine kinase ligand, a Rock kinase inhibitor, a P38 signal transduction inhibitor, a bone morphogenetic protein inhibitor and an MST1 / 2 kinase inhibitor. The invention also provides a method for culturing cells by adopting the culture medium for the mammary epithelial stem cells, and application and methods of amplified cell populations or organoids obtained by adopting the culture method in drug screening, toxicity testing and regenerative medicine.

Description

technical field [0001] The present invention relates to a medium for culturing epithelial stem cells in vitro, especially mammary gland epithelial stem cells, and a medium and a culturing method for culturing organoids comprising said stem cells. The present invention also relates to the use of the progeny of cells and organoids cultured by the culture medium and the culture method of the present invention in drug efficacy evaluation and screening, toxicity determination and regenerative medicine. Background technique [0002] Breast disease, especially breast cancer, is one of the most important diseases affecting women's health. In recent years, although people have made a lot of progress in the research on the classification and pathogenesis of breast diseases, the current standard treatment drugs for breast diseases, especially breast cancer, are still quite scarce, and there is even a lack of personalized precise drug guidance. The key to this problem is that there is ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/074C12Q1/02
CPCC12N5/0631G01N33/5017C12N2501/15C12N2501/33C12N2501/727C12N2501/155C12N2500/30G01N2500/10G01N33/5044G01N33/5073C12Q1/02C12N5/06
Inventor 刘青松刘飞扬王俊杰梅沪生王文超任涛王黎
Owner PRECEDO PHARMA CO LTD
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