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Culture medium for epithelial cells of esophageal squamous carcinoma, culture method and application thereof

A technology for esophageal squamous cell carcinoma and epithelial cells, applied in the field of medicine, can solve the problems of necrosis, complicated and time-consuming operation, high cost, etc., and achieves the effects of high efficiency, controllable culture cost, and improved success rate.

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

However, a variety of specific growth factors (such as Wnt proteins and R-spondin family proteins) need to be added to the culture medium of organoid technology, which is expensive and not suitable for popularization in the clinic for large-scale application
In addition, during the whole culture process of organoids, cells need to be embedded in extracellular matrix gel, and the plating steps of cell seeding, passage and drug sensitivity testing are cumbersome and time-consuming compared with 2D culture operations, and the technology formed The size of organoids is not easy to control, and some organoids tend to grow too large and cause internal necrosis
Therefore, organoid technology is less operable and applicable than 2D culture technology, requires professional technicians to operate, and is not suitable for large-scale and extensive clinical in vitro drug sensitivity testing (Nick Barker, Nat Cell Biol, 18(3 ): 246-54, 2016)

Method used

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  • Culture medium for epithelial cells of esophageal squamous carcinoma, culture method and application thereof
  • Culture medium for epithelial cells of esophageal squamous carcinoma, culture method and application thereof
  • Culture medium for epithelial cells of esophageal squamous carcinoma, culture method and application thereof

Examples

Experimental program
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preparation example Construction

[0084] [Preparation Example of MST1 / 2 Kinase Inhibitor]

[0085] In the present specification, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signal transduction. In general, MST1 / 2 kinase inhibitors, for example, bind to MST1 / 2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1 / 2 kinase inhibitors may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.

[0086] 1. Preparation of MST1 / 2 Kinase Inhibitor Compound 1

[0087] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide 1

[0088]

[0089] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (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 ...

Embodiment 1

[0102] Isolation of Primary Human Esophageal Squamous Cell Carcinoma Epithelial Cells

[0103] The tissue samples of esophageal squamous cell carcinoma were obtained from three cases of patients with esophageal squamous cell carcinoma who had been explained and obtained consent, and they were respectively sample numbers 0F0060, 0F0061, and 0F0062. The following is an example of a sample (No. 0F0060) for illustration.

[0104] The above tissue samples were collected within half an hour of the patient's surgical resection or biopsy. More specifically, under sterile conditions, tissue samples from non-necrotic sites were removed in a volume of 0.5 cm 3 Above, it was placed in pre-cooled 4mL tissue transport solution (see Table 1 for specific preparation), and the transport solution was contained in a 5mL plastic sterile cryopreservation tube with a cover (purchased from Guangzhou Jiete Biology), and the cold chain (0- 10°C) to the laboratory.

[0105] Table 1 Formula of tissue...

Embodiment 2

[0116] Optimization of culture medium for primary esophageal squamous cell carcinoma epithelial cells

[0117] (1) The role of different factors

[0118] extracellular matrix gel (manufactured by BD Biotechnology Co., Ltd.) Use serum-free DMEM / F12 medium at a dilution ratio of 1:100 to prepare an extracellular matrix dilution, add 500 μl / well of the extracellular matrix dilution to a 48-well culture plate to completely cover it Bottom of culture plate wells. Place in a 37°C incubator for 1 hour. After 1 hour, the extracellular matrix dilution was removed to obtain a Matrigel-coated culture plate.

[0119] Preparation of basal medium (abbreviated as BM): add 0.2 volume % Primocin (purchased from Invivogen, the concentration is 50 mg / mL) to the commercially available DMEM / F-12 medium to obtain a final concentration of 100 μg / mL, and prepare Get BM.

[0120] Next, different types and different concentrations of additive factors (Table 3) were added to the basal medium (BM) ...

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Abstract

The invention provides a primary cell culture medium for culturing primary esophageal squamous carcinoma epithelial cells and containing a combination of an MST1 / 2 kinase inhibitor and a ROCK kinase inhibitor, and a culture method using the primary cell culture medium. According to the culture method, the primary cells are cultured on a culture vessel coated with extracellular matrigel by using the primary cell culture medium, so that the primary cells are rapidly proliferated. The cell model obtained by the primary cell culture medium and the primary cell culture method can be used for evaluating and screening curative effects 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 esophageal squamous cell carcinoma epithelial cells in vitro, and also relates to a method for evaluating and screening the curative effect of the cultured cells in drugs and apply. Background technique [0002] Esophageal cancer is one of the most common gastrointestinal malignancies in the world. According to the latest statistics from the National Cancer Center, esophageal cancer ranks sixth among the top ten malignant tumors in men, and ranks second in women. In many regions of the world, the local incidence rate is increasing, and my country is a high-incidence area of ​​esophageal cancer, with an annual incidence of 246,000 and an average annual death toll of about 150,000, accounting for 21.8% of the national tumor mortality rate. The death rate of cancer ranks 4th in China (2019 Nationa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693C12N5/0625G01N33/5011C12N2501/727C12N2501/11C12N2500/25C12N2501/12C12N2501/15C12N2503/02G01N2500/10C40B30/06G01N33/5044C12N5/0679C12N2533/90C12N2501/105C12N2501/115
Inventor 刘青松胡洁王文超陈程任涛王黎
Owner PRECEDO PHARMA CO LTD
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