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Culture medium and culture method for primary cells of esophageal squamous carcinoma

A technology for esophageal squamous cell carcinoma and primary cells, applied in the biological field, can solve the problems of long culture period, complicated operation, expensive reagents, etc., achieve the effect of reducing culture cost, simple processing method, and meeting experimental needs

Active Publication Date: 2021-08-13
PRECEDO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the method in the above-mentioned non-patent literature 2 cannot be stably cultivated for a long time, the cultivation period of the method in the non-patent literature 3 and the non-patent literature 4 is longer, and the method in the non-patent literature 5 The cost is relatively high, and the reagents used for organoid culture in Non-Patent Document 6 are too expensive, and the operation is complicated during the culture process, which is not conducive to large-scale application
[0005]And because the pathological type of esophageal cancer abroad is mainly esophageal adenocarcinoma, the methods in foreign literature are basically based on the research of esophageal adenocarcinoma, so it is not suitable for culturing esophageal adenocarcinoma squamous cell carcinoma

Method used

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  • Culture medium and culture method for primary cells of esophageal squamous carcinoma
  • Culture medium and culture method for primary cells of esophageal squamous carcinoma
  • Culture medium and culture method for primary cells of esophageal squamous carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Promoting Cell Proliferation of Esophageal Squamous Cell Carcinoma Primary Cell Medium Containing Different Components

[0050] (1) Preparation of culture medium with different components

[0051] According to the ingredients in Table 1, the culture medium with different components was prepared, and the proliferation-promoting effect on esophageal squamous cell carcinoma cells was investigated.

[0052] 10 μM Y27632 (purchased from MCE) and 100 mg / mL Primocin were added to the DMEM / F12 medium (purchased from Corning) as the basal medium (hereinafter sometimes referred to as BM), and different factors were gradually added on this basis. No.1 medium was obtained by adding insulin (purchased from Gibco Company) on the basis of BM, and No.2 medium was obtained by adding hydrocortisone (purchased from Sigma Company) on the basis of No.1 medium. No.3 medium is obtained by adding non-essential amino acid (NEAA) (purchased from Gibco Company) on the basis of No.2 med...

Embodiment 2

[0075] Example 2 Effect of Multifactor Combination on the Proliferation of Esophageal Squamous Cell Carcinoma Primary Cell Culture Medium

[0076] In the basal medium of Example 1 (DMEM / F12 medium + 10 μM Y27632 + 100 mg / mL Primocin) were separately added 1:50 ratio N2 additive, 20 μg / mL insulin, 7 μg / mL bovine pituitary extract, 2 mM gluten Aminoamide, 400 μM non-essential amino acids, 0.4 μg / mL hydrocortisone, and 5 ng / mL insulin-like growth factor 1 were used to prepare the culture medium of this example.

[0077] In the same manner as in Example 1, endoscopic tissue samples 57, 58, 59, 60, and 61 were separated to obtain primary cells of esophageal squamous cell carcinoma, and the culture media prepared in this example and the culture medium in Example 1 were used. The obtained primary cells of esophageal squamous cell carcinoma were cultured in BM medium and No.7 medium (EM) respectively, and the living cell density was 1×10 4 piece / cm 2 Seed in 48-well plate (10,000 ce...

Embodiment 3

[0079] Example 3 Effects of adding other factors on the proliferation of esophageal squamous cell carcinoma primary cells in the primary cell culture medium of esophageal squamous cell carcinoma of the present invention

[0080] 10ng / mL fibroblast growth factor 2 (FGF2), 10ng / mL fibroblast growth factor 10 (FGF10), and 10ng / mL fibroblast growth factor were added separately in the No.7 medium (EM) of embodiment 1. Factor 7 (FGF7) or 10 ng / mL FGF2, 10 ng / mL FGF10 and 10 ng / mL FGF7 were added together to prepare the medium of this example.

[0081] In the same manner as in Example 1, endoscopic tissue sample 62 and sample 63 were separated to obtain primary cells of esophageal squamous cell carcinoma, and each medium prepared in this example and No.7 medium (EM) in Example 1 were used to separate The obtained primary cells of esophageal squamous cell carcinoma were cultured according to the living cell density of 1×10 4 piece / cm 2 Seed in a 48-well plate (10,000 cells per well)...

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Abstract

The invention provides a culture medium and a culture method for rapidly amplifying primary cells of esophageal squamous carcinoma in vitro and application of the culture medium and the culture method in medicine screening. The culture medium for the primary cells of the esophageal squamous carcinoma contains an initial culture medium, an Rho protease inhibitor, antibiotics, insulin, an N2 additive, an insulin-like growth factor 1, non-essential amino acid, optional hydrocortisone, optional glutamine and optional bovine pituitary extract.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a culture medium and a culture method for rapidly expanding esophageal squamous cell carcinoma primary cells in vitro. Background technique [0002] Esophageal cancer is one of the most common gastrointestinal malignancies in the world. In the pathological classification of esophageal cancer, there are obvious differences between foreign countries and China. More than 90% of esophageal cancers abroad are adenocarcinomas, while more than 90% of domestic esophageal cancers are squamous cell carcinomas. Since the NCCN guidelines are based on the guidance given by foreign case studies, leading to There are certain differences in the use of NCCN to guide the drug use of domestic esophageal cancer patients. Therefore, it is urgent to establish a Chinese primary cell sample bank to study the pathogenesis of esophageal cancer in vitro and develop new drugs for the treatment of es...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693G01N33/5011C12N2509/00C12N2501/734C12N2501/33C12N2501/105C12N2500/32C12N2501/39C12N2500/84C12N2502/02C12N2503/02G01N2500/10C12N5/0679
Inventor 刘青松胡洁陈程王文超王黎
Owner PRECEDO PHARMA CO LTD
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