Special medium used for in-vitro culture of esophagus cancer tumor organoid based on human esophagus cancer tissue and culture method

An in vitro culture and esophageal cancer technology, applied in the field of tumor organoid culture, can solve the problems of slow growth, easy death, and reduced vitality, and achieve the effect of improving success rate, increasing growth speed, and good vitality

Pending Publication Date: 2016-12-07
张云霞
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The application of the original organoid culture technology to the culture of human esophageal cancer organoids has the following problems: the first-generation culture takes a long time, and it takes 2 to 6 weeks to complete the first-generation culture; the viability of organoids decreases rapidly after passage, and the growth rate is obvious Slow down; recovery efficiency is low after cryopreservation, death is easy, vigor is reduced, and growth rate is slowed down; the success rate of culture is not high, and the fresh specimens obtained by surgery are less than 60%; for biopsy specimens, the original organoid culture technology is almost ineffective nourish

Method used

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  • Special medium used for in-vitro culture of esophagus cancer tumor organoid based on human esophagus cancer tissue and culture method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The preparation of embodiment one special culture medium

[0072] The culture medium of the present invention contains

[0073] One or several receptor tyrosine kinase ligands: including human or animal epidermal growth factor, the concentration ranges from 2ng / mL to 100ng / mL; human or animal hepatocyte growth factor, the concentration ranges from 10ng / mL to 200ng / mL; human or animal basic fibroblast growth factor, the concentration range is 5ng / mL to 100ng / mL;

[0074] Rho-related Frizzled kinase inhibitor: selected from Fasudil, Y-27632, RKI-1447 and GSK429286A;

[0075] Multiple vitamins and hormones: putrescine, concentration range 0.1 μg / mL to 100 μg / mL; epinephrine, concentration range 10 ng / mL to 10 μg / mL; niacinamide, concentration range 1 μg / mL to 2 mg / mL; A acetate, the concentration range is 10ng / mL to 500ng / mL;

[0076] Antioxidant: selected from reduced glutathione; vitamin C; vitamin E; beta-mercaptoethanol (0.01mM to 1.5mM);

[0077] Agonists of pathw...

Embodiment 2

[0078] Example 2 Preparation of ECRM-I, ECRM-II, ECRM-III medium for rapid expansion of primary esophageal cancer cells and EDM-1 medium for scale-up culture and drug-sensitive detection

[0079] The formulation of ECRM-I includes DMEM / F12 medium supplemented with 2.5% FBS, 1x B27 supplement, ascorbic acid, vitamin A acetate, epinephrine, putrescine, nicotinamide, insulin, hydrocortisone, human activin A, FGF2 , Wnt3a, EGF, retinoic acid and Y27632.

[0080] The formulation of ECRM-II medium includes DMEM / F12 medium supplemented with 2.5% FBS, 1x B27 supplement, nicotinamide, epinephrine, BMP4, EGF, Wnt3a, IGF, sodium butyrate, putrescine, vitamin A acetate, insulin , dexamethasone, β-mercaptoethanol, FGF2, human activin A, retinoic acid and Y27632.

[0081] The formulation of ECRM-III medium includes DMEM / F12 medium, supplemented with 1% BSA, 1x B27 supplement, mold hydrogen peroxide, vitamin A acetate, nicotinamide, epinephrine, sodium butyrate, insulin, dexamethasone, putr...

Embodiment 3

[0083] Example 3 Preparation of Tissue Digestive Solution

[0084] Optimizing surgical and biopsy tissue digests for maximum yield while maintaining tumor viability.

[0085] The formula of EM-BS-1 tissue enzymatic hydrolysis solution is as follows: add 2.5% FBS, type IX collagen (1 μg / mL-10 μg / mL), neutral proteolytic enzyme (50 μg / mL-500 μg / mL) to DMEM medium .

[0086] The formula of EM-BS-2 tissue enzymatic solution is as follows: add 2.5% FBS, 1x penicillin and streptomycin, type IV collagenase (0.01mg / mL-1mg / mL), hyaluronidase (0.001 mg / mL-0.2mg / mL) and DNase I (0.001mg / mL-0.2mg / mL).

[0087] The formula of EM-3 enzymatic hydrolysis solution is as follows: add 2.5% FBS, 1x penicillin and streptomycin, XI collagenase (20U / mL-200U / mL), dispase neutral proteolytic enzyme I (1μg / mL-100μg / mL) and DNase I (0.01mg / mL-1mg / mL).

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Abstract

The invention relates to the field of culture of tumor organoids, and in particular relates to a special medium used for in-vitro culture of esophagus cancer tumor organoid based on human esophagus cancer tissue and a culture method. The special medium comprises a receptor tyrosine kinase ligand, an Rho-associated coiled-coil protein kinase inhibitor, vitamin and hormone, an antioxidant, and an agonist for inhibiting cell differentiation pathway. According to the method, through the step-by-step culture technology adopting conditional dedifferentiation, tumor cells rapidly amplify within a short time to form the organoid. Through the step-by-step culture technology, primary cells rapidly form the organoid, meanwhile, adequate cells are formed within the effective time, so that the follow-up experiment operation can be conveniently carried out, and therefore, the culture speed and success rate of the organoid are improved.

Description

technical field [0001] The invention relates to the field of culturing tumor organoids, in particular to a special culture medium and a culturing method for culturing esophageal cancer tumor organoids in vitro based on human esophageal cancer tissues. Background technique [0002] Esophageal cancer is a common malignant tumor of the digestive tract, accounting for 2% of all malignant tumors. About 200,000 to 300,000 people die of esophageal cancer every year in the world. Esophageal cancer should rank first among digestive system tumors in my country, and ranks very high in the entire tumor ranking, about four or five. Compared with other countries, the incidence of esophageal cancer in my country is relatively high, and nearly half of the world's esophageal cancer occurs in China. my country is the country with the highest morbidity and mortality rate of esophageal cancer, accounting for more than half of the world's number of esophageal cancer cases and deaths every year...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/30C12N2500/38C12N2500/44C12N2500/46C12N2501/11C12N2501/115C12N2501/12C12N2501/16C12N2501/41C12N2501/415C12N2501/727C12N2501/81
Inventor 张云霞
Owner 张云霞
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