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Culture medium and culture method of pleural mesothelioma organ

A culture method and organoid technology, applied in the directions of biochemical equipment and methods, 3D culture, culture process, etc., can solve the problem of less research on pleural mesothelioma organoid culture method, test process, operation steps, culture conditions and culture medium The formulation does not have too many reports and other problems to achieve the effect of reducing cytotoxicity and inhibitors, reducing the risk of microbial contamination, and improving the success rate and survival rate

Inactive Publication Date: 2022-01-04
ZHEJIANG CANCER HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although a variety of tumor tissues can be successfully cultured into organoids in vitro using different methods and under different culture conditions, there are very few studies on the culture methods of pleural mesothelioma organoids, especially the specific test procedures and operations. There are not many reports on the steps, culture conditions and medium formulations

Method used

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  • Culture medium and culture method of pleural mesothelioma organ
  • Culture medium and culture method of pleural mesothelioma organ

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Embodiment 1

[0028] A culture medium for pleural mesothelioma organoids, including basal medium Advanced DMEM / F12 and specific additive factors; the specific additive factors include components at the following final concentrations: B27 (without vitamin A), 1× ; N2, 1×; N-acetylcysteine, 2 μM; EGF, 50 ng / ml; Noggin, 50 ng / ml; R-spondin 1, 100 ng / ml; Wnt3a, 50 ng / ml; CHIR99021, 1 μM; μM; VEGFR, 10 ng / ml; PDGFR, 10 ng / ml; penicillin-streptomycin mixed solution, 1×; Primocin, 1mg / ml.

[0029] A method for culturing pleural mesothelioma derived from pleural effusion, comprising the following steps:

[0030] S1. Take fresh pleural effusion from a patient with pleural mesothelioma, centrifuge at 500 rpm for 5 minutes at 4°C, and remove the supernatant for later use;

[0031] S2. Take the above medium and extracellular matrix gel and mix according to the volume ratio of 1:1.5, then use the mixture to resuspend the cell pellet obtained in step S1, and then use a pipette to drop the gel mixed with...

Embodiment 2

[0036] A pleural mesothelioma culture medium, including basal medium Advanced DMEM / F12 and specific additive factors; the specific additive factors include components at the following final concentrations: B27 (without vitamin A), 1×; N2, 1×; N-acetylcysteine, 5 μM; EGF, 10 ng / ml; Noggin, 50 ng / ml; R-spondin 1, 500 ng / ml; Wnt3a, 100 ng / ml; CHIR99021, 1 μM; Thiazovivin, 5 μM; VEGFR , 10 ng / ml; PDGFR, 10 ng / ml; penicillin-streptomycin mixed solution, 1×; Primocin, 1mg / ml.

[0037] A method for culturing pleural mesothelioma derived from puncture tissue, comprising the following steps:

[0038] S1. Collect fresh biopsy tissue (about 2 mm in length) from pleural mesothelioma, wash it twice with PBS buffer containing antibiotics, cut the tissue into small pieces of 0.5 mm, and digest with trypsin at room temperature for 5 minutes , filter with a 70 μm filter to obtain a cell mass with a cell number of 3-50 cells, centrifuge at 500 rpm, 5 min, and 4°C to remove the supernatant for ...

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Abstract

The invention discloses a culture medium and a culture method of a pleural mesothelioma organ. The culture medium comprises a basic culture medium Advanced DMEM / F12 and a specific addition factor, the specific addition factor is prepared from the following components: B27 which does not contain vitamin A, N2, N-acetylcysteine, EGF (Epidermal Growth Factor), Noggin, R-spondin 1, Wnt3a, CHIR99021, Thiazovivin, VEGFR (Vascular Endothelial Growth Factor Receptor), PDGFR, Penicillin and Streptomycin mixed liquor and Primocin. The culture medium contains the least components required by pleural mesothelioma organoid culture, can effectively culture mesothelial cell-derived tumor tissues, does not contain FBS, saves the cost, and reduces the cytotoxicity and inhibitors caused by FBS at the same time. The pleural mesothelioma organoid cultured by the invention maintains the morphological structure and gene characteristics of the primary tissue.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a culture medium and a culture method of pleural mesothelioma organoids. Background technique [0002] Mesothelioma is a cancer that starts in the thin layer of tissue (mesothelial cells) that covers the surface of internal organs. The most commonly affected site is the pleura. The peritoneum is also present, but less commonly. Rarely in the pericardium or testicular sheath. Pleural mesothelioma (pleural mesothelioma) is a primary tumor derived from pleural mesothelial cells, which accounts for 5% of pleural tumors and is rare in clinical practice. Pleural mesothelioma can occur in any part of the visceral pleura and parietal pleura, 80% in the visceral pleura, 20% in the parietal pleura; it can occur at any age, usually between 40 and 60 years old; now the onset rate has an upward trend. [0003] More than 80% of pleural mesotheliomas are caused by exposure to asbestos....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/071
CPCC12N5/0693C12N5/0625C12N2500/32C12N2501/11C12N2501/998C12N2501/415C12N2501/727C12N2501/165C12N2501/135C12N2513/00C12N2535/00C12N2533/90C12N2509/00
Inventor 徐晓玲毛伟敏陈泽新范云徐艳珺李晖
Owner ZHEJIANG CANCER HOSPITAL
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