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Method for utilizing malignant pleural effusion for separately culturing primary cancer cells

A technology for primary tumor cells and malignant pleural effusion, applied in tumor/cancer cells, cell dissociation methods, animal cells, etc. The effect of solving culture failure and optimizing culture conditions

Inactive Publication Date: 2018-05-04
SHENZHEN RES INST OF WUHAN UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the problem in the prior art that the cultivation of primary tumor cells in malignant pleural effusion is prone to failure, the present invention provides a method based on the sample collection solution (the sample collection solution contains 2% penicillin / streptomycin and 100 μg / mL Nystatin The primary tumor cells of the pleural effusion were obtained by separating the erythrocyte lysate and the PBS buffer, and the primary tumor cells were cultured and subcultured using the KL medium

Method used

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  • Method for utilizing malignant pleural effusion for separately culturing primary cancer cells
  • Method for utilizing malignant pleural effusion for separately culturing primary cancer cells
  • Method for utilizing malignant pleural effusion for separately culturing primary cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] [Example 1] Isolation and culture of primary tumor cells from pleural effusion

[0054] (1) Preparation before the experiment: Add 10 mL of sample collection solution into a 50 mL sterile centrifuge tube to collect pleural effusion;

[0055] (2) Collect the patient's pleural effusion samples into the above centrifuge tubes, each tube can collect 30mL pleural effusion, collect 2 tubes from each patient, and transport them to the cell culture room under refrigerated conditions;

[0056] (3) Centrifuge at 1000rmp for 5min, discard the supernatant;

[0057] (4) Add 5 mL of erythrocyte lysate to remove red blood cells, mix gently, place at room temperature for 5 minutes, shake properly in the middle, centrifuge at 1000rmp, and discard the supernatant for 5 minutes;

[0058] (5) Add 15mL of PBS buffer to wash twice, centrifuge at 1000rmp, and discard the supernatant for 4min;

[0059] (6) Resuspend the cell pellet with 1mL of KL medium, inoculate the cells in a T25 culture ...

Embodiment 2

[0061] [Example 2] Subculture of primary tumor cells from pleural effusion

[0062] (1) When the primary tumor cells of pleural effusion cultured in T25 culture flask proliferate to an abundance of more than 85%, they can be digested and passaged, and the old medium is discarded;

[0063] (2) Rinse the cells with PBS buffer to remove cell secretions that hinder trypsin digestion, wash twice, and be gentle;

[0064] (3) Add 1ml, 0.05% trypsin-EDTA and incubate at 37°C for 2min, shake the cell culture flask lightly to make the digestion solution cover the entire cell layer, then pat;

[0065] (4) When 90% of the cells fall off from the culture flask, add 2 mL of PBS stop solution containing 10% fetal bovine serum to stop the digestion reaction;

[0066] (5) Transfer all the cell suspension that terminated the digestion to a 15mL centrifuge tube, rinse the culture bottle once with PBS buffer, and centrifuge to discard the supernatant;

[0067] (6) Resuspend the cells with 1mL o...

Embodiment 3

[0069] [Example 3] The growth curve drawing of primary tumor cells in pleural effusion

[0070] (1) When the primary tumor cells from pleural effusion cultured in T25 culture flasks proliferate to an abundance of more than 85%, they can be digested and centrifuged and planted in 96-well plates;

[0071] (2) When the primary tumor cells from pleural effusion digest the centrifuged seed plate, repeat the operation steps of Example 2 (2) to (5);

[0072] (3) Prepare a single cell suspension with KL medium, mix the trypan blue and the cell suspension 1:1 and measure the cell number and viability with a cell counter, inoculate 200 μL of the cell suspension in each well on a 96-well plate, and the number of cells is 6000 cells / well, set up 12 multiple holes per day, put in 37°C CO 2 Culture in the incubator for 1 to 7 days respectively;

[0073] (4) Remove the solution in the plate every day, and add 100 μL of serum-free DMEM medium containing 10 μL CKK-8 detection reagent to each...

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Abstract

The invention discloses a method for utilizing a malignant pleural effusion for separately culturing primary cancer cells. The method comprises the steps of putting a pleural effusion sample into a sample collecting liquid, and centrifuging to remove a supernatant; adding a red blood cell lysis buffer for removing a red blood cell, adding a PBS buffer solution for washing the cells, and centrifuging to remove a supernatant; using a KL culture medium for resuspending a cell precipitate, inoculating in a culture flask, and culturing in a culture box. When the cells are proliferated to more than85 percent of abundance, the cells are washed through the PBS buffer solution, pancreatin-EDTA is digested, and a stop buffer neutralizes digestion reaction; the cells are centrifugally collected, areresuspended through the KL culture medium, and inoculated in the cell flask according to the proportion so as to be subcultured. The primary cancer cells of the pleural effusion obtained by separatedculture through the method provided by the invention can be applied to related research of physiology, pharmacy, new drug research and development and the like, exploration of pathogenesis of relateddiseases of a lung cancer, detection of drug sensitivity of different drugs, screening of anti-cancer drugs, new drug research and development, and establishment of a personalized treatment program aiming at a patient.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for separating and culturing primary tumor cells by using malignant pleural effusion and an application of the primary tumor cells obtained by the method. Background technique [0002] 1. Causes of malignant pleural effusion [0003] At present, malignant pleural effusion is mostly caused by the progression of malignant tumors, which is a common complication of advanced malignant tumors. The tumor involves the pleura, which increases the surface permeability, or the lymphatic drainage is blocked, or it is accompanied by obstructive pneumonia involving the pleura. Can cause exudative pleural effusion, which can be seen in pleural mesothelioma, lung cancer, breast cancer, lymphoma, etc. For this reason, correct diagnosis of malignant tumors and their tissue types, and timely and reasonable and effective treatment are of great significance for relieving sym...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693C12N2509/00G01N33/5017
Inventor 李晖陈雨叶立娜吴小婷张康蔡铠红魏高斌曾志宏
Owner SHENZHEN RES INST OF WUHAN UNIVERISTY
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