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Method for preparing suspension tumor cell organoids from malignant pleural effusion

A tumor cell and organoid technology, applied in the field of the preparation of suspended tumor cell organoids, can solve the problems of high culture cost, low culture success rate, loss of anti-anoikis properties of suspended tumor cells, etc., and achieves a high degree of fit.

Active Publication Date: 2021-08-27
科途(固安)生物科技有限公司
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Problems solved by technology

[0004] In the prior art, tumor cells separated from malignant pleural and ascites can be mixed with extracellular matrix (such as 3D-matrigel), and tumor cells can be cultured in vitro under the condition of adding various growth factors and compound culture medium, but this method There are defects such as low culture success rate and high culture cost, and often the cultured suspension tumor cells lose the anti-anoikis characteristics of suspended malignant tumor cells in malignant pleural ascites

Method used

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Examples

Experimental program
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Effect test

Embodiment

[0053] 1. Collection of malignant pleural effusion

[0054] Under B-ultrasound positioning, aseptic puncture, take 300 mL of malignant pleural and ascites, and add heparin, so that the concentration of the heparin contained in the malignant pleural and ascites is 50 U / mL.

[0055] 2. Isolation of the first dissociated cells

[0056] In the biological safety cabinet, the malignant pleural ascites obtained in step 1 was filtered through a nylon microporous filter with a pore size of 70-100 μm, so that the clot part and the liquid part of the malignant pleural ascites were separated. Centrifuge the liquid part at 4 °C and 500 rpm / min for 30 min, discard the supernatant to obtain free cell pellets; resuspend the obtained free cell pellets in PBS without calcium and magnesium ions, wash the cells, and place in 4 Centrifuge at 1000 rpm / min for 10 min, and repeatedly wash the free cell pellet 2-3 times to obtain the first free cell.

[0057] 3. Isolation of the second dissociated c...

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Abstract

The present disclosure relates to a method for preparing suspended tumor cell organoids from malignant pleural ascites, characterized in that the method comprises: a. obtaining free cells in malignant pleural ascites, wherein the free cells include first free cells and second free cells Cells, the first free cells are derived from the liquid part of the malignant pleural effusion, and the second free cells are derived from the clot part of the malignant pleural effusion; b. Obtain the monocytes and tumor cells contained in the free cells to obtain raw cells; c. Mix the raw material cells with agarose gel and medium for pre-cultivation to obtain pre-cultured cells; d. Mix the pre-cultured cells with agarose gel and medium for 12‑15 days to obtain suspension tumor cell organoids . The biological effects of the suspended tumor cell organoids cultured by the disclosed method are highly consistent with the biological characteristics of the suspended tumor cells in malignant pleural ascites, and have the characteristics of malignant tumor cells resistant to anoikis.

Description

technical field [0001] The present disclosure relates to the technical field of biomedicine, in particular to a method for preparing suspended tumor cell organoids using malignant pleural and ascites fluid. Background technique [0002] Malignant pleural effusion refers to the abnormal increase of body cavity fluid caused by diffuse lesions in the thoracic cavity and visceral parietal pleuraperitoneum caused by malignant tumors or cancerous lesions that occur in the whole body or in the thoracic and abdominal cavity. Malignant pleural effusion is a common clinical symptom in patients with lung cancer, breast cancer, gastric cancer, colorectal cancer, ovarian cancer and other cancers due to tumor metastasis to the thoracoabdominal cavity. Tumor cells in pleural effusion can continue to proliferate and grow in suspension in vitro, and have the ability to resist apoptosis. The rate of apoptosis (anoikis) after detaching from the extracellular matrix is ​​less than 1%, and more ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0786C12N5/09
CPCC12N5/0645C12N5/0693
Inventor 孙志坚康平李程
Owner 科途(固安)生物科技有限公司
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