Method for fast and efficient separation and purification of tumor cells from malignant pleural effusion and ascites

A technology for the separation and purification of tumor cells, which is applied in the field of fast and efficient separation and purification of tumor cells from malignant pleural effusion, which can solve the problems of separation and purification steps, the inability to conduct further research, and the small amount of cell collection, so as to achieve small cell damage , Tumor cells are in good condition, and the effect is reproducible

Inactive Publication Date: 2015-10-07
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods all have different defects, such as the small amount of cell collection, separation and purification steps, low degree of purification, and the need for special separation and purification reagents such as tumor cell separation liquid and lympho

Method used

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  • Method for fast and efficient separation and purification of tumor cells from malignant pleural effusion and ascites
  • Method for fast and efficient separation and purification of tumor cells from malignant pleural effusion and ascites
  • Method for fast and efficient separation and purification of tumor cells from malignant pleural effusion and ascites

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0030] Example 1

[0031] 1. Main experimental materials

[0032] (1) Source of pleural fluid: pleural effusion of patients with lung adenocarcinoma.

[0033] (2) PBS solution: potassium dihydrogen phosphate (KH 2 PO 4 ): 0.27g, disodium hydrogen phosphate (Na 2 HPO 4 ): 1.42g, sodium chloride (NaCl): 8g, potassium chloride (KCl) 0.2g, add about 800mL of deionized water to fully stir to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.2-7.4, and finally dilute to 1L. Prepared 24 hours before the experiment, sterilized at high temperature and autoclaved, and sealed at room temperature.

[0034] (3) Calf serum: Hangzhou Sijiqing Company.

[0035] (4) 50ml centrifuge tube: American BD company.

[0036] (5) Sterile glass Pasteur pipette: American Fasher company

[0037] (6) Centrifuge: Eppendrof, Germany

[0038] 2. Operation steps

[0039] (1) Aseptically extract 500ml of pleural effusion from lung cancer patients, take 200ml from it, put them into 4 50ml centrifuge ...

Example Embodiment

[0042] Example 2

[0043] The difference from Example 1 is:

[0044] 2. Operation steps

[0045] (1) Aseptically extract 500ml of pleural effusion from lung cancer patients, take 200ml from it, put them into 4 50ml centrifuge tubes, and centrifuge at 1500r / min for 5 minutes at room temperature to obtain cell pellet.

[0046] (2) Collect and mix the cell pellet with 20ml PBS liquid to obtain a tumor cell suspension. Take a sterile 50ml centrifuge tube and add 30ml calf serum. The specific viscosity of the serum (relative to distilled water) is 1.8. Use a Pasteur pipette to gently add the mixed tumor cell suspension to the serum along the tube wall to divide it into two layers, with the tumor cell suspension layer on top and the serum layer on the bottom, and the height is not less than 4cm.

[0047] (3) Leave the centrifuge tube added with the tumor cell suspension layer for 5 minutes. Use a Pasteur pipette to suck 5ml of serum from the bottom of the centrifuge tube. Take care not to...

Example Embodiment

[0048] Example 3

[0049] The difference from Example 1 is:

[0050] 2. Operation steps

[0051] (1) Aseptically extract 500ml of pleural effusion from lung cancer patients, take 200ml from it, put them into 4 50ml centrifuge tubes, and centrifuge at 1500r / min for 5 minutes at room temperature to obtain cell pellet.

[0052] (2) Collect and mix the cell pellet with 20ml PBS liquid to obtain a tumor cell suspension. Take a sterile 50ml centrifuge tube and add 20ml calf serum. The specific viscosity of the serum (relative to distilled water) is 1.6. Use a Pasteur pipette to gently add the mixed tumor cell suspension to the serum along the tube wall to divide it into two layers, with the tumor cell suspension layer on top and the serum layer on the bottom, and the height is not less than 4cm.

[0053] (3) Leave the centrifuge tube with the tumor cell suspension layer to stand for 6 minutes, and use a Pasteur pipette to suck 5ml of serum from the bottom of the centrifuge tube. Take care...

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Abstract

The invention discloses a method for fast and efficient separation and purification of tumor cells from malignant pleural effusion and ascites. The method includes the steps of: (1) conducting aseptic operation to collect malignant pleural effusion and ascites, conducting subpackaging in sterile centrifuge tubes, and performing centrifugation at room temperature to obtain cell precipitate; (2) conducting re-suspension on the cell precipitate with an aseptic PBS liquid to obtain a tumor cell suspension, adding the tumor cell suspension onto the serum in the centrifuge tubes along the pipe walls of the centrifuge tubes to form a two-layer solution in the centrifuge tubes, with the upper layer being a tumor cell suspension layer and the lower layer being a serum layer; and (3) standing the centrifuge tubes added with the tumor cell suspension in step (2) for 5-8min, then sucking the serum at the tube bottoms by a pasteur pipet without destroying liquid layering, placing the sucked serum in a new centrifuge tube, adding aseptic PBS liquid, conducting centrifugation at room temperature, and discarding the supernatant, thus obtaining purified tumor cells. The method provided by the invention is suitable for separation and purification of tumor cells in various malignant pleural effusion and ascites, and avoids the limitations of existing separation and purification methods.

Description

technical field [0001] The invention relates to a method for separating tumor cells, in particular to a method for rapidly and efficiently separating and purifying tumor cells from malignant pleural ascites. Background technique [0002] Pleural effusion is a common clinical symptom in respiratory medicine. It is divided into two types: exudative and exudative. It can be caused by inflammatory lesions, neoplastic lesions, systemic diseases, and certain organ lesions. Among them, malignant pleural effusion is mainly caused by malignant tumors of thoracic organs, especially common in advanced lung cancer, with an incidence rate as high as 60%. And often progressive. The main cause of the disease is the invasion and obstruction of capillaries and lymphatic vessels by pleural metastatic nodules. Therefore, the pleural effusion contains a large amount of protein and formed blood components, and bloody pleural effusion accounts for about 75%. [0003] Ascites is excessive fluid ...

Claims

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Application Information

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IPC IPC(8): C12N5/09
Inventor 陈豫钦喻鹏久
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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