Simple single circulating tumor cell separation method and apparatus

A tumor cell and separation method technology, applied in the field of simple separation of a single circulating tumor cell, can solve the problems of high performance requirements of instruments and equipment, difficult to popularize and apply, and high price.

Pending Publication Date: 2016-06-15
上海张江转化医学研发中心有限公司 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These two methods have high requirements on the performance of corresponding ins

Method used

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  • Simple single circulating tumor cell separation method and apparatus
  • Simple single circulating tumor cell separation method and apparatus
  • Simple single circulating tumor cell separation method and apparatus

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Embodiment approach

[0075] A preferred embodiment of the present invention is as follows:

[0076] A method for isolating a single circulating tumor cell in peripheral blood by using a circulating tumor cell (CTC) negative enrichment technique and immunofluorescence staining technique combined with a single cell separation device, which includes the following steps:

[0077] (1) 7.5ml of peripheral blood sample was collected by anticoagulant tube, and there was no hemolysis and coagulation phenomenon before the experiment;

[0078] (2) Perform density gradient centrifugation in a horizontal centrifuge to collect mononuclear cell aggregates; add immunomagnetic beads coupled to the leukocyte surface antigen CD45 into the mononuclear cell suspension to separate leukocytes, thereby achieving the purpose of negatively enriching CTCs;

[0079] (4) Harvest the cells to be tested, and perform immunofluorescence staining on the cells according to different experimental purposes:

[0080] a. To perform su...

Embodiment 1

[0093] Example 1 A method for isolating living single circulating tumor cells for subsequent culture

[0094](1) ACD anticoagulant tube collects 7.5ml of peripheral blood samples from tumor patients, and enriches them within 48 hours, and checks before the experiment that there is no hemolysis and coagulation phenomenon. In addition, 2 ml of the patient's peripheral blood was collected with a procoagulant tube, and the serum was separated to be used as a subsequent cell culture additive.

[0095] (2) Negative enrichment of circulating tumor cells in peripheral blood.

[0096] Balance the blood collection tube, mix well by inverting head to tail, and centrifuge at room temperature for 7 minutes (800g). Discard the supernatant to 5 mm above the brownish-red precipitate.

[0097] Add 3ml of cell density gradient centrifugal separation medium into a 50ml centrifuge tube, and slowly add the blood cell mixture after separating the plasma to the top layer of the separation medium a...

Embodiment 2

[0108] Example 2 A method for isolating a single circulating tumor cell for genome analysis

[0109] (1) ACD anticoagulant tube collects 7.5ml of peripheral blood samples, enriches them within 48 hours, and checks for no hemolysis and coagulation before the experiment.

[0110] (2) Negative enrichment of circulating tumor cells in peripheral blood.

[0111] Balance the blood collection tube, mix well by inverting head to tail, and centrifuge at room temperature for 7 minutes (800g). Discard the supernatant to 5 mm above the brownish-red precipitate.

[0112] Add 3ml of cell density gradient centrifugal separation medium into a 50ml centrifuge tube, and slowly add the blood cell mixture after separating the plasma to the top layer of the separation medium along the liquid surface of the tube wall. Use 1ml of 1×CTC buffer to wash the wall of the original blood sample tube, and transfer the washing solution to the top layer of the solution in centrifuge tube B. Centrifuge for ...

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Abstract

The present invention discloses a simple single circulating tumor cell separation method and apparatus. In particular, the present invention discloses a single circulating tumor cell (CTC) separation method, first, an enriched CTC sample is obtained by negative enrichment of a peripheral blood sample; a first-fluorescent-labeled anti-epithelial cell marker antibody and a second-fluorescent-labeled anti-CD45 antibody are respectively used for fluorescent staining of the enriched CTC sample, and the stained enriched CTC sample is diluted; and single cell isolation is performed on first fluorescent positive and second fluorescent negative target cells. The present invention also discloses a single circulating tumor cell (CTC) separation apparatus applicable to the single circulating tumor cell (CTC) separation method. CTC negative enrichment technology is used for the CTC enrichment, compared with a positive magnetic bead enrichment method, disturbance to the target cells is less, and the picked and separated cells can completely maintain cell activity, can be used for nucleic acid and protein analysis, and can also be used in cell culture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a simple method and device for isolating single circulating tumor cells. Background technique [0002] Tumor metastasis is the main cause of death in cancer patients. In recent years, the role of circulating tumor cells (CirculatingTumorCell, CTC) in the process of malignant tumor metastasis has attracted increasing attention. In the process of tumor metastasis, cancer cells break off from the primary tumor and enter the blood circulation system, and a small number of cells survive to become circulating tumor cells, and further develop into metastatic tumors in distant organs. Therefore, the detection of tumor cells in peripheral blood indicates the possibility of tumor metastasis. [0003] The counting of CTCs in the blood of cancer patients has been gradually used to evaluate the efficacy of cancer treatment. Genomic analysis of CTC will help us better understand the biological ...

Claims

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Application Information

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IPC IPC(8): G01N1/40G01N1/30
Inventor 张桢珍楼敬伟许骋吴云鸣吴唯唯易静丁飞飞
Owner 上海张江转化医学研发中心有限公司
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