Cell separation device and cell separation method

A separation device and cell technology, applied in the fields of clinical medicine and biological sciences, can solve the problems of poor cell separation effect, save sorting time, reduce difficulty, and achieve the effect of separation

Active Publication Date: 2019-06-28

SHENZHEN DAKEWE BIOENG

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AI-Extracted Technical Summary

Problems solved by technology

[0006] In order to solve the technical problem of poor cell separation effect in the prior art, th...

Abstract

The invention discloses a device for separating cells. The device comprises an inner tube and an outer tube. A flange which extends outwardly is arranged at an opening end of the inner tube, so that the inner tube can be nested in an opening end of the outer tube; a bottom of the inner tube is separated from a bottom of the outer tube by a preset distance, so that single cells which enter the outer tube and are not captured by three-dimensional antibody nets can be accommodated; the bottom of the inner tube is of a porous structure, and the porous structure is used for separating the single cells which are not captured by the three-dimensional antibody nets from the cells which are captured by the three-dimensional antibody nets; the single cells which are not captured by the three-dimensional antibody nets can enter the outer tube via the porous structure, and three-dimensional antibody nets and the cells which are captured by the three-dimensional antibody nets are entrapped in the inner tube by the porous structure. The device for separating the cells has the advantage that specific types of cells can be captured by the three-dimensional antibody nets, and accordingly the cells can be effectively separated from one another.

Application Domain

Bioreactor/fermenter combinationsBiological substance pretreatments +11

Technology Topic

AntibodyEngineering +3

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Examples

Experimental program(3)

Example Embodiment

[0027] Example 1

[0028] The enrichment of T lymphocytes (CD3+T) cells in peripheral blood mononuclear cells includes the following steps:

[0029] a. Separate peripheral blood mononuclear cells (PBMCs) from 50ml of whole blood by density gradient centrifugation, adjust the cell density to 1×107 cells/ml with phosphate buffer solution (PBS) to prepare a single cell suspension of PBMCs;

[0030] b. Add FP001 (a modified glycan, purchased from Nissan Chemical Industries, LTD, catalog number: 385-07981) with CD3 monoclonal antibody (anti-CD3mAb) and PBS solution at a ratio of 1:5 to a 50ml cone In a centrifuge tube, gently invert and mix evenly to prepare 20ml of solidified anti-CD3mAb three-dimensional antibody network solution;

[0031] c. Add the three-dimensional antibody net solution to the inner tube of the cell separation device, and at the same time add the separated PBMCs to the inner tube, gently invert to mix, and incubate at room temperature for 20 minutes;

[0032] d. Centrifuge at 500g centrifugal force for 10 minutes, add 5ml PBS solution to transfer the three-dimensional antibody net and cells retained by the screen to another conical centrifuge tube;

[0033] e. Dilute the harvested supernatant by adding more than 3 times the volume of PBS solution, centrifuge at 800g centrifugal force for 10 minutes, and collect the cell pellet to be the enriched CD3+ T cells;

[0034] f. Flow cytometry analysis of the purity of the enriched CD3+ T lymphocytes, from figure 2 It can be seen that the average purity is 92±3%.

Example Embodiment

[0035] Example 2

[0036] The removal of CD45+ cells from peripheral blood mononuclear cells includes the following steps:

[0037] a. Separate peripheral blood mononuclear cells in 30ml whole blood by density gradient centrifugation, adjust the cell density to 1×107 cells/ml with PBS solution;

[0038] b. Add the solidified CD45 monoclonal antibody FP001 and PBS solution in a ratio of 1:6 to a 50ml conical centrifuge tube, gently invert and mix well to prepare 15ml anti-CD45mAb three-dimensional antibody network solution;

[0039] c. Add the three-dimensional antibody net solution to the inner tube of the cell separation device, and at the same time add the separated PBMCs to the inner tube, gently invert to mix, and incubate at room temperature for 20 minutes;

[0040] d. Centrifuge at 500g centrifugal force for 10 minutes, and collect the cell pellet in the outer tube to be the PBMCs after removing CD45+ cells;

[0041] e. Analysis of CD45+ cell removal rate by flow cytometry, from image 3 It can be seen that the removal rate is 84±3%. Example 3

Example Embodiment

[0041] e. Analysis of CD45+ cell removal rate by flow cytometry, from image 3 It can be seen that the removal rate is 84±3%. Example 3

[0042] The enrichment of circulating tumor cells in whole blood includes the following steps:

[0043] a. Heparin anticoagulation draws 10ml of peripheral blood from tumor patients;

[0044] b. Add the solidified epithelial cell adhesion molecule monoclonal antibody (anti-EpCAM mAb) FP001 and PBS solution at a ratio of 1:4 into a 50ml conical centrifuge tube, gently invert and mix evenly to prepare 30ml solidified anti- EpCAM mAb three-dimensional antibody network solution;

[0045] c. Add the three-dimensional antibody net solution to the inner tube of the cell separation device, and at the same time add the separated PBMCs to the inner tube, gently invert to mix, and incubate at room temperature for 20 minutes;

[0046] d. Centrifuge for 10 minutes at a centrifugal force of 500g, add 5ml PBS to the inner tube to transfer the three-dimensional antibody net and cells retained by the screen to another conical centrifuge tube;

[0047] e. Dilute the harvested supernatant by adding more than 3 times the volume of PBS solution, centrifuge at 800g centrifugal force for 10 minutes, and collect the cell pellet to be the enriched EpCAM-positive circulating tumor cells.

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Classification and recommendation of technical efficacy words

achieve separation

Cell separation device and cell separation method

A separation device and cell technology, applied in the fields of clinical medicine and biological sciences, can solve the problems of poor cell separation effect, save sorting time, reduce difficulty, and achieve the effect of separation

AI-Extracted Technical Summary

Problems solved by technology

Abstract

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Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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