Cell sorting system based on endonuclease specific recognition

An endonuclease and sorting system technology, which is applied in the field of cell sorting systems based on the specific recognition of endonucleases, can solve the problems of small cell damage, low yield, and the sorting process is not gentle enough, and achieves cell The effect of low damage, high cell yield and survival rate

Active Publication Date: 2015-04-08
CYAGEN BIOSCI INC
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

[0007] The above technologies have the following disadvantages: Although the flow cytometry separation technology can sort the target cells, ① the cells must be able to be labeled by fluorescent molecules; ② can only detect single cells in suspension; ③ the sorting process is not gentle enough, causing great damage to the cells; ④It may contaminate the cells; ⑤It is not conducive to the sorting of large volume samples
Although immunomagnetic bead sorting technology can obtain target cells through positive and negative screening, ① cannot complete "one-time combination" to se

Method used

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  • Cell sorting system based on endonuclease specific recognition
  • Cell sorting system based on endonuclease specific recognition
  • Cell sorting system based on endonuclease specific recognition

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Embodiment 1

[0067] Cell therapy dominated by immune cell therapy has a great effect in cancer treatment. The main cell types used in immune cell therapy are: DC cells, NK cells and CIK cells. Due to the different culture conditions, these cells need to be separated and then cultured to achieve a good expansion effect. The purpose of this embodiment is to separate these three kinds of cells in the blood at one time, and the specific separation scheme is as follows:

[0068] 1 Experimental materials

[0069] ①CD3, CD56 and HLA-DR antibodies were purchased from Biolegend;

[0070] ②Single-stranded nucleotide fragments modified with sulfhydryl and biotin, synthesized at Jinweizhi Company;

[0071] ③ Endonucleases BamH I, EcoR I, Hind III were purchased from NEB Company;

[0072] ④Streptavidin magnetic beads were purchased from Dongguan Hannuo Biotechnology Co., Ltd.;

[0073] ⑤Cell culture medium X-VIVO TM 15Chemically Defined, Serum-free Hematopoietic Cell Medium, purchased from U.S. Lo...

Embodiment 2

[0103] 1 Experimental method

[0104] ① Separate NK cells from human peripheral blood mononuclear cells by flow cytometry and the sorting method in Example 1, culture them, and observe the cell morphology.

[0105] ② MTT method to detect cell viability, the steps are as follows:

[0106] a) Cell inoculation: inoculate a cell suspension of appropriate concentration into a 96-well plate at a volume of 100 μL-200 μL / well, and set 10 duplicate wells. In addition, blank control wells were set up with culture medium.

[0107] b) Cell culture: 5% CO 2 , and incubate the cultured cells at 37°C.

[0108] c) Color development: after culturing, add MTT solution (5 mg / mL) with 10% volume of the culture solution in the well, and incubate for 2-4 hours.

[0109] d) The culture was terminated, and the supernatant was discarded after centrifugation (250g×4min).

[0110]e) Add 100 μL DMSO to each well, place on a plate shaker and shake for 5-10 minutes to fully melt the crystals. Set zer...

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Abstract

The invention relates to a cell sorting system based on endonuclease specific recognition. The cell sorting system based on the endonuclease specific recognition comprises the following components: a first single-stranded nucleotide, a second single-stranded nucleotide, an antibody, a cell sorting medium and a restriction endonuclease, wherein the first single-stranded nucleotide and the second single-stranded nucleotide are complementary, and a complementary region contains a recognition site of the restriction endonuclease; the first single-stranded nucleotide, the second single-stranded nucleotide, the antibody and the cell sorting medium are all modified, the modified first single-stranded nucleotide can be connected with the modified antibody, and the modified second single-stranded nucleotide can be connected with the modified cell sorting medium. The invention also provides a cell sorting method applying the cell sorting system based on the endonuclease specific recognition, and a kit and method for sorting DC (dendritic) cells, NK (natural killer) cells and CIK (cytokine-induced killer) cells from cell samples. By adopting the cell sorting technology provided by the invention, multiple cells can be sorted at the same time, sorting of a large number of delicate cells can be carried out, and the cell yield and survival rate are high.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a cell sorting system based on endonuclease specific recognition. Background technique [0002] There are two main types of cell sorting techniques: [0003] ① Flow cytometry technology [0004] This is achieved by isolating droplets containing single cells. The cells to be tested are combined with monoclonal antibodies and fluorescent dyes to make suspension samples, and the samples enter the flow chamber under a certain gas pressure, and are arranged in a single row under the cell-free buffer package. Under the high-frequency oscillation of the ultra-high-frequency piezoelectric crystal in the flow chamber, the liquid flow is broken into uniform droplets, and the cells to be tested are included in the droplets. These droplets are charged with positive or negative charges. When the charged droplets pass through the electric field, they are deflected under the action of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/34G01N33/68C12N5/0784C12N5/0783
CPCC12N5/0639C12N5/0646C12N2509/00
Inventor 蓝田J·盖茨郑敦武
Owner CYAGEN BIOSCI INC
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