Method for mixed cultivation of multiple immune cells

A technology of mixed culture and immune cells, applied in the field of mixed culture of various immune cells, can solve the problems of limiting the activity of killing tumor cells, low proliferation rate of immune cells, and inability to form multiple killing activities

Inactive Publication Date: 2017-03-08
BEIJING YULONG SHENGSHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, only two kinds of cells are mixed and cultured, which cannot form multiple killing activities, which limits the activity of killing tumor cells, and the proliferation rate of immune cells is low.

Method used

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  • Method for mixed cultivation of multiple immune cells
  • Method for mixed cultivation of multiple immune cells
  • Method for mixed cultivation of multiple immune cells

Examples

Experimental program
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Effect test

Embodiment 1

[0035] A method for mixed culture of various immune cells, comprising the following steps:

[0036] DC-CIK cells, DC-CTL cells, and DC-NK cells were mixed and cultured at a cell number ratio of 13:2:4. The medium was composed of KBM581 culture solution and solute, and the solute and its culture medium in KBM581 The concentration in the solution is 40IU / mL interleukin 2 and 90IU / mL interleukin 15. After 3 days of mixed culture in the medium, half of the medium was changed; after 6 days of culture, half of the medium was changed again, and 200U / mL rhIL was added. -4, continue culturing for 8 days to obtain the immune cell population.

[0037] The preparation of the DC-CIK cells, DC-CTL cells, and DC-NK cells includes the following steps,

[0038] 1) Take a bottle of lung cancer A549 cells, use MEM + 10% FBS + double antibody + 1mol / L glutamine as the medium, culture to a monolayer, digest with ET digestion solution, stop digestion with stop solution, collect cells, 1000 rpm Se...

Embodiment 2

[0042] A method for mixed culture of various immune cells, comprising the following steps,

[0043] DC-CIK cells, DC-CTL cells, and DC-NK cells were mixed and cultured at a cell number ratio of 17:4:6. The culture medium was composed of KBM581 culture fluid and solute, and the solute and its culture medium in KBM581 The concentration in the solution was 400IU / mL interleukin 2 and 600IU / mL interleukin 15. After 4 days of mixed culture in the medium, half of the medium was changed; after 7 days of culture, half of the medium was changed again, and rhIL-450U / mL was added. , and continue culturing for 9 days to obtain the immune cell population.

[0044] The preparation of the DC-CIK cells, DC-CTL cells, and DC-NK cells includes the following steps,

[0045] 1) Take a bottle of lung cancer A549 cells, use MEM + 10% FBS + double antibody + 1mol / L glutamine as the medium, culture to a single layer, digest with ET digestion solution, stop digestion with stop solution, collect cells,...

Embodiment 3

[0049] A method for mixed culture of various immune cells, comprising the following steps,

[0050] DC-CIK cells, DC-CTL cells, and DC-NK cells were mixed and cultured at a cell number ratio of 15:3:5. The medium was composed of KBM581 culture medium and solute, and the solute and its culture medium in KBM581 The concentration in the solution was 250IU / mL interleukin 2 and 400IU / mL interleukin 15. After 4 days of mixed culture in the medium, half of the medium was changed; after 7 days of culture, half of the medium was changed again, and 400U / mL rhIL was added. -4, continue culturing for 8 days to obtain the immune cell population.

[0051] The preparation of the DC-CIK cells, DC-CTL cells, and DC-NK cells includes the following steps,

[0052] 1) Take a bottle of lung cancer A549 cells, use MEM + 10% FBS + double antibody + 1mol / L glutamine as the medium, culture to a single layer, digest with ET digestion solution, stop digestion with stop solution, collect cells, 1200 rpm...

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Abstract

The invention discloses a method for mixed cultivation of multiple immune cells. The method comprises the following steps: conducting mixed cultivation on prepared DC-CIK cells, DC-CTL cells and DC-NK cells at a ratio of (13-17) to (2-4) to (4-6) in accordance with the quantity of the cells in a culture medium for 3-4 days, and changing half amount of the culture medium, wherein the culture medium consists of a KBM581 culture solution and a solute, and the solute consists of 40-400IU / mL of interleukin 2 and 90-600IU / mL of interleukin 15 in accordance with concentrations in the KBM581 culture solution; and after conducting the mixed cultivation for 6-7 days, changing half amount of the culture medium once again and supplementing 50-400U / mL of rhIL-4, and continuing the cultivation, so that an immune cell population is obtained. According to the method provided by the invention, by conducting mixed cultivation on the prepared DC-CIK cells, DC-CTL cells and DC-NK cells at a certain proportion, and by obtaining an optimal mixing proportion and an optimal cultivation mode through tests, the immune cell population, which is consistent in life cycle and has multiple killing activities, is obtained, so that the anti-virus activity and a cell proliferation rate of the immune cells are greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mixed culture method of various immune cells. Background technique [0002] Dendritic cells (DC) are derived from precursor cells in the bone marrow. They are known to have the strongest antigen-presenting ability in the body, 1000 times stronger than ordinary antigen-presenting cells. After being loaded with tumor antigens, they can stimulate T lymphocytes differentiate into cytotoxic T lymphocytes with anti-tumor activity and specifically kill tumor cells. [0003] Cytokine-induced killer cells (cytokine-induced killers, CIK) are a kind of anti-tumor and anti-virus effector cells, which can be induced and proliferate in large quantities in vitro. [0004] Cytotoxic T lymphocytes (cytotoxic lymphocytes, CTL), also known as TC cells (cytotoxic T cells), is a subdivision of white blood cells, a specific T cell, specialized in secreting various cytokines to participate i...

Claims

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Application Information

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IPC IPC(8): C12N5/0784C12N5/0783
CPCC12N5/0639C12N5/0638C12N5/0646C12N2500/05C12N2500/90C12N2501/22C12N2501/2302C12N2501/2315C12N2501/24C12N2501/25C12N2502/1114C12N2502/1121C12N2502/1164
Inventor张晓南陈虎吴芳春马一盖冯凯贾绍昌王蔚
OwnerBEIJING YULONG SHENGSHI BIOTECH CO LTD