Method for carrying out in-vitro efficient amplification on natural killer (NK) cells, and application of method

Abstract

The invention discloses a method for carrying out in-vitro efficient amplification on natural killer (NK) cells, and application of the method, aiming at solving the problems that the NK cells amplified by the traditional local method are small in amplification times, have cytokine dependency and cell aging, and the like. The method is characterized by comprising the following steps: 1, separating peripheral blood mononuclear cells (PBMCs) from human peripheral blood by using the traditional Ficoll-Paque density gradient centrifugation method; 2, carrying out 100 Grays irradiation treatment on artificial antigen presenting cells, and then storing by means of liquid nitrogen cryopreservation; 3, carrying out co-culture on the PBMCs and the artificial antigen presenting cells, subjected to the irradiation treatment, in a cell culture flask T75 of an RPMI 1640 medium, wherein the mass ratio of the PBMCs to the artificial antigen presenting cells is equal to 1: 2, and 50IU / mL of interleukin 2 is added into the RPMI 1640 medium; replacing the medium with a fresh medium every 2 to 3 days; 4, calculating the number of total cells in the cell culture flask T75 every 7 days, adding the same number of artificial antigen presenting cells subjected to the irradiation treatment into the cell culture flask T75 for stimulating again, and continuously culturing for 21 days to obtain the amplified NK cells. Therefore, after the method is adopted, a great deal of high-purity and high-quality NK cells can be obtained.

Description

technical field

[0001] The invention relates to the fields of cell culture and immunotherapy, in particular to a method for efficiently expanding NK cells in vitro and its application. Background technique

[0002] Natural killer cells are differentiated and developed from hematopoietic stem cells. They are a kind of human immune cells. They are effector lymphocytes that have cytolytic effects and can secrete immune regulatory factors. They are called "the first anti-tumor and anti-infection cells in the human body" in medicine. line of defense". Unlike T lymphocytes and B lymphocytes, NK cells can kill target cells (including bacteria, virus-infected host cells and tumor cells) without prior exposure to antigens, and their killing effects are not MHC-restricted, which makes NK cells It plays a huge role in the process of anti-tumor response and immune surveillance of diseased cells and cancerous cells. A large number of clinical experimental data show that NK cell therapy...

Examples

Embodiment Construction

[0043] The present invention will be described in further detail below in conjunction with the accompanying drawings.

[0044] Isolation of PBMCs by Density Gradient Centrifugation

[0045] Step1. Take 5 mL of peripheral blood from a 40-year-old middle-aged volunteer and place it in a sterile centrifuge tube, add 0.5 mL of sodium citrate and mix well;

[0046] Step2. Add 5mL1640 cell culture medium to mix again and dilute;

[0047] Step3. Take 5mL of lymphocyte separation solution and place it in a centrifuge tube, slowly add the blood diluted in Step1 to the separation solution along the tube wall with a dropper, and keep the blood and the separation solution separated in two phases;

[0048] Step4. Centrifuge the centrifuge tube at 1800rpm / min for 25min;

[0049] Step5. Insert the mononuclear cell layer along the tube wall with a capillary pipette, wash out the mononuclear cells and place them in another sterile centrifuge tube;

[0050] Step6. Add 5 times the volume of 1...

technical field

[0001] The invention relates to the fields of cell culture and immunotherapy, in particular to a method for efficiently expanding NK cells in vitro and its application. Background technique

[0002] Natural killer cells are differentiated and developed from hematopoietic stem cells. They are a kind of human immune cells. They are effector lymphocytes that have cytolytic effects and can secrete immune regulatory factors. They are called "the first anti-tumor and anti-infection cells in the human body" in medicine. line of defense". Unlike T lymphocytes and B lymphocytes, NK cells can kill target cells (including bacteria, virus-infected host cells and tumor cells) without prior exposure to antigens, and their killing effects are not MHC-restricted, which makes NK cells It plays a huge role in the process of anti-tumor response and immune surveillance of diseased cells and cancerous cells. A large number of clinical experimental data show that NK cell therapy...

Examples

Embodiment Construction

[0043] The present invention will be described in further detail below in conjunction with the accompanying drawings.

[0044] Isolation of PBMCs by Density Gradient Centrifugation

[0045] Step1. Take 5 mL of peripheral blood from a 40-year-old middle-aged volunteer and place it in a sterile centrifuge tube, add 0.5 mL of sodium citrate and mix well;

[0046] Step2. Add 5mL1640 cell culture medium to mix again and dilute;

[0047] Step3. Take 5mL of lymphocyte separation solution and place it in a centrifuge tube, slowly add the blood diluted in Step1 to the separation solution along the tube wall with a dropper, and keep the blood and the separation solution separated in two phases;

[0048] Step4. Centrifuge the centrifuge tube at 1800rpm / min for 25min;

[0049] Step5. Insert the mononuclear cell layer along the tube wall with a capillary pipette, wash out the mononuclear cells and place them in another sterile centrifuge tube;

[0050] Step6. Add 5 times the volume of 1...

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