Culture method of placental blood NK (natural killer) cells

A culture method and NK cell technology, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of difficult clinical use of NK cells, high operating conditions and high cost, and achieve easy promotion and application, and simple operation , low cost effect

Inactive Publication Date: 2019-03-22
药鼎(北京)国际细胞医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods are relatively high in terms of operating conditions and co

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  • Culture method of placental blood NK (natural killer) cells
  • Culture method of placental blood NK (natural killer) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A culture method for placental blood NK cells, the steps are as follows:

[0044] 1.1 Placental blood NK cell culture medium

[0045] 1) Basal medium: VIVO-15, GT-T581;

[0046] 2) Additives: FBS, IL-2, IL-12, IL-15, CD16 monoclonal antibody;

[0047] 1.2 Placental blood NK cell method

[0048] 1) Coating: Take a T25 culture flask, add 1 mL LD-PBS, add 3 μg CD16 monoclonal antibody, put it in a 37°C incubator for coating for 4 hours.

[0049] 2) PBMC separation: Ficoll density gradient method was used to separate and extract PBMC cells.

[0050] 3) Adjust the cell density to about 1×10 with VIVO-15 medium 6 / mL inoculated into the coated T25 culture flask, 5mL / bottle.

[0051] 4) Add FBS 250μL on day 0, add IL-2: 2500IU, IL-12: 12.5ng, IL-15: 100ng.

[0052] 5) On the 4th day, coat a T75 culture flask, add 3mL LD-PBS, add 9μg CD16 monoclonal antibody, and put it into a 37°C incubator for coating for 4 hours. Take out the T75 bottle, pour off the coating solution,...

Embodiment 2

[0064] A culture method for placental blood NK cells, the steps are as follows:

[0065] 1.1 Placental blood NK cell culture medium

[0066] 1) Basal medium: VIVO-15, GT-T581;

[0067] 2) Additives: FBS, IL-2, IL-12, IL-15, CD16 monoclonal antibody;

[0068] 1.2 Placental blood NK cell method

[0069] 1) Coating: Take a T25 culture flask, add 1 mL LD-PBS, add 3 μg CD16 monoclonal antibody, put it in a 37°C incubator for coating for 4 hours.

[0070] 2) PBMC separation: Ficoll density gradient separation was used to extract PBMC cells.

[0071] 3) Adjust the cell density to about 0.6×10 with VIVO-15 medium 6 / mL inoculated into the coated T25 culture flask, 5mL / bottle.

[0072] 4) Add FBS 260μL on day 0, add IL-2: 2500IU, IL-12: 13.5ng, IL-15: 90ng.

[0073] 5) On the 4th day, coat a T75 culture flask, add 3mL LD-PBS, add 9μg CD16 monoclonal antibody, and put it into a 37°C incubator for coating for 4 hours. Take out the T75 bottle, pour off the coating solution, transfe...

Embodiment 3

[0080] A culture method for placental blood NK cells, the steps are as follows:

[0081] 1.1 Placental blood NK cell culture medium

[0082] 1) Basal medium: VIVO-15, GT-T581;

[0083] 2) Additives: FBS, IL-2, IL-12, IL-15, CD16 monoclonal antibody;

[0084] 1.2 Placental blood NK cell method

[0085] 1) Coating: Take a T25 culture flask, add 1 mL LD-PBS, add 3 μg CD16 monoclonal antibody, put it in a 37°C incubator for coating for 4 hours.

[0086] 2) PBMC separation: Ficoll density gradient separation was used to extract PBMC cells.

[0087] 3) Adjust the cell density to about 2×10 with VIVO-15 medium 6 / mL inoculated into the coated T25 culture flask, 5mL / bottle.

[0088] 4) Add FBS 250μL on day 0, add IL-2: 2500IU, IL-12: 12.5ng, IL-15: 100ng.

[0089] 5) On the 4th day, coat a T75 culture flask, add 3mL LD-PBS, add 9μg CD16 monoclonal antibody, and put it into a 37°C incubator for coating for 4 hours. Take out the T75 bottle, pour off the coating solution, transfer...

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PUM

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Abstract

The invention relates to the field of cell culture, in particular to a culture method of placental blood NK (natural killer) cells. The method comprises the steps as follows: inoculating a culture vessel coated with a CD16 monoclonal antibody with PBMC cells separated from placental blood, and performing culture, wherein components including FBS, IL-2, IL-12 and IL-15 are added to a culture systemon the basis of a VIVO-15 base culture medium; performing enlarged culture after 4-4.5 days of culture, wherein components including FBS, IL-2, IL-12 and IL-15 are added to a culture system for enlarged culture on the basis of a GT-T581 base culture medium. The culture method of the placental blood NK cells is easy to operate and low in cost, more NK cells meeting the clinical use standard can becultured, and a good foundation is laid for application of the placental blood NK cells.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for culturing placental blood NK cells. Background technique [0002] NK cells, also known as natural killer cells, are large granular lymphocytes, derived from bone marrow, distributed in peripheral blood, liver and spleen, and a small amount in lymph nodes and other tissues, accounting for 5% to 15% of peripheral blood lymphocytes. important immune cells. NK cells are an important mechanism of the body's initial defense and have strong cytotoxic activity. At the same time, it plays an immune regulatory role, rapidly improving immunity and rehabilitation quality. Scientists identified NK cells for the first time in 1975. This type of cells can directly kill tumor cells in the absence of T and B cells. Unlike cytotoxic CD8+ T cells, NK cells can directly kill MHC-negative tumor cells without pre-sensitization when killing tumor cells, which makes NK cells widely used in ado...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 周海涛
Owner 药鼎(北京)国际细胞医学技术有限公司
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