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NK cell culture solution and cell culture method

A cell culture and NK cell technology, applied in the field of cells, can solve the problems of increasing cost, increasing separation steps, and easy introduction of exogenous cells, etc., to reduce risks, simplify separation steps, optimize cytokine combination and culture The effect of the process

Active Publication Date: 2016-12-14
湖南丰晖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In view of this, the present invention provides a cell culture solution, a cell culture method and its application, which are used to solve the need to use trophoblast cells in the prior art, which is easy to introduce exogenous cells; or to purify NK cells to increase separation Steps to increase the cost of technical defects

Method used

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  • NK cell culture solution and cell culture method
  • NK cell culture solution and cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] A cell culture solution for culturing NK cells, comprising: culture solution A and culture solution B.

[0050] The preparation of culture medium A is as follows: add OKT3, CD16, IL-2, IL-15 and 4-1BBL to the basal medium and mix well; among them, OKT3, CD16, IL-2, IL-15 and 4- The final concentrations of 1BBL in culture solution A were: 50ng / mL, 50ng / mL, 100U / mL, 50ng / mL and 50U / mL.

[0051] The preparation of culture medium B is as follows: add IL-2, IL-15 and 4-1BBL to the basal medium and mix well; wherein, the final concentration of IL-2, IL-15 and 4-1BBL in culture medium B Concentrations are: 100U / mL, 50ng / mL and 50U / mL.

[0052] The above-mentioned cytokines and basal medium are commercially available. The basal medium is the NK cell serum-free medium produced by Beijing Youkang Biotechnology Co., Ltd., and OKT3, CD16, IL-2, IL-15 and 4-1BBL were purchased from Beijing Tong Lihaiyuan Biotechnology Co., Ltd.

Embodiment 2

[0054] Under sterile conditions, collect 100 mL of peripheral blood from normal volunteers with a blood collection bag, dilute it with a mixed solution of 0.9% normal saline and peripheral blood at a ratio of 1:1 under an ultra-clean workbench, and blow it evenly with a straw to obtain diluted blood. ;

[0055] Take another new 50mL centrifuge tube, add lymphocyte separation medium, and slowly add the diluted blood to the surface of lymphocyte separation medium according to the ratio of diluted blood: lymphocyte separation medium of 2:1, so that the gap between the two Form a clear interface, and then centrifuge at 2000r / min for 20min at room temperature;

[0056] After taking it out, it can be seen that the liquid in the tube is divided into four layers. From top to bottom, they are plasma (including platelets), middle cloud layer cells (ie mononuclear cells), lymphatic separation fluid, red blood cells and granulocytes. Use a straw to carefully suck out the middle cloud laye...

Embodiment 3

[0058] Take lymphocytes, add NK cell serum-free medium to suspend the cells, and perform cell counting, then use NK cell serum-free medium to adjust the cell density to 2×10 6 / mL, transferred to two cell culture flasks, then added culture solution A, diluted to a cell density of 0.5×10 6 / mL-1.5×10 6 / mL or so, placed at 37°C, 5% CO 2 cultured in a cell culture incubator;

[0059]On the 3rd, 5th, and 7th day of cell culture, supplement culture solution A to maintain the cell density at 0.5×10 6 / mL-1.5×10 6 / mL at 37°C, 5% CO 2 cultured in a cell culture incubator;

[0060] After the medium was supplemented on the seventh day, the cells were transferred to cell culture bags for culture, and then culture solution B was supplemented on the 9th, 11th, 13th, 15th, 17th, and 19th days of culture, so that the cell density was 0.5×10 6 / mL-1.5×10 6 / mL, continue at 37°C, 5% CO 2 Cells were cultured in a cell culture incubator until the 21st day, and the cells were collected....

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Abstract

The invention belongs to the technical field of cells, and particularly relates to a cell culture solution and cell culture method and application thereof. The cell culture solution comprises a first culture solution and a second culture solution, the first culture solution contains cell factors such as OKT3, CD16, IL-2, IL-15 and 4-1BBL, and the second culture solution contains cell factors such as IL-2, IL-15 and 4-1BBL. The invention further provides the cell culture method. The method comprises the steps that cells are inoculated into the first culture solution for culture, and then the first culture solution is supplemented on the next day; the second culture solution is supplemented when culture is conducted on the ninth day for continuous culture, and the second culture solution is supplemented on the next day. According to the cell culture solution and cell culture method and application thereof, the cell factor combination and culture technological process is optimized, the culture period is shortened, only 14-21 days are needed for culture, and the purity of NK cells obtained through culture reaches 70% or above; the NK cells do not need to be purified, the steps are simple and convenient, and the operability is high.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to NK cell culture fluid and a cell culture method. Background technique [0002] Tumor, also known as cancer, is a new organism formed by the abnormal proliferation of cells in local tissues under the action of various tumorigenic factors, often manifested as a local mass. Tumor cells have abnormal morphology, metabolism and function. They grow vigorously, often showing continuous growth that is not controlled by the body, and eventually destroy the normal functions of various organs of the body and lead to the death of the body. Tumor is not a disease unique to humans. Almost all animals (except individual species) can form tumors, and even some plants can get "cancer". Therefore, tumors are common diseases among higher species. Tumor has the characteristics of high incidence, strong concealment and high lethality. With the continuous growth and aging of the global popu...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/078
CPCC12N5/0634C12N5/0646C12N2500/90C12N2501/2302C12N2501/2315C12N2501/25C12N2501/515C12N2501/599
Inventor 许澎
Owner 湖南丰晖生物科技有限公司
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