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Cell culture method

A cell culture and nuclear cell technology, applied in cell culture active agent, culture process, tissue culture and other directions, can solve the problems of high cost, poor activity, low purity and so on

Active Publication Date: 2017-08-22
深圳市沃英达生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, most in vitro expansion techniques can only expand NK cells dozens of times, and the purity is low, the activity is poor, and the cost is high, which is difficult to meet the needs of clinical use.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Prepare activation medium and proliferation medium, the steps are as follows:

[0043] 1. Preparation of activated medium

[0044]Serum-free RPMI1640 medium was used as the basal medium, and anti-CD16 monoclonal antibody, IL-2, ascorbic acid, TGF-β1 and thymosin were added to the basal medium, and mixed evenly to make anti-CD16 monoclonal antibody, IL-2, ascorbic acid The final concentrations of , TGF-β1 and thymosin in the activation medium were 60ng / ml, 250U / ml, 2ng / ml, 60ng / ml and 2ng / ml respectively. Sterilize with a 0.1μm membrane filter, and store in the dark at 4-8°C after aliquoting.

[0045] 2. Preparation of Proliferation Medium

[0046] Serum-free RPMI1640 medium was used as the basal medium, and IL-1α, IL-2, ascorbic acid, IL-21, IL-7 and 41-BBL were added to the basal medium. Mix well so that the final concentrations of IL-1α, IL-2, ascorbic acid, IL-21, IL-7 and 41-BBL in serum-free RPMI1640 medium are 50ng / ml, 500U / ml, 2ng / ml, 60ng / ml, respectively. m...

Embodiment 2

[0049] Cell culture, the steps are as follows:

[0050] Step 1, separating peripheral blood mononuclear cells (PBMC), the steps are as follows:

[0051] 50ml of peripheral blood was collected from healthy people. According to the method reported in the literature, peripheral blood PBMCs were separated using lymphocyte separation medium (purchased from Tianjin Haoyang). Carefully aspirate the cells in the middle cloud layer to separate the peripheral blood mononuclear cells (PBMCs), and wash them twice with normal saline. Add activation medium to resuspend PBMCs, and perform cell counting.

[0052] Step 2, the cultivation of NK cells, the steps are as follows:

[0053] Adjust the PBMC density with the activation medium prepared in Example 1 to be 2×10 6 cells / ml, and 56°C inactivated autologous plasma (plasma collected in step 1 in Example 2) was added, so that the volume percentage of autologous plasma in the activation medium was 4%. Transfer the PBMC NK cells and their m...

Embodiment 3

[0057] The peripheral blood mononuclear cells cultured to the 16th day were collected, and the ratio of CD3-CD56+CD16+ cells was detected by flow cytometry. figure 2 It is the flow cytometric detection result of NK cells on the 16th day obtained by adopting the method of the present invention. As shown in the results, the ratio of CD3-CD56+CD16+ of peripheral blood mononuclear cells obtained by the present invention reaches 75%, indicating that through 16 days of cultivation, the purity of NK cells (NK cells accounting for the percentage of total cells) cultivated in vitro is as high as 75%.

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Abstract

The invention belongs to the field of cell culture, and particularly relates to a cell culture method. The cell culture method comprises the following steps that 1, a peripheral blood mononuclear cell is separated; 2, the peripheral blood mononuclear cell separated in the first step is added into a plasma-containing activated culture medium to activate NK cells; 3, a proliferation culture medium is added into the peripheral blood mononuclear cell which is cultured for 10 days in the second step to promote proliferation of the NK cells. According to the cell culture method, the technical defect that the amplified cell volume is low when the NK cells are subjected to in vitro expansion can be effectively overcome.

Description

technical field [0001] The invention belongs to the field of cell culture, in particular to a cell culture method. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment model and a new treatment method that relies on autoimmunity to fight cancer. It collects immune cells from the patient's body, then cultures and expands them in vitro, and then infuses them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. Unlike surgery, radiotherapy, and chemotherapy that damage the immune system, cell therapy can greatly improve the anti-cancer immunity of patients, and remove blood, lymph, or cancer cells that have metastasized to other tissues that cannot be touched by surgery, even dormant cancer cells. Treatment is more thorough than that. [0003] NK cells are natural killer cells (natural killer cells, NK) are important immune cells in the body, not only related to an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/38C12N2501/15C12N2501/2301C12N2501/2302C12N2501/2307C12N2501/2321C12N2501/599C12N2501/998
Inventor 林洁璇王旭林词雄李陶朱刚
Owner 深圳市沃英达生命科学有限公司
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