In-vitro amplification method of iNKT cells

An in vitro amplification and cell technology, applied in the field of cell expansion, can solve the problems of amplification multiple, purity and killing function, low ethical risk, etc., and achieve the effect of avoiding ethical risks, increasing amplification multiple, and improving killing performance.

Inactive Publication Date: 2020-03-10
中冠赛尔生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For this reason, the embodiment of the present invention provides an in vitro expansion method of iNKT cells to solve the problems of existing expansion methods, such as low amplification factor, purity, and killing function, as well as ethical risks.

Method used

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  • In-vitro amplification method of iNKT cells
  • In-vitro amplification method of iNKT cells
  • In-vitro amplification method of iNKT cells

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0046] This embodiment is a method for extracting mononuclear cells in human peripheral blood. The extraction method includes the following steps:

[0047] (a) Take a sterile centrifuge tube, add 30ml of peripheral blood into the centrifuge tube, centrifuge at 2800rpm for 10min, and take the upper serum into the centrifuge tube, then put it in a water bath at 56°C for 30min, and then Centrifuge at 3500rpm for 20min, collect the supernatant into a clean centrifuge tube, store at 4°C for later use, and collect the lower layer of blood cells;

[0048](b) Add physiological saline to the blood cells to the whole blood volume, add hydroxyethyl starch to the blood cell solution at a volume ratio of 1:2 and mix well, settle for 30 minutes, take the supernatant into a centrifuge tube, and centrifuge at 2000rpm for 10 minutes , collect the precipitate and resuspend in 20ml saline;

[0049] (c) Slowly add the resuspended cell suspension into the supernatant in step (a) along the wall of...

Embodiment 2

[0051] Example 2 is a method for preparing DC cells, and the DC cells are prepared by the following method:

[0052] (a) mononuclear cells are obtained according to the method of Example 1;

[0053] (b) Add Corning88-551 medium to mononuclear cells and suspend them, adjust the inoculation density to 3×10 6 cells / ml, inoculate 10ml cell suspension into 90mm culture dish, at 37℃, 5% CO 2 Incubate for 4 hours in an incubator under certain conditions, then draw the supernatant into a blank centrifuge tube, and use PBS buffer to wash the culture bottle after absorbing the supernatant, and transfer the washing solution to a blank centrifuge tube containing the supernatant, Centrifugation and water washing, the centrifugation speed is 1500rpm, the time is 10min, and the water washing is washed twice with normal saline (the washing process should be light, so as not to wash down the monocytes);

[0054] (c) Add 10ml of Corning 88-551 blank medium to the washed centrifuge tube, and a...

Embodiment 3

[0057] This embodiment is a preparation method of iNKT cells, iNKT cells are prepared by the following method:

[0058] (a) Obtain mononuclear cells according to the method of Example 1, add Corning88-551 serum-free medium to the mononuclear cells and suspend, adjust the inoculation density to 3×10 6 cells / ml, inoculate 10ml cell suspension into 90mm culture dish, at 37℃, 5% CO 2 Incubate in an incubator under the conditions for 4 hours, then shake the culture dish slightly, draw the supernatant into a 50ml centrifuge tube, centrifuge at 2000prm for 10min, and collect the precipitated cells;

[0059] (b) Use Corning88-581 serum-free medium to resuspend and mix the pelleted cells, and the cell concentration is 2×10 6 cells / ml, then the cell suspension was transferred to the T-75 culture flask coated with CD3 (lug / ml) monoclonal antibody and CD16 (lug / ml) monoclonal antibody 5ml;

[0060] (c) Add IL-15, IL-2, IL-21 and α-galactosylceramide to the culture flask for cultivation,...

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Abstract

The embodiment of the invention discloses an in-vitro amplification method of iNKT cells. The amplification method comprises: mixing DC cells and iNKT cells to obtain mixed cells, and inoculating andculturing the mixed cells. According to the amplification method disclosed by the invention, a dendritic cell (as a full-time antigen presenting cell with the strongest body function) and iNKT cell mixed culture mode is adopted so that the iNKT cell proliferation rate can be increased, the capacity of the iNKT cell for releasing granzyme, perforin, IFN-gamma, IL-2, IL-4 and TNF can be improved, animmune system in the body of a patient is recovered while mutant cells in the body are killed, and then the killing capacity of the iNKT cell is improved.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of cell expansion, and specifically relates to an in vitro expansion method of iNKT cells. Background technique [0002] With the in-depth research on the mechanism of tumor occurrence and development and the mechanism of immune tolerance, it is gradually realized that the autoimmune system plays a very important role in the prevention and treatment of tumors. The immune system of the human body can play an immune monitoring function to identify and eliminate the mutant cells produced in the body, and remove them in time to avoid the occurrence of tumors in the body. [0003] Immunotherapy has gradually become the fourth mode of treatment for tumors after radiotherapy, chemotherapy and surgical resection, which has brought hope to conquer malignant tumors. Human invariant natural killer T cells (iNKT) connect innate immunity and specific immunity, play an important role as a bridge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0784
CPCC12N5/0646C12N2500/02C12N2500/46C12N2501/22C12N2501/2302C12N2501/2304C12N2501/2315C12N2501/2321C12N2501/24C12N2501/515C12N2501/599C12N2502/1121
Inventor 吴勇君江良旗王俊
Owner 中冠赛尔生物科技(北京)有限公司
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