Amplification method for NK cell without trophocyte
A technology of NK cells and trophoblasts, applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve problems such as difficulty in ensuring sustainability, achieve increased quantity and purity, high amplification efficiency, and cost savings Effect
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Embodiment 1
[0039] Example 1 Isolation of mononuclear cells from peripheral blood or umbilical cord blood
[0040] 1) In the biological safety cabinet, divide 30mL blood into 50ml centrifuge tubes, and centrifuge at 500g for 10min;
[0041] 2) After centrifugation, put the upper plasma layer into a new 50ml centrifuge tube, inactivate it in a water bath at 56°C for 30 minutes, centrifuge it at 2000g for 10 minutes, and save the supernatant for later use.
[0042] 3) Dilute the blood cells with 15mL normal saline at a ratio of 1:1, take 15mL of lymphocyte separation medium, and slowly add the mixed blood cells into the centrifuge tube containing lymphocyte separation medium at a ratio of 2:1 to make it appear Clear interface, centrifuge at 400g for 20min;
[0043] 4) After centrifugation, the liquid surface will be divided into 4 layers. From the bottom, there are red blood cells, lymphatic separation liquid layer, buffy coat layer and supernatant layer. Discard the supernatant layer, and...
Embodiment 2
[0046] Example 2 Cytokine combination optimization experiment
[0047] Cytokine combinations were divided into 3 groups: (1) autologous plasma medium group (on the basis of GT-T551H3 culture medium, add 1-10% autologous plasma, 200IU / mL IL-2); (2) co-culture Liquid group (on the basis of GT-T551H3 culture medium, add 1-10% autologous plasma, 200IU / mL IL-2, 50ng / mL IL-15 and 25ng / mL IL-21); (3) total After 7 days of culture in the mixed culture medium, it was replaced with the autologous plasma medium group.
[0048] On the 0th day of culture, the mononuclear cells obtained in Example 1 were respectively suspended in the corresponding culture medium, and the cell density was adjusted to 1.5×10 6 / mL, seeded in a 6-well plate, the final volume of each well is 2ml, the autologous plasma concentration is 5%, at 37°C, 5% CO 2 and saturated steam incubator.
[0049] On the third day of culture, add 1ml of the corresponding culture solution, the concentration of autologous plasma ...
Embodiment 3
[0055] The mass expansion culture of embodiment 3 NK cells
[0056] 1) Activation and stimulation of NK cells: Mononuclear cells were mixed with culture medium (on the basis of GT-T551H3 culture medium, containing 5% autologous plasma, 200IU / mL IL-2, 50ng / mL IL-15, 25ng / mL IL-21) was resuspended, and the cell density was adjusted to 1.5×10 6 / mL, after mixing, take 4mL and add it to a T25 culture flask. 2 and saturated water vapor incubator, add 2mL blended culture solution on the 3rd day, continue to culture for 7 days, and observe the cell morphology under the microscope.
[0057] 2) NK cell expansion culture: After 7 days of activation culture, the natural killer cells were pipetted with a 10mL pipette, then pipetted into a T75 cell culture bottle, and 8mL of fresh medium (based on the GT-T551H3 culture medium, containing 1% autologous plasma, 200IU / mL IL-2), 37℃, 5%CO 2 and saturated water vapor incubator for 2 days, and then supplemented every 2 to 3 days to maintain t...
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