NK cell culturing solutions and culturing method
A technology of NK cells and culture methods, applied in the direction of cell culture active agent, animal cells, culture process, etc., can solve problems such as hidden safety hazards and inability to carry out quality control, and achieve the effects of avoiding hidden safety hazards, increasing multiplication rate, and high killing activity
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Embodiment 1
[0067] This embodiment is the cultivation of NK cells
[0068] (1) Separation of mononuclear cells from peripheral blood:
[0069] (1.1) Whole blood separation and plasma processing
[0070] Collect 5ml of human peripheral venous blood, transfer it to a centrifuge tube and centrifuge at 2500rpm for 10min, transfer the upper layer of plasma to a new centrifuge tube, heat inactivate it in a water bath at 56.0°C for 30min, and store it in a refrigerator at 4°C for later use;
[0071] (1.2) Separation of peripheral blood mononuclear cells: add an equal volume of cleaning solution to the lower cell layer separated in (1.1) to dilute, and blow evenly. After adding 5ml Ficoll to the 15ml centrifuge tube, tilt the centrifuge tube at 45°, add the diluted cells to the upper layer of Ficoll along the tube wall at a uniform and slow speed, centrifuge at 2500rpm for 20min; suck out most of the cleaning solution and discard the residual plasma mixed layer. Absorb and transfer the remainin...
Embodiment 2
[0092] This embodiment is the cultivation of NK cells
[0093] The difference between this embodiment and embodiment 1 is: (2.4) regular bottle rotation and rehydration steps:
[0094] Add IL-2 and IL-15 to the lymphocyte serum-free medium, mix well to obtain culture medium C, wherein the final concentrations of IL-2 and IL-15 in culture medium C are 1000IU / ml, 50μg / ml respectively .
[0095] Add IL-2 to the lymphocyte serum-free medium, and mix well to obtain culture medium D, wherein the final concentration of IL-2 in culture medium D is 1000 IU / ml.
[0096] Day2: Supplement 5ml culture solution C;
[0097] Day4: Transfer all the cells in the T25 flask to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and wash twice with washing solution. Resuspend the cells with culture medium D, transfer them all to a new T75 bottle, and supplement culture medium D to a total volume of 40ml, and 1ml (1.1) of heat-inactivated plasma after centrifugation;...
Embodiment 3
[0101] This embodiment is the cultivation of NK cells
[0102] The difference between this embodiment and embodiment 1 is: (2.4) regularly turn the bottle and rehydration:
[0103] Add IL-2 to the lymphocyte serum-free medium, and mix well to obtain culture medium D, wherein the final concentration of IL-2 in culture medium D is 1000 IU / ml.
[0104]Day2: Supplement 5ml culture solution D;
[0105] Day4: Transfer all the cells in the T25 bottle to a new T75 bottle, and supplement the culture medium D to a total volume of 40ml, and 1ml (1.1) heat-inactivated plasma after centrifugation;
[0106] Day8: Transfer all the cells in the T75 bottle to a new T175 bottle, and supplement the culture medium D to a total volume of 100ml, and heat-inactivated plasma after centrifugation in 0.5ml (1.1);
[0107] Day11: Supplement 50ml of culture medium D.
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