NK cells using factor in vitro culture

1. An NK cell culture kit contains:

①serum-free medium 2 bottles (l000mL / bottle):

②NK reagent A, 1 branch:

③NK reagent B, 1 branch:

④NK reagent C, 1 branch:

⑤NK reagent D, 1 branch:

⑥NK reagent E, 1 branch:

Two (1 million IU/branch) rH IL-2 are needed in the culture system.

2. Raw materials

① Take peripheral blood (can be organized from cord blood, bone marrow, etc.)

②Required reagent supplies include:

0.4% trypan blue solution, normal saline, human lymphocyte separation solution (GE recommended), 4375px2 culture flask, GT-T610 (A) culture bag, 15ral/50ml/250ml centrifuge tube, pipette, 1ml sterility syringe

3. NK cell culture time process:
The day before: the cell activation bottle (NK Reagent A) should be processed in advance.
Day 1: Peripheral blood mononuclear cells isolated and induced (NK Reagent B)

Day 3: NK cells first rehydration, rehydration 50ml (NK Reagent C)
Day 5: NK cell second rehydration, rehydration 175ml (NK Reagent D)

Day 7: NK cell rehydration for the third time, bagging and rehydration 500ml (NK Reagent E)
Day 9-10: NK cells for the fourth rehydration, sub-package and rehydration 750ml
Day 12: NK cell fifth rehydration. Rehydration 500ml
Day 13: Quarantine
Days 14 and 15: Successful culture

(The success time of cell culture depends on the growth of specific cells. It can also be cultivated for one more day while maintaining the culture medium ph and cell viability.)

4. The specific steps are as follows: 

①Always handle the cell activation bottle on the first day.

②Add NK Reagent A (4 degree storage) to a 175 cm2 bottom area cell culture flask containing 12.5 ml of normal saline, and mix thoroughly for 30 sec - 1 min to allow the liquid to fully disperse at the bottom of the bottle overnight.

(NK reagent A needs to be thoroughly mixed with physiological saline and spread on the bottom of the bottle; the liquid can be directly absorbed after the next day, no need to clean)
③Peripheral blood mononuclear cells isolated and induced (Day 1)
④Take 100ml peripheral blood as an example. If the blood volume is different, it can also be adjusted according to the operating procedures.

⑤The patient's peripheral blood was pipetted with a small amount of raw blood (about 300 ul) or dropped into a plate for colonization. Then, centrifugation was carried out at normal room temperature.

⑥Transfer the upper plasma to the centrifuge, inactivate, centrifuge, and take the supernatant for use.

⑦Transfer the upper plasma to the centrifuge, inactivate, centrifuge, and take the supernatant for use.
⑧Pipette the peripheral blood mononuclear cells (PBMC) layer and try to absorb the cell layer at the junction of the two fluids. Add saline to mix and centrifuge. The cells were washed several times in the same manner.
⑨After discarding the supernatant, resuspend the cells in serum-free medium and aspirate a small number of cells.
⑩According to the cell density of 1-2*10^6/ml (optimally 5 *10^6/ml), wash the centrifuge tube with medium, merge into the culture flask, and add 1 NK reagent B. The patient inactivated plasma 1.25 ml (5%) to ensure that the final volume of the medium is about 25 ml. The remaining plasma was sealed at 4 ° C for later use.

Note: The time for the bottle to be taken out is 5 minutes before the cells are added, and the other time should be placed at 4 degrees for use.
Reagents A, B, C, D, E are taken. After the first suction of the stock solution, flush the vial with the appropriate medium and then suck it out to minimize the loss.
⑪Configuration of activation medium: One bottle of serum-free medium was added with 1 million IU of IL-2 at a final concentration of 1000 IU/mL. 
Note: The activated medium is returned to room temperature before each use.
⑫The first rehydration of NK cells (Day 3), supplemented with 5% of patient inactivated plasma and NK Reagent C, ensuring a final volume of 75 mL.

⑬The second rehydration of NK cells (Day 5), supplemented with 5% of patient inactivated plasma and NK Reagent D to ensure a final volume of 250 ml.

⑭The third rehydration of NK cells and their bagging (Day 7), the cells at the bottom of the flask were slightly blown off before the cells were transferred to the bag, and then the cell suspension in the flask and its 500 mL activation culture were carried out. The base was loaded into a cell culture bag while adding 1 NK Reagent E, and all remaining plasma, to ensure a final volume of 750 ml.

⑮The fourth rehydration of NK cells (Day 9), mainly for the bagging operation, the culture system was expanded from 750 ml to 1500 ml.
⑯The fifth rehydration of NK cells (Day! 2 days). Mainly for rehydration operation, divide the remaining 500 mL of the amplification solution into 2 culture bags to ensure that the volume per bag is 1000 ml.

⑰Quarantine, cell culture on the 13th day, the cell suspension was tested for bacteria and endotoxin. Use a 5 ml syringe to extract the least cell suspension from the culture flask and the bag, add it to the upright plate, place it in a 35 °C constant temperature biochemical incubator, record the date and culture items. Inverted after overnight, for 48 hours.

⑱Harvest, under normal conditions, 1000 ml of cell suspension were harvested on days 13 and 14, respectively. If the advance or delayed return is due to clinical needs, it can be advanced or delayed accordingly. (NK cells are recommended for use within 2-4 hours to ensure their activity)