Precautions for NK-92 cell culture

Characteristics of NK-92 cells

The growth characteristics of NK-92 cells are suspension growth. After receiving the cells, observe whether the bottle body is intact, there is no leakage, whether the culture medium is clear and transparent, and observe the cells under a microscope;

Wipe the back of the cell culture bottle with 75% alcohol and let it stand upright for 2-4 hours to allow the cells to settle to the bottom; After the cells have settled, carefully aspirate the upper part of the culture medium from the bottle, leaving the bottom cells and about 3ml of culture medium in the original bottle;

Collect cell sediment by centrifuging the extracted cell culture medium at a speed of 1000 rpm for 3 minutes, or depending on the actual situation of your centrifuge. NK-92 cells are sensitive to centrifugation and the speed should not be too high; After culturing with 2-3ml of NK-92, resuspend the obtained cell pellet and return it to the original bottle for further overnight cultivation. The T25 culture volume is 5-6 milliliters;

NK-92 cells can be supplemented with IL-2 at a concentration of 200U/ml.

The culture bottle has an airtight cap, which must be loosened to prevent it from falling off, allowing the cells to fully breathe; Place the culture bottle horizontally, loosen the bottle mouth for ventilation, and incubate overnight;

Observe cells under a microscope, and perform passaging based on cell density and medium consumption. It is not recommended to perform centrifugation directly, as the cells may not reach their optimal state after transportation bumps and various treatments. Try not to blow away the cell clusters, shake the culture bottle gently after adding the solution.

Precautions for cell culture

NK-92 cells grow in suspension, with most cells gathering into clusters and a few cells scattered. There are also many dead cells and cell debris in the intercellular space. During the cultivation process, black particles and small black spots can often be observed, which are probably carried by the cells. Generally, a small number of black spots do not proliferate and will not affect cell growth. If black spots proliferate and grow, it is suspected that the cells are contaminated and may affect cell growth, even causing cell death.

NK-92 cells are sensitive to IL-2. The degradation of IL-2 in the culture medium will lead to a deterioration of the cell state. The long-term storage temperature for IL-2 is -20 ℃. After thawing, it should be stored in a refrigerator at 4 ℃ for no more than two weeks. The prepared fresh culture medium should be used up as soon as possible, preheated before use, and stored at 4 ℃ in the refrigerator after use;

NK-92 cells are sensitive to centrifugation. During normal cultivation, the liquid exchange cycle is 2-3 days. It is recommended to alternate between half volume liquid exchange and centrifugal liquid exchange methods, that is, after 2-3 times of half volume liquid exchange, centrifuge the full volume liquid exchange once. Try to minimize the number of centrifugation cycles. It is generally not recommended to centrifuge when the cell state is poor or the cell volume is small.

Do cell clusters need to be blown apart?

The clumps need to be blown apart, but there is no need to deliberately blow them apart into individual pieces;

After normal passaging or centrifugation and liquid exchange, control the number and intensity of blows. Even if the cells are dispersed into individual cells, they will still aggregate within 1-2 days; When the cell cluster is too large, it needs to be dispersed;

When cells are frozen, they need to be dispersed as much as possible, otherwise it will affect the freezing effect.

Fluid change method

(1) Fluid replenishment method: Add an appropriate amount of fresh culture medium every 2-3 days (depending on the volume of the culture container and the original cell suspension, T25 bottles can be replenished with 1-2 milliliters of fluid at a time), and at the same time, add 200U/ml IL-2. After 2-3 refills, centrifuge and replace all the media.

(2) Half liquid exchange method: Taking the T25 bottle (containing 5ml of culture medium) as an example, stand the bottle upright and let it stand for a period of time (lightly tap the bottle before standing it upright to let the cell clusters lightly adhere to the bottom float). Wait for the cells to sink to the bottom (observe the cell sinking situation with the naked eye), carefully aspirate 2.5ml of supernatant and transfer it to a centrifuge tube. Centrifuge at 1000rpm for 3-5 minutes, and resuspend the fine precipitate with 2.5ml of fresh culture before returning it to the original bottle for further cultivation. When cells grow, the aggregated cell clusters gradually increase, and normal cell clusters appear white and transparent under a microscope. If there is too much cell aggregation and the middle of the cell cluster appears dark, passage can be performed.

Passage method

Gently disperse the cell clusters using a pipette (usually 2-3 times, the blowing force should not be too strong, otherwise a large number of dead cells and cell fragments will appear), divide them evenly into two bottles, and add an equal amount of complete culture medium to each bottle. Cells have high nutritional requirements, and excessive cell count can affect cell growth; When the cell mass is too large, it needs to be blown away slightly. Pay attention to controlling the number of blows, and do not blow it all away.

Cell cryopreservation

It is recommended to use a freezing solution ratio of 90% FBS+10% DMSO, and NK-92 cells can be supplemented with IL-2 appropriately; Try to blow away the cells as much as possible and pay attention to controlling the blowing force.

Cell revival

After recovery, resuspend the cells in 6ml of fresh complete culture medium; After centrifugation, remove the supernatant and resuspend the cells in 1~2mL of complete culture medium, taking care to gently blow the cells a few times.

Place the bottle vertically in the incubator to increase local cell density, while keeping the bottle cap breathable;

Cell growth requires a stable environment, which can be observed once a day. Do not frequently take out the incubator for observation