This protocol can significantly improve the expansion of functional, non-exhaustive, memory-like NK cells ex vivo compared to other feeder cells based on expansion system. This is a simpler method compared to other protocols using feeder cells in which we skip the NK cell isolation step prior to NK cell expansion. This protocol can provide a sufficient number of the NK cell and CAR-NK for immunotherapy.

This technique is highly reproducible. However, using fresh, new starting materials and taking care not to disturb the NK cells during the first few days of expansion is crucial. Begin by identifying and sectioning the viable tissue areas using sterile surgical equipment to obtain lymphocytes.

Then, place the sectioned tissues in 30 milliliters of HBSS without calcium or magnesium and keep the tissue on ice until ready for isolation. Working inside a biosafety cabinet, mince the tissue into less than 0.5 centimeter cubes with sterile razor blades and forceps. Place the minced tissue pieces, not more than 4 grams, in the tissue dissociator tubes and add 10 milliliters of collagenase IV to the tissue pieces.

To mince the tissue thoroughly, treat the tissue dissociator tubes into a tissue dissociator at 37 degrees Celsius. After removing the tubes from the tissue dissociator, triturate the minced tissue through a 40-micron nylon cell strainer using the backend of a 5 milliliter syringe. Discard the large undigested fragments.

Spin down the collected eluent at 400G for 5 minutes at room temperature and aspirate the supernatant before resuspending the cell pellets in 30%polyvinylpyrrolidone-coated silica to remove the fat cells. Spin down the cells as described before resuspending the cell pellet in 9 milliliters of R-10 media. To separate lymphocytes from red blood cells and polymorphonuclear cells, carefully layer the cell suspension over 4 milliliters of Ficoll or lymphocyte separation media.

Separate the layers by centrifuging at 400G for 23 minutes at room temperature with the acceleration and brakes off. Then, carefully decant the upper-medium layer and harvest the interphase containing tissue-infiltrating lymphocytes. Rinse the cells with 10 milliliters of media and proceed for analysis or primary natural killer, or NK, cells expansion protocol.

Wash the peripheral blood mononuclear cells, or PBMCs, and 100 gamma-irradiated 221-mIL-21 cells separately by centrifugation at 400G for 5 minutes with 10 milliliters of R-10 media. After centrifugation, save 1 x 10 to the 6th PBMCs for flow cytometry and mix 5 x 10 to the 6th PBMCs with 10 x 10 to the 6th 100 gamma-irradiated 221-mIL-21 cells in a special 6-well plate. Add 30 milliliters of R-10 media supplemented with human interleukin-2 and human interleukin-15 to the same 6-well plate and incubate the plate at 37 degrees Celsius with 5%carbon dioxide, with replacing the media every 3 to 4 days.

While in incubation, record the total peripheral blood natural killer, or PBNK, cell number viability and perform flow cytometry every 3 to 4 days to calculate the NK cell expansion rate. Add 1.8 x 10 to the 6th 293T cells in 11 milliliters of the D-10 media per treated 100 millimeter plate and incubate 293T cells at 37 degrees Celsius with 5%carbon dioxide. In a 1.70 milliliter tube, mix 470 microliters of the reduced serum media with 30 microliters of the transfection reagent.

In a separate 1.70 milliliter tube, add 2.5 micrograms of pRDF plasmid, 3.75 micrograms of Pegpam3 plasmid, and 2.5 micrograms of CAR construct in SFG vector into the reduced serum media to make the final volume of 500 microliters. Mix the contents of the tube with the 1.70 milliliter tube prepared earlier in a dropwise manner. After 15 minutes of incubation at room temperature, add 1 milliliter of the mixture from the tube to the 293T cell plate in a dropwise manner and place the plate at 37 degrees Celsius with 5%carbon dioxide for 48 to 72 hours.

Dilute the retronectin protein with PBS to a final concentration of 50 to 100 micrograms per milliliter. Add 500 microliters of the diluted retronectin into each well of an untreated 24-well plate. Then, seal the plate using parafilm and incubate the plate at 4 degrees Celsius overnight.

The next day, centrifuge the retronectin plate at 2, 103G for 30 minutes at 4 degrees Celsius and discard the supernatant. After blocking each well of the 24-well plate with 1 milliliter of R-10 medium, incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 1 hour. Filter the previously-transfected 293T cells using a 0.45 micron filter to collect the retrovirus supernatant.

Aliquot 2 milliliters of the filtered retrovirus supernatant into each well of the 24-well retronectin plate. Centrifuge the 24-well retronectin plate at 2, 103G for 2 hours into a pre-warmed centrifuge at 32 degrees Celsius. While the plates are being centrifuged, collect the expanded PBNK cells from Day 0 and count the cells using Trypan Blue.

Dilute the expanded PBNK cells with R-10 media supplemented with interleukin-2 and interleukin-15 to the concentration of 2.5 x 10 to the 5th to 5 x 10 to the 5th cells per milliliter. After centrifugation of the 24-well retronectin plate, partially aspirate the retrovirus supernatant from each well. Then, add 2 milliliters of the diluted expanded PBNK cells to each well.

Centrifuge the plate at 600G for 10 minutes at 32 degrees Celsius before incubating the plate at 37 degrees Celsius with 5%carbon dioxide for 48 to 72 hours. Transfer the cells from the 24-well plate into a 50 milliliter centrifuge tube to centrifuge the tube at 400G for 5 minutes. After resuspending the pellet with 1 milliliter of R-10 media, transfer the resuspended cells to a special 6-well plate containing 30 milliliters of R-10 media supplemented with interleukin-2 and interleukin-15.

Incubate the special 6-well plate at 37 degrees Celsius and 5%carbon dioxide with refreshing media and calculate the NK cell expansion rate every 3 to 4 days. The PBNK cells expanded by 221-mIL-21 were shown to expand nearly 5 x 10 to the 4th folds. And the NK cell purity was maintained at around 85%throughout the 21-day expansion.

Prior to the expansion, the robustness of the 221-mIL-21 expansion system was examined by staining PBMCs for anti-CD56 and anti-CD3, which showed a cell purity of 7.09%for NK cells and a high percentage of T cells. On Day 4 of PBMCs and 221-mIL-21 coculture, the NK purity was checked before CAR-NK transduction. The CAR-NK cells stained for anti-CD56, anti-CD3, and anti-human-IgG showed a high NK cell population on Day 7 and a high CAR transduction efficiency of approximately 70%was observed.

On the 18th day, the CAR expression in various subsets was checked with flow cytometry. Following NK cell expansion, cells can be used for in-vitro functional assays or for in-vivo experiments. Researcher can use this protocol to expand NK and CAR-NK for patients with functional NK deficiency as well as other species, such as dog.

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CAR NK Cell Expansion