TITLE: Obtaining of NK cell clones using IL-2 and gene-modified K562 cells expressing membrane-bound IL-21
AUTHORS: Maria Streltsova, Sofya Erokhina, Leonid Kanevskiy, Dean Lee, William Telford, Elena Kovalenko

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here we present the simple and effective method for obtaining clones of NK cells, sorted according to a given set of surface markers. The lifespan of clones grown by this method can reach 14 weeks and cloning efficiency is up to 50%, depending on the original NK cell subset. Obtained clones are functionally active; they are able to lyse target cells and produce IFNγ. The principle of method includes single cell sorting and further clonal expansion in the presence of IL-2 and gene-modified K562 feeder cells. Our method allows to expand the progeny of a single NK cell up to 10-20×10</span><span style = "vertical-align:super;">6 </span><span>NK cells suitable to study phenotype, proliferative and functional activity of a certain NK cell clone.</span></div><div class = "text-block">The work was supported by Russian Science Foundation, grant #16-15-00309.</div></div>

[STEPS]
?. [NK cell isolation]
Resuspend PBMC with ice-cold separation buffer in 15 or 50 ml Falcon™ tube. Determine cell number.
All further steps should be done in sterile conditions and as fast as possible. Keeping cells cold and using pre-cooled solutions is needed to prevent capping of antibodies and non-specific labelling. The NK Cell Isolation Kit, human (Miltenyi Biotec) is used for negative magnetic NK cell separation.
Separation buffer:Dilute 0.5% BSA (Serva) and 2 mM EDTA (Serva) in PBS. Adjust pH to 7.2. Sterilize by filtering through 22-μm Millipore filter and store up to 2 months at 4°C.
?. [NK cell isolation]
Centrifuge at 200×g for 10 min at 4°C. Remove the supernatant by decanting or with an automatic pipette.
?. [NK cell isolation]
Resuspend the pellet in 40 μl of separation buffer per 107 of total cells and transfer the suspension into a new Falcon™ 15 ml polypropylene conical centrifuge tube.
This step allows to avoid the intrusion of non-labelled cells from the previous 15/50 ml Falcon™ tube walls into the separation column.
?. [NK cell isolation]
Add 10 μl of NK Cell Biotin-Antibody Cocktail (The NK Cell Isolation Kit, human, by Miltenyi Biotech) per 107 of total cells and mix gently.
?. [NK cell isolation]
Incubate for 5 min at 4°C.
?. [NK cell isolation]
Add 30 μl of separation buffer and 20 μl of NK Cell MicroBead Cocktail (The NK Cell Isolation Kit, human, by Miltenyi Biotech) per 107 of total cells.
?. [NK cell isolation]
Mix gently and incubate for 10 min at 4°C.
?. [NK cell isolation]
Adjust the volume of the separation buffer up to 500 μl  per 108 of total cells.
?. [NK cell isolation]
Install MACSTM LD column into the magnetic field of a MidiMACS Separator and rinse it with 3 ml of separation buffer.
It is convenient to begin rinsing the column, when you have put the cells into the ice for 10 min (step 7), as rinsing takes approximately the same time.Other MACSTM columns (MS, LS) can be used for this procedure, but with the LD column, we obtained NK cells with the highest purity.
?. [NK cell isolation]
Prepare a new Falcon™ 15 ml polypropylene conical centrifuge tube and put it under the column to collect flow-through containing unlabeled NK cells. Apply cell suspension onto the column.
?. [NK cell isolation]
Wash the column two times with 3 ml of separation buffer. The buffer should be added only when the column reservoir is empty.
?. [NK cell isolation]
Take the 15 ml tube from under the column, mix well and determine cell number.
?. [Fluorescence-activated single cell sorting]
Centrifuge the tube with NK cells at 200×g for 10 min at 25°C. Remove the supernatant by decanting or with automatic pipette.
?. [Fluorescence-activated single cell sorting]
Resuspend the pellet in 100 μl of separation buffer per 5×106 of total cells and transfer suspension into a new Falcon® 5mL round bottom polypropylene tube, with snap cap.
If needed, add larger volume of separation buffer and take some cells to prepare controls, e.g., unstained control or single-stain controls, in the additional 5 ml polypropylene centrifuge tubes.
?. [Fluorescence-activated single cell sorting]
Add anti-CD56 and anti-CD3 monoclonal antibodies (and any other antibodies needed)  according to the manufacturer's instructions into the experimental tube and control tubes.
It is possible to expand the panel of antibodies for staining and subsequent sorting of different NK cell subpopulations.
?. [Fluorescence-activated single cell sorting]
Mix gently and incubate for 30 min on ice.
?. [Fluorescence-activated single cell sorting]
Add 1 ml of staining solution, centrifuge at 300×g for 4 min at 4°C, and decant the supernatant. Repeat one more time.
?. [Fluorescence-activated single cell sorting]
Resuspend the pellet in 300-500 μl of separation buffer per 5×106 of total cells.
?. [Fluorescence-activated single cell sorting]
Sort the cells into the 96 well plates, prepared in advance (see below “Preparation of plates for cell sorting” and "Preparation of feeder cells"), using “single cell” mode on your cell sorter.
?. [Fluorescence-activated single cell sorting]
Cultivate the plates with cells in a CO2-incubator for 2-3 weeks.
It is important not to move the plates during this period. Stirring of the medium can adversely affect the growth of the clones.
?. [Fluorescence-activated single cell sorting]
After 2 weeks of cultivation in a CO2-incubator, a visual assessment of the clone growth is carried out using optical microscope.
Cells should be attached to the bottom on the plate and located in the center forming a circular clump. If the accumulation of cells is barely noticeable, the plates should be returned to the CO2-incubator for one more week.
?. [Preparation of feeder cells]
Irradiate feeder cells with γ radiation (100 Gy). Cells can be used immediately or frozen in 90% FCS (HyClone) containing 10% DMSO (Sigma) in a refrigerator (-150°C – -135°C) or in liquid nitrogen.
Using non-irradiated feeder cells will result in the death of NK cells due to much faster proliferation of K562 cells and subsequent lack of nutrients in the medium. Therefore, if you are not sure about the quality of feeder cell irradiation, you can cultivate 5×105 K562-mbIL21 cells in cell culture flask for 3-5 days. The number of irradiated cells should not increase.
?. [Preparation of feeder cells]
Cultivate the K562mbIL-21 feeder cells using RPMI-1640 medium in 25cm² cell culture flask at the concentration of 2-6×105 cells/ml (to maintain cell growth in log-phase).
?. [Preparation of plates for cell sorting]
Add 100 unit/ml of recombinant IL-2 (Sigma) and 104 of irradiated feeder cells per ml into warm AIM-V medium (Gibco).
?. [Preparation of plates for cell sorting]
Put 200 μl of the medium into central 60 wells of a 96-well plate.
?. [Preparation of plates for cell sorting]
Put 200 μl of the RPMI-1640 medium into 36 marginal wells.
Marginal wells are prone to rapid evaporation. This leads to early death of NK cells clones. Therefore, wells with medium only should always surround the wells with cultivated cells. This approach minimizes evaporation from the central wells.
?. [Preparation of plates for cell sorting]
During cell sorting, the medium in the plates should be warm. For this purpose, the prepared plates can be put in a CO2-incubator for a few hours.