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Human NK Cell Proliferation Protocol

1. Human NK Cells Isolation

Thaw cryopreserved PBMCs and perform cell counting and viability assessment (viability should be >95%). Label and isolate Human NK cells according to the manufacturer's instructions using the separation magnetic beads (Human NK Cell Isolation Kit, Column-Based, Cat. No. S0K1009). Collect the isolated Human NK cells for subsequent culture.

2. Human NK Cell Expansion

Day 0 - Initial Seeding

Complete culture medium composition: KBM581 basal medium, 5% heat-inactivated human AB serum, 1% penicillin-streptomycin, 55 μM β-mercaptoethanol

Resuspend the isolated Human NK cells in complete medium at a density of 1 × 10⁶ cells/mL. Seed the cells at 1 mL per well into a 24-well plate. Immediately add recombinant human cytokines IL-15 (100 ng/mL), IL-18 (100 ng/mL), IL-2 (200 ng/mL) and 2.5 μg/mL soluble anti-human CD56 antibody. Place the 24-well plate in a humidified incubator at 37°C with 5% CO₂ to initiate culture."

Day 3 - First Medium Change

Gently pipette to resuspend the cells and collect the cell suspension. Centrifuge at 300 × g for 5 minutes at room temperature. Discard the supernatant and resuspend the cell pellet in fresh complete medium. Adjust the cell density to 5 × 10⁵ cells/mL. Add recombinant human cytokines IL-15 (100 ng/mL), IL-18 (100 ng/mL), IL-2 (200 ng/mL) and 2.5 μg/mL soluble anti-human CD56 antibody. Transfer 3 mL per well into a new 6-well plate and continue culture.

Day 5 - Maintenance

Observe the cell status. If cells appear healthy with minimal debris, gently pipette to resuspend all cells, then transfer the cell suspension to an appropriate tube. Count and assess viability. Resuspend cells in fresh complete medium and adjust the cell density to 5 × 10⁵ cells/mL. Add recombinant human cytokines IL-15 (100 ng/mL), IL-18 (100 ng/mL), IL-2 (200 ng/mL), and 2.5 μg/mL soluble anti-human CD56 antibody. Seed 3 mL per well into a new 6-well plate and continue culture.

If significant debris is observed, centrifuge the collected cells at 300×g for 5 minutes at room temperature, resuspend the pellet in fresh medium, adjust the density to 5 × 10⁵ cells/mL, add recombinant human cytokines IL-15 (100 ng/mL), IL-18 (100 ng/mL), IL-2 (200 ng/mL) and 2.5 μg/mL soluble anti-human CD56 antibody, and seed 3 mL per well into a new 6-well plate for continued culture.

Day 7 - Continued Expansion

Repeat the Day 5 procedure. If the cell proliferation fold-change is high and the culture volume is large, consider transferring the cells to a T25 flask for culture.

Day 9 - Scale-up

Repeat the Day 5 procedure. If cells have already been transferred to a T25 flask, an alternative partial medium change may be performed: Stand the T25 flask upright and allow cells to settle for 5-10 minutes. Carefully aspirate 1/3 to 1/2 of the supernatant without disturbing the cells. Add fresh complete medium supplemented with recombinant human cytokines IL-15 (100 ng/mL), IL-18 (100 ng/mL), IL-2 (200 ng/mL), and 2.5 μg/mL soluble anti-human CD56 antibody. Gently mix to ensure uniform distribution and adjust the final cell density to 5 × 10⁵ cells/mL. Continue culture

Day 11 – Harvest

Gently pipette to ensure all cells are resuspended. Transfer the cell suspension to an appropriate tube. The expanded cells can now be used for downstream assays or analysis. For certain donor samples, culture may be extended beyond 14 days to achieve larger-scale NK cell expansion.

Store in separate containers to minimize freeze-thaw cycles.