Figure 1. ImmunoCult™ NK Cell Expansion Protocol

Human natural killer (NK) cells are isolated from blood or leukapheresis using EasySep™ selection. The NK cells are cultured in ImmunoCult™ NK Cell Expansion Medium, on plates coated with ImmunoCult™ NK Cell Expansion Coating Material. After 3 days, fresh medium is added to the culture. On day 7, and again on day 10 or 11, expanding NK cells are harvested and replated on freshly coated plates. Expanded NK cells were harvested on day 14 for use in downstream assays.

Figure 2. CD56+CD3− NK Cells Expand Over 14 Days in Feeder- and Serum-Free Culture Conditions

Isolated human CD56+CD3− NK cells were cultured using ImmunoCult™ NK Cell Expansion Kit for 14 days (Figure 1). Cells were harvested and analyzed for expression of characteristic NK cells markers, including CD56, CD3, CD16, CD94, KIR, NKG2D, NKp46, NKp30, and NKp44 by flow cytometry. Staining for killer cell immunoglobulin-like receptor (KIR) molecules was performed using two different antibody clones, HP-MA4 and 180704, which recognize distinct KIR molecules. Dead cells were excluded by light-scatter profile and DRAQ7™ staining. (A - H) Representative flow cytometry plots. (I) The average frequencies of viable CD56+CD3− and CD56+CD16+ NK cells on day 14 were 87 ± 1% and 75 ± 2%, respectively. The average fold expansion of CD56+CD3− cells was 89 ± 17. Results shown represent mean ± SEM (n = 34).

Figure 3. Expanded NK Cells Are Functional, Killing K562 Cells in Co-Culture

Isolated CD56+CD3− NK cells were expanded as described in Figure 1. Expanded NK cells were co-cultured with Incucyte® Cytolight Rapid Dye-labeled K562 cells at 1:1 ratio of NK:K562 cells at 37°C for 4 hours. Incucyte® Caspase-3/7 Dye, a caspase-inducible dye, was added to the co-culture to detect caspase-induced apoptosis of the K562 cells. Images were obtained every hour using the Incucyte® imaging system and then analyzed to determine % killing (# apoptotic K562 cells ÷ # total labeled K562 cells). After 4 hours, an average of 48 ± 2.4% K562 cells were killed (n = 9). Data represent mean ± SEM.

Figure 4. Expanded NK Cells Degranulate and Produce Cytokines After Stimulation

Isolated CD56+CD3− NK cells were expanded for 14 days (Figure 1). Expanded NK cells were left unstimulated (control) or were stimulated with either phorbol 12-myristate 13-acetate (PMA) and ionomycin or K562 cells at a ratio of 1:1 effector:target cells. CD107a antibody was added, and cultures were incubated at 37°C for 4 hours. After the first hour, Monensin and Brefeldin A were added. Cells were assessed for surface CD56, CD107a, and intracellular IFN-γ and TNF-α expression by flow cytometry. (A-C) Representative histograms of CD107a, IFN-γ, and TNF-α expression of unstimulated (grey filled), PMA and ionomycin-stimulated (orange), and K562-stimulated (purple) NK cell samples. (D) The average frequency of NK cells expressing surface CD107a, a marker of degranulation, was 23 ± 5% for the unstimulated control, 88 ± 5% after stimulation with PMA and ionomycin, and 74 ± 6% after stimulation with K562 cells. (E) The average frequency of NK cells expressing intracellular IFN-γ was 10 ± 2% for the unstimulated control, 75 ± 4% for cells stimulated with PMA and ionomycin, and 48 ± 4% for cells co-cultured with K562 cells. (F) The average frequency of NK cells expressing intracellular TNF-α was 6 ± 4% for the unstimulated control, 85 ± 1% cells stimulated with PMA and ionomycin, and 45 ± 4% for cells co-cultured with K562 cells. Data represent mean ± SEM (n = 6 - 13).