Ex vivo Expansion of NK-92 Cells

NK-92 cells were donated by the Bioengine Co., China. The cells were seeded at 1 × 105 cells/ml in serum-free medium in the presence of 1,000 U/ml human recombinant IL-2 (Peprotech, USA). Cell cultures were maintained at 37 °C in a humidified incubator with 21% O2 and 5% CO2 in an atmosphere of nitrogen. NK-92 cells were counted every 2 days during the culture process, and the culture medium was sufficiently mixed before sampling. Then, fresh medium and IL-2 were added to maintain the cell density at 1 × 105 cells/ml. The kinetics of NK-92 cell growth was calculated according to the following equations:

Expansion fold of total cells:

$$Y=frac{{N}_{t}}{{N}_{0}}$$

(1)

where Y is the expansion fold of the cells, Nt is the number of the cells at indicated time t and N0 is the number of cells at time t0.

The specific growth rate:

$$mu =frac{ln{N}_{2}-ln{N}_{1}}{{t}_{2}-{t}_{1}}$$

(2)

where μ is the specific growth rate of NK-92 cells, N1 is the number of the cells at time t1 and N2 is the number of the cells at time t2.

Experimental design and data analysis of optimization of vitamin concentrations

The concentrations of niacinamide (A) (Sigma-Aldrich, Germany, Catalog # N0636), riboflavin (B) (Sigma-Aldrich, Germany, Catalog # R9504) and inositol (C) (Sigma-Aldrich, Germany, Catalog # I7508) in the serum-free medium designed in the present study based on chemically defined EM were optimized for best cell expansion through 3-factor and 5-level CCD using Design Expert 8.0 software. Cell expansion after 8 days was selected as the response variable (Y). The concentration range of the factors was as follows: 16.50–40.00 µM niacinamide, 0.26–1.31 µM riboflavin and 26.97–238.75 µM inositol. An experimental design of 20 runs with 3 factors varying over 5 levels is summarized in Table 1. Optimum values of three variables were obtained after the response surface analysis.

Table 1 Central composite design and experimental data used for the response surface analysis

Immunophenotype analysis of expanded NK-92 cells

Approximately 7 × 105 NK-92 cells were collected by centrifugation. After rinsing twice with antibody diluent, the cells were stained with PE-conjugated anti-human CD56 antibody (BD, USA, Catalog # 555,516) and FITC-conjugated anti-human CD3 antibody (BD, USA, Catalog # 555,332) for 30 min at 4 °C in the dark. Then, the cells were washed twice with antibody diluent and resuspended in 500 µl of antibody protection solution. Sample phenotypes were analysed by flow cytometry (BD, USA).

Cytotoxicity assays of expanded NK-92 cells

Cytotoxicity of ex vivo expanded NK-92 cells was assessed by killing of K562 cells measured with cell counting kit-8 (CCK8) (Dojindo, Japan). NK-92 effector cells (E) target tumour K562 cells (T). Briefly, three experimental groups included a target cell group with (T), an effector cell group with (E) and an experimental group with cells at an E:T ratio of 5:1. The cells were suspended in 100 µl of the medium and seeded into 96-well microplates. After incubation for 4 h at 37 °C in an incubator, 10 µl CCK8 solution was added into each well, and the cells were incubated for another 2 h before detection of absorbance at 450 nm using a microplate reader. The cytotoxicity of NK-92 cells against K562 cells was calculated as follows:

$$cytotoxicity, %=frac{{N}_{1}-({N}_{3}-{N}_{2})}{{N}_{1}}times 100 %$$

(3)

where N1, N2 and N3 represent the absorbance of the target cell group, effector cell group and experimental group, respectively.

The CD107a expression of cultured cells was assayed to evaluate cell degranulation. NK-92 cells were co-cultured with K562 cells at an E: T ratio of 5:1 for 4 h and then stained with mouse anti-human CD107a PE-Cy7-conjugated antibodies (BD, USA, Catalog # 561,348) for 30 min at 4 °C in dark. Besides, to carry out the intracellular granzyme B and perforin of NK-92 cells in cultures, cells were fixed, permeabilized and stained with antibodies of V450-granzyme B (BD, USA, Catalog # 563,389) and V450-perforin (BD, USA, Catalog # 563,393). The expression of CD107a, granzyme B and perforin were measured by flow cytometry.

Detection and calculation of kinetic parameters

The culture supernatant was collected at specific timepoints by centrifugation to analyse the concentrations of glucose and lactic acid using a glucose assay kit and a lactate assay kit (Jiancheng Bioengineering Institute, China). The absorbance values were detected by a microplate reader. Relevant kinetic parameters were determined as follows:

Specific glucose consumption rate:

$${Q}_{gluc}=frac{{C}_{1}-{C}_{2}}{{int }_{{t}_{1}}^{{t}_{2}}Af(t)dt}$$

(4)

where C1 represents the concentration of glucose at time t1, C2 represents the concentration of glucose at time t2 and ({int }_{{t}_{1}}^{{t}_{2}}Afleft(tright)dt) is the integral of the number of NK92 cells A from t1 to t2.

Specific lactic acid production rate:

$${q}_{lac}=frac{{K}_{2}-{K}_{1}}{{int }_{{t}_{1}}^{{t}_{2}}Af(t)dt}$$

(5)

where K1 represents the concentration of lactic acid at time t1, K2 represents the concentration of lactic acid at time t2 and ({int }_{{t}_{1}}^{{t}_{2}}Afleft(tright)dt) is the integral of the number of NK-92 cells (A) from t1 to t2.

The yield coefficient of lactate to glucose:

$${Y}_{lac/gluc}=frac{{q}_{lac}}{{Q}_{gluc}}$$

(6)

Determination of activities of enzymes of glucose metabolism

The enzyme activities of phosphofructokinase (PFK), glucose-6-phosphate dehydrogenase (G6PDH), pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH) in NK-92 cells were detected with PFK, G6PDH, PDH and LDH assay kits, respectively, following the manufacturer’s instructions (Comin Biotechnology, China).

Extracellular flux assays

A Seahorse XFe 96 analyzer (Agilent Technologies, USA) was used to measure the OCR and ECAR of NK-92 cells cultured in various media. NK-92 cells (4 × 104 per well) were seeded in Seahorse XF96 cell culture microplates, which were coated with polylysine overnight and washed twice using sterile water before plating the cells for the assay. The cells were cultured in the assay medium and incubated for an hour at 37 ℃ in an incubator without CO2. The glycolysis assay medium was glucose-free, and the mitochondrial assay medium contained 2 mM glutamine, 1 mM pyruvate and 10 mM glucose. For glycolytic stress tests, 10 mM glucose, 2 µM oligomycin and 30 mM 2-deoxyglucose were injected during the measurements. For the mitochondrial stress tests, 2 µM oligomycin, 0.5 µM FCCP and 2 µM rotenone/antimycin were sequentially added to the wells.

Detection of intracellular metabolites

NK-92 cells were collected by centrifugation, and the intracellular metabolites ATP, NADP(H) and GSH were assayed using the corresponding assay kits, including an ATP assay kit, a NADP(H) assay kit and a GSH assay kit, respectively (Beyotime, China).

Statistical analysis

The values are presented as the mean ± standard error. The significant differences were assessed by Student’s t test (two samples, one-tailed). P < 0.05 was considered statistically significant.

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