nk 92 mi cell line  (ATCC)

Journal: bioRxiv

Article Title: A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

doi: 10.64898/2026.01.21.700863

Figure Lengend Snippet: Toxicity of different concentrations of the cryopreservation medium STEM-CELLBANKER ® EX (SCB) towards K562 cells, primary NK cells, and NK-92 cells; cell viability was assessed via flow cytometry after 24 h incubation. A: Relative viability of K562 cells incubated with 0–100% SCB at 37°C and room temperature (RT) (n = 3 independent experiments, each 1 technical replicate); statistical analysis: two-way ANOVA with Tukey post hoc test within RT and 37°C groups. B: Relative viability of primary NK cells with 0–100% SCB at RT, 37°C, and 37°C + 500 U/mL IL-2 (left, n = 1 experiment, 1 technical replicate; no statistics performed) and with 0–25% SCB at 37°C (right, n = 2 donors/experiments, each 3 technical replicates); statistical analysis: two-way ANOVA with Tukey post hoc test. C: Relative viability of NK-92 cells with 0–100% SCB (left, n = 1 experiment, 3 technical replicates) and 0– 25% SCB (right, n = 1 experiment, 3 technical replicates) at 37°C; statistical analysis: one-way ANOVA with Tukey post hoc test. Bars represent mean ± SEM (ns = not significant; *p < 0.05; **p < 0.01; ****p < 0.0001).

Article Snippet: NK-92 cells (ATCC ® CRL-2407TM, American Type Culture Collection, Manassas, VA, USA) were cultured in MEM-α (Sigma-Aldrich) with 12.5% fetal calf serum (FCS; PAN-Biotech), 12.5% horse serum (PAN-Biotech), 100 U/mL penicillin plus 100 μg/mL streptomycin (Sigma-Aldrich), and 100 U/mL recombinant human Interleukin-2 (IL-2) (Proleukin S, Novartis, Basel, Switzerland).

Techniques: Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

doi: 10.64898/2026.01.21.700863

Figure Lengend Snippet: Analysis of specific lysis of washed versus non-washed target cells using bead-based absolute counting or viability-based calculation across effector-to-target (E:T) ratios. A: Schematic of the analysis workflow; after flow cytometric acquisition, specific lysis was calculated using either counting beads to derive viable targets per defined volume (bead-based) or the numbers of live and dead targets (viability-based). Created in BioRender. Kaltschmidt, C. (2025) https://BioRender.com/x49u069 B: Specific lysis in assays using primary NK (pNK) cells as effectors (left: viability-based; right: bead-based; n = 5 donors/experiments, each 2–3 technical replicates). C: Specific lysis in assays using NK-92 cells as effectors (left: viability-based; right: bead-based; n = 1 experiment, 3 technical replicates). Statistical analysis: two-way ANOVA with Sidak post hoc test. Bars represent mean ± SD (*p < 0.05; ***p < 0.001; ****p < 0.0001).

Article Snippet: NK-92 cells (ATCC ® CRL-2407TM, American Type Culture Collection, Manassas, VA, USA) were cultured in MEM-α (Sigma-Aldrich) with 12.5% fetal calf serum (FCS; PAN-Biotech), 12.5% horse serum (PAN-Biotech), 100 U/mL penicillin plus 100 μg/mL streptomycin (Sigma-Aldrich), and 100 U/mL recombinant human Interleukin-2 (IL-2) (Proleukin S, Novartis, Basel, Switzerland).

Techniques: Lysis

Journal: bioRxiv

Article Title: A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

doi: 10.64898/2026.01.21.700863

Figure Lengend Snippet: Cellular cytotoxicity assay using ready-to-thaw target cells without washing. CFSE-stained K562 targets were frozen in 40 µL aliquots of SCB with 20% FCS using isopropanol thermal buffering, thawed by direct addition of pre-warmed medium, and co-incubated with NK-92 cells for 4 h; EDTA and propidium iodide were then added for conjugate dissociation and dead-cell staining. A: Relative (left) and absolute (right) viability of target cells with-out NK-92 before cryopreservation, after thawing, and after the cytotoxicity assay (n = 1 experiment, 4–5 technical replicates: 5 before cryopreservation; 4 for both post-thaw conditions); statistical analysis: ordinary one-way ANOVA with Tukey post hoc test (both). B: Consistency of viable recovery (left) enables adjustment of pre-cryo-preservation target-cell density to achieve the desired post-thaw cell count (right) (n = 1 experiment, 4–5 technical replicates: 5 before cryopreservation; 4 after thawing); statistical analysis: unpaired t test (both). C: Specific lysis measured with NK-92 effectors by viability-based calculation (upper) and by volumetric absolute counts (lower) (n = 1 experiment, 4–5 technical replicates per E:T ratio: 4 for 0:1 and 9:1; 5 for 1:1 and 3:1). Bars represent mean ± SEM. (*p < 0.05; ****p < 0.0001).

Article Snippet: NK-92 cells (ATCC ® CRL-2407TM, American Type Culture Collection, Manassas, VA, USA) were cultured in MEM-α (Sigma-Aldrich) with 12.5% fetal calf serum (FCS; PAN-Biotech), 12.5% horse serum (PAN-Biotech), 100 U/mL penicillin plus 100 μg/mL streptomycin (Sigma-Aldrich), and 100 U/mL recombinant human Interleukin-2 (IL-2) (Proleukin S, Novartis, Basel, Switzerland).

Techniques: Cytotoxicity Assay, Staining, Incubation, Preserving, Cell Characterization, Lysis

Journal: Frontiers in Immunology

Article Title: Construction of anti-HER2 affibody-directed CAR-NK and its synergistic effects with doxorubicin-loaded nanodrug against HER2-positive breast cancer

doi: 10.3389/fimmu.2025.1692107

Figure Lengend Snippet: Synergistic antitumor effect of HER2-targeted CAR-NK cells and hyaluronic acid (HA)- chitosan (CS)-based doxorubicin-loaded nanoparticles for the treatment of breast cancer. Recombinant HER2 lentivirus is used to transduce NK92-MI cells, generating HER2-specific CAR-NK cells. Positively charged CS and negatively charged HA self-assemble into nanoparticles loaded with the chemotherapeutic drug doxorubicin. This dual therapeutic system is administered intravenously into breast tumor-bearing mice. After injection, both HER2 CAR-NK cells and HA-CS nanoparticles circulate through the bloodstream and accumulate at the tumor site. The nanoparticles target tumor cells via HA–CD44 interaction and undergo endocytosis, releasing doxorubicin intracellularly to induce tumor cell cytotoxicity. Meanwhile, HER2 CAR-NK cells specifically recognize and kill HER2-positive tumor cells. The combination therapy exerts a synergistic antitumor effect through both immune-mediated and chemotherapeutic mechanisms.

Article Snippet: Human natural killer cell NK-92MI was purchased from ATCC (Manassas, VA, USA).

Techniques: Recombinant, Transduction, Injection

Journal: Frontiers in Immunology

Article Title: Construction of anti-HER2 affibody-directed CAR-NK and its synergistic effects with doxorubicin-loaded nanodrug against HER2-positive breast cancer

doi: 10.3389/fimmu.2025.1692107

Figure Lengend Snippet: The expression of HER-2 in different breast cancer cell lines and the killing effect of three kinds of CAR-NK on various breast cancer cells. (A) Using western blot to detect the expression of HER-2 on the surface of three breast cancer cells, the antibody used anti-HER-2 and anti-GAPDH monoclonal antibodies. (B–D) Cell lysis efficiency of effector cell (NK-92MI, scFv CAR-NK, Affi1 CAR-NK, Affi2 CAR-NK) and target cell (MCF-7, BT-474, SK-BR-3) incubate for 6 hours at E:T 1:1, 5:1 and 10:1. Data are presented as the mean ± SD, and significant differences between groups were measured by using two-way analysis of variance (ANOVA). * P<0.05, **P <0.01, ***P < 0.001, ns not significant, n=3.

Article Snippet: Human natural killer cell NK-92MI was purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Bioprocessing, Lysis

Journal: Frontiers in Immunology

Article Title: Construction of anti-HER2 affibody-directed CAR-NK and its synergistic effects with doxorubicin-loaded nanodrug against HER2-positive breast cancer

doi: 10.3389/fimmu.2025.1692107

Figure Lengend Snippet: Time-lapse imaging of Affi1 CAR-NK–mediated killing of HER2-positive tumor cells. Affi1 CAR-NK cells (effector) and SK-BR-3 cells (target) were co-cultured at an E:T ratio of 1:1, and live-cell imaging was performed at the indicated time points (0 min, 42 min, 1 h 14 min, 2 h 50 min, 2 h 56 min, and 3 h 28 min). White arrows indicate Affi1 CAR-NK cells, and red arrows indicate SK-BR-3 tumor cells. Progressive reduction of target cells over time reflects Affi1 CAR-NK cytotoxic activity. Scale bar = 50 μm.

Article Snippet: Human natural killer cell NK-92MI was purchased from ATCC (Manassas, VA, USA).

Techniques: Imaging, Cell Culture, Live Cell Imaging, Activity Assay

Journal: Frontiers in Immunology

Article Title: Construction of anti-HER2 affibody-directed CAR-NK and its synergistic effects with doxorubicin-loaded nanodrug against HER2-positive breast cancer

doi: 10.3389/fimmu.2025.1692107

Figure Lengend Snippet: Effects of different doses of irradiation on the proliferation of Affibody 1 CAR-NK cells and the killing function of CAR-NK cells after irradiation. (A) Changes in the number of Affi1 CAR-NK cells after different doses of radiation (0, 2, 5, 10 Gy) during different times (0, 24, 48, 72, 96 hours) (B) CCK8 was used to measure the change of cell activity of Affi1 CAR-NK after irradiation (C) Killing of target cells MCF-7 by effector cells after irradiation with different doses (0, 2, 5, 10 Gy) (D) Killing of target cells SK-BR-3 by effector cells after different doses of irradiation (0, 2, 5, 10 Gy). Data are presented as the mean ± SD, and significant differences between groups were measured using two-way analysis of variance (ANOVA). ****P < 0.0001 ns not significant, n=3.

Article Snippet: Human natural killer cell NK-92MI was purchased from ATCC (Manassas, VA, USA).

Techniques: Irradiation, Activity Assay

Journal: Frontiers in Immunology

Article Title: Construction of anti-HER2 affibody-directed CAR-NK and its synergistic effects with doxorubicin-loaded nanodrug against HER2-positive breast cancer

doi: 10.3389/fimmu.2025.1692107

Figure Lengend Snippet: Affi1 CAR-NK cell killing after doxorubicin nano-drug treatment (A) MCF-7 was used as the target cell, and DNP was added in advance to incubate for 12h. After that, effector cells Affi1CAR-NK were added at different effector-to-target ratios (1:1, 5:1, 10:1), and the cytotoxicity was detected by LDH after 6h. (B) SK-BR-3 was used as the target cell, and DNP was added in advance to incubate for 12h. After that, effector cells Affi1CAR-NK were added at different effector-to-target ratios (1:1, 5:1, 10:1), and the cytotoxicity was detected by LDH after 6h. Data are presented as the mean ± SD, and significant differences between groups were measured using two-way analysis of variance (ANOVA). * P<0.05, ****P < 0.0001 ns not significant, n=3.

Article Snippet: Human natural killer cell NK-92MI was purchased from ATCC (Manassas, VA, USA).

Techniques: