CZ.02.1.01/0.0/0.0/18_069/0010060), MH CZ DRO FNOs/2020, and the Institutional Development Plan of University or college of Ostrava, allocated by The Ministry of Education, Youth and Sports (project no. myeloma cell collection by more than 20%. Time-lapse imaging of mice with established human multiple myeloma xenografts revealed that combination therapy of selected and pretreated NK cells with Daratumumab offered tumor volumes 43-fold smaller than control ones. Combination therapy with an allogeneic source of fully functional NK cells could be beneficial in future clinical settings to circumvent monoclonal antibodies low therapeutic efficiency due to NK cell dysfunctionality in MM patients. 0.05. 3. Results 3.1. Isolation and Phenotypic Characterization of Expanded PBNK Cells PBNK cells were isolated from three healthy donors using unfavorable selection with the untouched NK cell isolation kit (Miltenyi, Bergisch Gladbach, Germany). In all cases, we obtained a similar quantity of PBNK cells, accounting for ~5% of mononuclear cells (Physique 1a). Expansion of the NK cells was performed without feeder cells in the NK MACS Medium (Miltenyi, Bergisch Gladbach, Germany) supplemented with IL-2 and IL-15. Cell proliferation and viability were tracked for six weeks, reaching the maximum cell number in the fifth week, with an average fold growth of 486 157.8 (Determine 1b) and a median of 14,050 302 million PBNK cells from a single donor. The producing doubling times were 101 h, 47 h, and 40 h for PBNK cells from donors 1, 2, and 3, respectively. Next, along the time of growth, we conducted the phenotypic characterization of the three expanded PBNK cell donors by circulation cytometry using antibody panels to protect non-NK lineage and NK cell lineage markers. During the six TPO agonist 1 weeks of growth, we observed a lack of expression of non-NK lineage markers and positive expression of the unique markers related to NK cell lineage in all three expanded PBNK cell donors (Physique 1c). Interestingly, most of the different markers remained stable during the six weeks of the growth period, except for the NK phenotype marker NKG2C, which significantly and progressively increased over time in all three donors. The NKG2C receptor is an activating receptor in NK cells and, together with the inhibitory receptor NKG2A, belongs to the family of TPO agonist 1 the C-type lectin receptors . The ligand for these receptors is the non-classical HLA-E molecule, frequently overexpressed in TPO agonist 1 different types of tumor cells [27,28]. Open in a separate window Physique 1 Isolation of NK cells, growth to clinically relevant PBNK cell concentrations, and phenotype analysis. (a) A representative table with the yields of PBNK cells from three different donors using a unfavorable selection approach. (b) Summary graph showing the median growth fold of the three PBNK cell donors L1CAM antibody isolated and expanded in the feeder-free NK MACS medium. (c) Representative graphics of PBNK cell purity obtained by FACS at the TPO agonist 1 indicated quantity of days post-expansion. Data in (b,c) are means SEM of the three expanded PBNK cell donors. Collectively, our results indicate the TPO agonist 1 feasibility and reproducibility in the isolation and growth of PBNK cells, achieving highly purified and clinically relevant numbers of NK cells after five weeks of growth, applying a straightforward feeder-free method of growth. 3.2. Functional Characterization of Expanded PBNK Cells Next, we evaluated the ability of the PBNK cells isolated and expanded from three different donors to mediate activity against different tumor cells. For this purpose, we performed a bioluminescence-based cytotoxicity assay  based on the co-culture of the expanded PBNK cells with several human tumor target cell lines stably transduced to express luciferase. The specific lysis of target cell lines by PBNK cells was measured in weeks two, four, and six of growth. As shown in Physique 2a, we observed cytotoxicity against the five targeted tumor cell lines for the different expanded PBNK cell donors, achieving maximum cytotoxicity in the fourth week of growth. In week six, the.