PBMCs from healthy donors (buffy coats provided by DRK Blutspendedienst, Frankfurt, Germany) were enriched using density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, Taufkirchen, Germany). NK cells from PBMCs were enriched using a negative selection kit following the manufacturer’s instructions (EasySep™ Human NK Cell Enrichment Kit; STEMCELL Technologies, Cologne, Germany). NK cells were expanded and activated in NK-MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with human IL-15 (Peprotech, Rocky Hill, CT) for 15 days. The NK cell population purity was validated to be above 95% after enrichment (day 0), during expansion (day 7) and on the day of experiments (day 15) using a flow cytometric staining panel. NK cells were defined as CD56 and CD16 double-positive and CD3 negative.
Nk macs
Manufactured by Miltenyi Biotec
Sourced in Germany
The NK MACS is a specialized laboratory equipment designed for the isolation and enrichment of natural killer (NK) cells from biological samples. This device utilizes magnetic cell separation technology to efficiently separate and purify NK cells from a heterogeneous cell population. The core function of the NK MACS is to enable researchers and scientists to study and work with highly pure NK cell samples, which is crucial for various applications in the field of immunology and cell biology.
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Lab products found in correlation
Histopaque 1077, by Merck Group (1 mentions)
Human il 15, by Thermo Fisher Scientific (1 mentions)
Easysep human nk cell enrichment kit, by STEMCELL (1 mentions)
Pbmcs, by Lonza (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Heat inactivated human serum, by Merck Group (1 mentions)
Human nk cell negative selection kit, by STEMCELL (1 mentions)
Nk supplement, by Miltenyi Biotec (1 mentions)
Cytation5 biotek plate reader, by Agilent Technologies (1 mentions)
Cytotox glo, by Promega (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Easysep magnet, by STEMCELL (1 mentions)
Iscove modified dulbecco media (imdm), by Corning (1 mentions)
Rhil 12, by Miltenyi Biotec (1 mentions)
6 well tissue culture plate, by Corning (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Texmacs, by Miltenyi Biotec (1 mentions)
6 protocols using nk macs
1
Expansion and Activation of NK Cells
2
Expansion and Isolation of Primary NK Cells
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PBMCs were purchased from Lonza (Morristown, NJ, USA). NK MACS (Miltenyl Biotec, Bergisch Gladbach, Germany) medium was used for cell culture. Primary NK (pNK) cells were expanded and maintained in the presence of IL-2 (200 IU/mL) and IL-15 (10 ng/mL; PeproTech, Cranbury, NJ, USA), and co-cultured with K562 feeder cells obtained from ATCC (Manassas, VA, USA). Following 2 weeks of expansion, pNK cells were isolated using an NK cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions.
3
ADCC Evaluation of BCA101 in EGFR-Expressing Cells
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ADCC of BCA101 was assessed using TGFβ pretreated NK cells and A431 (EGFRhigh) or MCF7 (EGFRlow) cell lines, procured from ATCC. NK cells were isolated from frozen or fresh human peripheral blood mononuclear cells (PBMC) using Human NK-cell negative selection kit (Stemcell Technologies Inc.) and EasySep magnet (Stemcell Technologies Inc.). About 1 million enriched NK cells were left untreated or cultured with 1 ng/mL of TGFβ (BPS Biosciences), TGFβ plus cetuximab or BCA101 (56 nmol/L) for 72 hours in a 12-well plate in NK MACS (Miltenyi Biotec) media containing 1% pen strep (GIBCO, Thermo Fisher Scientific), 1% heat-inactivated human serum (Sigma-Aldrich), and 1% NK supplement (Miltenyi Biotec). These pretreated NK cells were used as effectors in ADCC assay. BCA101 (56 nmol/L) was added to target tumor cells and preincubated for 1–2 hours, after which, pretreated NK cells were added at a ratio of 1:5 (5,000 target cells: 25,000 NK cells) in a 96-well plate. The assay plates were incubated for around 24 hours. Cytotoxicity was measured by evaluating protease release using CytoToxGlo (Promega). Luminescence readout was taken using a plate reader (Cytation5 BioTek plate reader) and data were analyzed using GraphPad Prism software (version9). Data are plotted as percentage change in cytotoxicity over human IgG control group.
4
Generating HPC-NK Cells from Human Progenitors
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UCB collection at delivery was approved (see “Compliance with ethical standards”). HPC-NK cells were generated as described [11 (link)] with the following minor modifications. Cells were cultured for 5–7 weeks in 6-well tissue culture plates (Corning, 3506), using NK MACS Basal medium and supplement (NK MACS, Miltenyi Biotec, 130–114-429) complemented with 10% human serum (HS, Sanquin) during expansion (day 0–14) and 2–10% HS during differentiation (from day 14). HPC-NK cells (> 70% CD56+) were used directly or cryopreserved. Cryopreserved HPC-NK cells were thawed and used after 5–7 days of culture in NK MACS containing 10% HS, 50 ng/ml recombinant human (rh)IL-15 (Immunotools, 11340155) and 0.2 ng/ml rhIL-12 (Miltenyi Biotec, 130–096-704). For experiments, HPC-NK cells were resuspended in Iscove's Modified Dulbecco's Medium (IMDM, Gibco, 21980–032) supplemented with 10% fetal calf serum (FCS, Integro, 5–45900 or Corning, 35–079-CV) (IMDM10), except assays with primary AML samples (10% HS), proliferation assays, and some serial killing experiments in microwells (NK MACS medium + 10% HS or FCS, respectively).
5
Optimizing NK Cell Expansion from PBMC
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For the culture medium optimization, PBMCs were cocultured with irradiated K562mbIL15 cells in a 1:1.5 ratio in the indicated growth medium: RPMI-1640 (RPMI, Lonza, Basel, Belgium), stem cell growth medium (SCGM, Cellgenix, Freiburg, Germany), NK MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) or TexMACS GMP medium (TexMACS, Miltenyi Biotec, Bergisch Gladbach, Germany). All media were supplemented with 10% human AB serum (Sigma, St. Louis, MO, USA) and IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany) at 10 IU/mL for the first week and 100 IU/mL thereafter. Fresh medium was added every 2 days to a final concentration of 1–2 × 106 cell/mL.
To explore the feasibility of expanding NK cells from the CD45RA+ fraction, PBMCs or CD45RA+ cells from mobilized or non-mobilized apheresis were cocultured with irradiated K562mbIL15 in a 1:1.5 ratio, using complete TexMACS.
To determine the best NK cell source and aAPC, either PBMC or CD45RA+ cells were cocultured with irradiated K562mbIL15 or K562mbIL21 in a 1:1.5 ratio, using complete TexMACS.
Total cell expansion, percentage of NK cells and other lymphocyte subpopulations and viability of the cultures were monitored every week by flow cytometry. Cytotoxicity of expanded NK cells was tested against different tumor target cells between days 14 and 21 of NK cell expansion.
To explore the feasibility of expanding NK cells from the CD45RA+ fraction, PBMCs or CD45RA+ cells from mobilized or non-mobilized apheresis were cocultured with irradiated K562mbIL15 in a 1:1.5 ratio, using complete TexMACS.
To determine the best NK cell source and aAPC, either PBMC or CD45RA+ cells were cocultured with irradiated K562mbIL15 or K562mbIL21 in a 1:1.5 ratio, using complete TexMACS.
Total cell expansion, percentage of NK cells and other lymphocyte subpopulations and viability of the cultures were monitored every week by flow cytometry. Cytotoxicity of expanded NK cells was tested against different tumor target cells between days 14 and 21 of NK cell expansion.
6
Isolation and Expansion of NK Cells
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Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor's buffy coat or leukapheresis (OPBG Hospital, Rome, Italy) by a density-gradient technique (Ficool-Histopaque (Eurobio;France); the healthy donors had signed a written informed consent, in accordance with rules set by the Institutional Review Board of OPBG (Approval of Ethical Committee N_969/2015 prot. N_669LB). CD56 + CD3 neg NK cells, isolated with an NK isolation Kit (Miltenyi Biotec, Inc., San Diego, CA, USA), and expanded with NK Cell Activation/Expansion Kit (adapted protocol from Navarro et al. 46 ) (Miltenyi Biotec, Inc., San Diego, CA, USA) and recombinant human interleukin 2 (IL2, 500 U/ml; R&D; USA) or recombinant interleukin 15 (IL15 10 U/ml; R&D; USA). Activated NK cells were transduced in 24-well plates pre-coated with recombinant human RetroNectin (Takara-Bio. Inc; Japan) using retroviral supernatant. Enriched NK cells were cultured in GMP-compliant media (NK MACS Miltenyi Biotec, Inc., San Diego, CA, USA).
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