Application protocol

NK cells expansion from human PBMCs or isolated NK cells

Many downstream applications require expansion of NK cells prior to analysis. In vitro cultivation of NK cells often results in low expansion rates, exhausted phenotype due to long-term expansion, and overgrowth of conventional T cells if present in the initial culture. NK MACS Medium has a defined formulation that enables expansion of NK cells from peripheral blood mononuclear cells (PBMCs) or isolated NK cells. Expanded cells are fully functional and ready for downstream applications.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For isolation of PBMCs

Isolation of PBMCs using Ficoll-Paque™

  • PE buffer: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
    Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
  • Ficoll-Paque (ρ = 1.077 g/mL)
  • 50 mL conical centrifuge tubes  

For isolation of NK cells

  • NK Cell Isolation Kit, human (# 130-092-657)
  • PBE Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Degas buffer before use, as air bubbles could block the column.
  • (Optional) Pre-Separation Filters (30 μm) (# 130-041-407) to remove cell clumps
  • MACS MultiStand (# 130-042-303)
  • Choose the appropriate MACS Separator and MACS Columns:
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
MS1×1072×108MiniMACS™, OctoMACS™
LS1×1082×109MidiMACS™, QuadroMACS™
autoMACS2×1084×109autoMACS Pro
Note: When using this kit the unwanted cell fraction is labeled and the target cells remain unlabeled. Depending on the target cell frequency, the labeled fraction can therefore represent the majority of the total cells. To avoid blocking of the column, do not exceed the max. number of labeled cells per column. Estimate the number of labeled cells in the sample, split the sample if necessary and use the appropriate number of separation columns.

For NK cell expansion

  • NK MACS Medium (# 130-114-429)
  • Human IL-2 IS, premium grade (# 130-097-744, # 130-097-745, # 130-097-746)
  • Human AB serum or autologous plasma. Availability is country-specific.
  • 24-well, 12-well and 6-well cell culture plates
  • (Optional) T25, T75 and T175 flasks

For flow cytometry analysis

  • CD3-APC (# 130-109-462)
  • CD56-PE (# 130-113-312)
  • CD45-VioBlue® (# 130-110-637)
  • CD19-APC-Vio®770 (# 130-113-643)
  • CD14-FITC (# 130-110-518)
  • 7-AAD Staining Solution (# 130-111-568)
  • (Optional) CD16-PE-Vio615 (# 130-107-710)
Matching products:

Protocol


View timeline

This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step. 

Things to prepare in advance

PE buffer: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).

Isolation of human PBMCs using FicollPaque™

Note:

  • The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g., heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)).
  1. Dilute cells with 2–4× the volume of PE buffer.
    Note: The more diluted the blood sample, the better the purity of the mononuclear cells.
  2. Carefully layer 35 mL of diluted cell suspension over 15 mL of Ficoll-Paque™ in a 50 mL conical tube.
  3. Centrifuge at 400×g for 30–40 minutes at 20 °C in a swinging-bucket rotor without brake.
  4. Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.
  5. Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.
  6. Fill the conical tube with PE buffer, mix, and centrifuge at 300×g for 10 minutes at 20 °C. Carefully remove supernatant completely.
  7. For removal of platelets, resuspend the cell pellet in 50 mL of PE buffer and centrifuge at 200×g for 10–15 minutes at 20 °C. Carefully remove the supernatant completely.
    Note: This step will increase the purity of the target cells in the subsequent MACS® Cell Separation.
  8. Repeat step 7. Most of the platelets will remain in the supernatant upon centrifugation at 200×g.
  9. Resuspend cell pellet in an appropriate amount of PE buffer and proceed to "Isolation of NK cells".
    ▲ Note: PBMCs may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum. Do not store cells longer than one day in the refrigerator. Wash at least once before proceeding to magnetic labeling and resuspend cells in an appropriate buffer. For details see MACS Cell Separation Reagents data sheets.  

Isolate pure and fully functional NK cells using the NK Cell Isolation Kit, human. This indirect magnetic labeling system isolates untouched NK cells from human PBMCs by magnetically labeling  and depleting non-NK cells (i.e., T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes, and erythroid cells) with a cocktail of biotin-conjugated antibodies and the NK Cell MicroBead Cocktail. Follow the protocol in the kit data sheet.

Download the data sheet

NK Cell Isolation Kit, human

Things to prepare in advance

PBE Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Degas buffer before use, as air bubbles could block the column.

Surface staining of PBMCs or isolated NK cells

Notes:

  • Volumes given in the data sheet for each antibody are for up to 1×10⁶ or 1×107 nucleated cells. Follow the instructions provided.
  1. Determine cell number.
  2. Centrifuge cell suspension at 300×g for 10 minutes. Discard supernatant.
  3. Resuspend according to the data sheets of the antibodies used.
  4. Add antibodies according to the dilution listed in the antibody data sheets.
  5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
  6. Wash cells by adding 1−2 mL of PBE buffer per 1×10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Discard supernatant.
  7. Proceed immediately to "Flow cytometry analysis".

Flow cytometry analysis

Notes:

  •  Perform the purity analysis with a cell sample taken before cell separation and a cell sample taken after isolation.
  • When working with PBMCs as starting material, analyze only the original sample. 

After staining the samples, perform the flow cytometry analysis as follows:

  1. Identify lymphocytes by SSC/CD45 gating.
  2. Exclude dead cells from the analysis using the live/dead cell exclusion marker 7AAD
  3. Use the SSC/FSC gate to identify different populations:
Marker on y-axisMarker on x-axisIdentified cell population
CD14CD56CD14+ and CD14+CD56+ (monocytes)
CD3CD19CD3CD19+ (B cells)
CD3CD56CD3CD56+ (NK cells)
CD3CD14CD3+CD14 (T cells)
CD3CD56CD3+CD56+ (NKT cells)

Ex vivo cultivation is an attractive option to increase NK cell numbers for functional analyses, including anti-tumor potential. Miltenyi Biotec NK MACS® Medium addresses the major challenges of culturing NK cells with a uniquely designed formulation that expands fully functional NK cells from either PBMCs or isolated NK cells. The medium is xeno-free and enables superior NK cell expansion with minimal growth of unwanted cells, like B, T, or DC cells.
Note: supplementation with serum or autologous plasma is necessary.

Things to prepare in advance

Complete and expansion NK MACS Medium

  1. Before NK MACS Medium can be used, NK MACS Basal Medium and NK MACS Supplement (provided) must be mixed.
  2. Thaw NK MACS Supplement at 2–8 °C.
  3. Add 1% of NK MACS Supplement to the NK MACS Basal Medium, e.g., add 1 mL of supplement to 100 mL of basal medium.
  4. Add 5% AB serum. Mix well. This is complete medium.
    ▲ Note: Only prepare the amount of complete medium which is used within 3 weeks. Complete medium can be used up to 3 weeks when stored at 2–8 °C. Do not freeze.
  5. Prior to use for expansion, add 500 IU/mL of IL-2. This is expansion NK MACS medium.

Preparation of NK cells or PBMCs for culture

  1. Determine the total number of cells.
  2. Wash cells by adding 5–10 volumes complete medium (see "Things to prepare in advance") to the cells and centrifuge at 300×g for 10 minutes. Discard supernatant. 
  3. Resuspend cells at a concentration of 1×10⁶ cells/mL in expansion NK MACS medium (see "Things to prepare in advance").
  4. Pipet 300 µL of cell suspension into each well of a 24 well-plate (roughly 3×105 cells/well).
    ▲  Recommended: use residual volume for cell count determination.

Expansion process

Notes:

  • IMPORTANT: during NK cell cultivation, do not disturb the cells for the first week at all. After 7 days, do not change the medium. Instead, add fresh expansion NK MACS Medium (see "Things to prepare in advance")
  • Sampling or changing a flask: NK cells attach to the bottom of a plate or flask. Carefully scratch cells off the surface with a 1000 µL tip pipette and resuspend cells at least 5 times. Always handle cells gently. After 10 days, aggregates may appear. This is not unusual.
  • NK cell expansion is donor-dependent. The estimated volumes listed below are an example and may vary. NK cells can also be expanded for longer than 14 days, though success is also donor-dependent.

When adding expansion NK MACS medium to culture plates or flasks, please note:

  • Upon reaching a well volume of 1 mL, switch to a 12-well plate (around day 7 or 10)
  • Upon reaching a well volume of 2.5 mL, switch to a 6-well plate (this step can also be skipped and cells transferred directly to a T25 flask)
  • Upon reaching a flask volume of 5–6 mL, switch to a T25 flask (usually around day 10 or 12)
  • Upon reaching a flask volume of 7 mL, switch to a T75 flask (usually around day 12 or 14)
  • Upon reaching a flask volume of 30 mL, switch to a T175 flask (usually around day 14–17)

Day 1–6 

Do not disturb cells.


Day 7 

  1. Take a 50 µL sample of cells to determine cell count (w/o stain, just PI/7-AAD + Scatter).
  2. If the cell density is less than 1×106 cells/mL, double the culture volume with fresh expansion NK MACS medium (in the case illustrated by this protocol, add 250 µL).
  3. If the cell density is greater than 1×106 cells/mL, add enough fresh expansion NK MACS medium to dilute cells to a final concentration of 4–5×105 cells/mL (e.g., if cell density on day 7 is 1.5×106 cells/mL, add 500 µL of fresh medium to the existing culture volume of 250 µL. Total volume will then be 750 µL and final cell density 5×105 cells/mL).

Day 10 

  1. Take a 50 µL sample of cells to determine cell count (w/o stain, just PI/7-AAD + Scatter).
  2. Add enough fresh expansion NK MACS medium to achieve a final cell concentration of 4–5×105 cells/mL.

Day 12 

  1. Take a 50 µL sample of cells to determine cell count (w/o stain, just PI/7-AAD + Scatter).
  2. Add enough fresh expansion NK MACS medium to achieve a final cell concentration of 4–5×105 cells/mL.

Day 14 

Take a 50 µL sample of cells to determine NK cell count and perform cell analysis (e.g., proceed to "Cytotoxicity assay")

View details

Superior expansion of NK cells. NK cells can be expanded using PBMCs as starting material, or NK cells isolated from whole blood, buffy coats, or PBMCs. Using NK MACS Medium results in 3 times greater fold expansion of NK cells compared to other media or TexMACS™ Medium for T cells. Illustrated are NK cell fold expansion from (A) PBMCs (n=3) or (B) isolated NK cells (n=3) using 5% AB serum and 500 UI/mL of IL-2.

View details

Limited expansion of non-NK cells. When PBMCs are used as starting material, NK MACS Medium favors expansion of NK cells compared to other media or TexMACS Medium for T cells. The defined formulation limits growth of unwanted T (CD3+), NKT (CD3+CD56+), and other cells (e.g., CD3CD56). Illustrated is the cell composition of PBMC cultures after 14 days of expansion in different culture media.

NK cells cultivated in NK MACS® Medium retain their natural cytotoxicity against the K-562 cell line and are suitable for any downstream applications, including cytotoxicity assays and flow cytometry analysis. 

View details

Retained cytotoxicity of expanded NK cells. NK cells were expanded for 9 days in NK MACS Medium starting from (A) PBMCs and (B) isolated NK cells and their cytotoxicity against K-562 cells was measured by flow cytometry at different effector-to-target (E:T) ratios.

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.