Ex Vivo Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells

Published: July 31, 2023

Abstract

Source: Ma, M., et al. Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line. J. Vis. Exp. (2022).

This video demonstrates a technique for the expansion of natural killer (NK) cells directly from peripheral blood mononuclear cells (PBMCs) using a feeder cell line. PBMCs are co-cultured with genetically modified lymphoblastoid feeder cells expressing membrane-bound interleukin-21 (mIL-21), and the culture is supplemented with the cytokines interleukin-2 (IL-2) and interleukin-15 (IL-15). Together, this cocktail of ligands leads to the prolonged expansion of NK cells with high purity.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. NK cell expansion from liver tissues (Day 0), as shown in Figure 1.

NOTE: Initial cell number and viability are strongly correlated with the time since organ removal and the initial tissue sample amount. However, if tissues are placed in 30 mL of Hank's Balanced Salt Solution (HBSS) and kept on ice or in the fridge at 4 °C overnight, NK cells can still be expanded at high purity and viability up to 24 h later.

  1. Identify viable tissue areas to obtain lymphocytes from tissues and sections using sterile surgical equipment.
  2. Place tissues in 30 mL of HBSS (w/o calcium or magnesium) and keep on ice until ready to prepare for isolation.
  3. Mince the tissue into <0.5 cm cubes using sterile razor blades or scissors and forceps inside a biosafety cabinet.
  4. Prepare a 1x collagenase IV solution (1 mg/mL) by diluting a 10x stock in HBSS (10x Collagenase IV: 10 mg/mL or ~200 U/mL).
  5. Place the minced tissue pieces in the tissue dissociator tubes. Fill the tubes with no more than 4 g of tissue and immerse the tissue pieces in ~10 mL of 1x collagenase IV.
    NOTE: Using DNase I is not recommended as it may slightly decrease NK viability and yield. Please refer to the Table of Materials for the specific tissue dissociator tubes used.
  6. Place the tissue dissociator tubes into a tissue dissociator and blend at 37 °C to mince the tissue thoroughly.
    NOTE: For liver tissue, this may take over 30 min. For more friable tissue, around 15 min may be sufficient. Please refer to the Table of Materials for specific tissue dissociator tubes and the tissue dissociator used.
  7. Remove the tissue dissociator tubes and triturate through a 40 µm nylon cell strainer using the backend of a 5 mL syringe. Collect the eluent and discard the large undigested fragments.
  8. Spin down the eluent at 400 x g for 5 min at room temperature. Aspirate the supernatant.
  9. Resuspend the cell pellets in 30% polyvinylpyrrolidone (PVP)-coated silica to remove fat cells that will otherwise contaminate the final lymphocyte fraction.
    1. To prepare 1x PVP-coated silica, use 9:1 dilution of PVP-coated silica in 10x PBS.
      NOTE: Refer to the Table of Materials for the specific PVP-coated silica used.
    2. To prepare 30% PVP-coated silica, dilute 1x PVP-coated silica with PBS/HBSS.
  10. Spin down the cell pellet at 400 x g for 5 min at room temperature. Aspirate the supernatant.
  11. Resuspend the cell pellet in 9 mL of R-10 media.
    NOTE: Refer to the Table of Materials for the composition of R-10 media
  12. Carefully layer the cell suspension over 4 mL of Ficoll or lymphocyte separation media to separate lymphocytes from red blood cells and polymorphonuclear cells.
  13. Separate the layers by centrifuging at 400 x g for 23 min at room temperature with the acceleration and brakes off or at the lowest setting. Carefully decant the upper-medium layer and harvest the interphase containing tissue-infiltrating lymphocytes.
  14. Rinse the cells with 10 mL of media and proceed to cell counting, flow cytometry, aliquoting, and freezing of cells, or primary NK expansion protocol.

2. Primary NK cell expansion from PBMCs (or CB or organ tissues) (Day 0), as shown in Figure 2.

  1. Thaw the frozen PBMC and the frozen, irradiated feeder cells in a 37 °C water bath.
  2. Wash the PBMC and 100 Gamma-irradiated (Gy-irradiated) 221-mIL-21 cells by centrifugation at 400 x g for 5 min with 10 mL of R-10 media separately.
  3. Save 1 x 106 cells of PBMC for flow cytometry.
    NOTE: The initial NK cell purity is an important factor in calculating the NK cell expansion rate.
  4. Resuspend the cells in 1 mL of R-10 media. Count the cells using Trypan Blue.
  5. Mix 5 x 106 cells of PBMC with 10 x 106 cells of 100 Gy-irradiated 221-mIL-21 cells in a special 6-well plate (see Table of Materials).
  6. Add 30 mL of R-10 media supplemented with human IL-2 (200 U/mL) and human IL-15 (5 ng/mL) (see Table of Materials).
  7. Incubate the special 6-well plate at 37 °C with 5% CO2.
  8. Replace the media with R-10 media supplemented with human IL-2 (200 U/mL) and human IL-15 (5 ng/mL) to maintain NK cells every 3-4 days.
    NOTE: Maintain less than 20 x 106 cells per well for further expansion at each media change. For the best viability, ensure that the total cell number in each well does not exceed 100 x 106 cells.
  9. Record the total cell number, viability and perform flow cytometry every 3-4 days to calculate the NK cell expansion rate.

Representative Results

Figure 1
Figure 1: Diagram of NK cell expansion from solid human organ samples. Briefly, the obtained human liver samples are minced into small cubes for mechanical digestion. Dissociated cells are then isolated using PVP-coated silica and Lymphocyte Separation Media. Further, the NK cells are expanded using the expansion protocol described in Figure 2.

Figure 2
Figure 2: Schematic workflow of CAR-NK cell generation from PBMCs. Briefly, 221-mIL21 feeder cells were irradiated at 100 Gy prior to coculturing with PBMCs supplemented with IL-2 and IL-15 on Day 0. In parallel, 293T cells were transfected with the retrovirus packaging system to produce CAR retrovirus that was then transduced into the expanded PBNK cells in the presence of IL-2 and IL-15. Primary CAR-NK cells were harvested on Day 7 and continued expansion for 21 days. This figure has been modified from Yang et al.

Açıklamalar

The authors have nothing to disclose.

Materials

6-well G-REX plate Wilson Wolf 80240M For NK cell expansion
AF647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) Jackson ImmunoResearch 109-606-088 For flow cytometry
Collagenase IV Sigma C4-22-1G For TILs isolation
Cryopreserve media Corning 35-010-CV 90%
Ingredient: Fetal Bovine Serum (FBS)
Cryopreserve media Sigma D2050 10%
Ingredient: Dimethyl sulfoxide (DMSO)
gentleMACS C-tubes Miltenyi Biotec 130-093-237 For TILs isolation
gentleMACS Octo Miltenyi Biotec Quote For TILs isolation
Hank's Balanced Salt Solution (HBSS – w/o calcium or magnesium) ThermoFisher 14170120 For TILs isolation
Human IL-15 Peprotech 200-15 For NK cell expansion
Human IL-2 Peprotech 200-02 For NK cell expansion
Irradiated 221-mIL21 feeder cells Generated in Dr. Dongfang Liu's lab Frozen in cryopreserve media
Lymphocyte Separation Media Corning 25-072-CV For TILs isolation
PE anti-human CD3 clone HIT3a Biolegend 300308 For flow cytometry
PE/Cy7 anti-human CD56 (NCAM) clone 5.1H11 BioLegend 362509 For flow cytometry
Percoll GE Healthcare 17089101 For TILs isolation
Peripheral blood mononuclear cells (PBMCs) New York Blood Center Isolated from plasma of healthy donors, frozen in cryopreserve media
R-10 media VWR 45000-404
Ingredients: RPMI 1640
R-10 media VWR 45000-304 1%
Ingredient: L-glutamine
R-10 media VWR 45000-652 1%
Ingredient: Penicillin Streptomycin
R-10 media Corning 35-010-CV 10%
Ingredient: Fetal Bovine Serum (FBS)
Trypan Blue Corning 25-900-Cl For cell counting

Etiketler

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Bu Makaleden Alıntı Yapın
Ex Vivo Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells. J. Vis. Exp. (Pending Publication), e21557, doi: (2023).

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