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

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.