Natural Killer Cell Isolation from Liver Tissue for Selective Expansion

Overview

This video demonstrates the isolation of lymphocytes, including natural killer cells or NK cells, from human liver tissue for selective expansion. Enzymatic and mechanical digestion, followed by density gradient separation, promotes lymphocyte isolation, including NK cells. The collected lymphocytes can be further used for the selective expansion of NK cells.

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.

Representative Results

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. 

Materials

Name	Company	Catalog Number	Comments
Collagenase IV	Sigma	C4-22-1G	For TILs isolation
Hank's Balanced Salt Solution (HBSS - w/o calcium or magnesium)	ThermoFisher	14170120	For TILs isolation
Lymphocyte Separation Media	Corning	25-072-CV	For TILs isolation
Phosphate-Buffered Saline (PBS)	Corning	21-040-CV	For transduction
R-10 media Ingredients: RPMI 1640	VWR	45000-404
R-10 media Ingredient: L-glutamine	VWR	45000-304	1%
R-10 media Ingredient: Penicillin Streptomycin	VWR	45000-652	1%
R-10 media Ingredient: Fetal Bovine Serum (FBS)	Corning	35-010-CV	10%