This article details a protocol for isolating natural killer (NK) cells from human liver tissue. The process involves enzymatic and mechanical dissociation, followed by density gradient centrifugation to separate various cell types.
To isolate natural killer cells, or NK cells — specialized white blood cells involved in innate immunity, begin with human liver tissue containing diverse cell populations, including fat-containing cells, erythrocytes, granulocytes, lymphocytes, and other associated cells entrapped within a collagen-rich extracellular matrix or ECM.
Mince the tissue into small pieces and transfer them to a tube containing collagenase — a proteolytic enzyme. Collagenase cleaves the collagen, disrupts the matrix, and loosens the ECM-bound cells.
Insert the tube into a tissue dissociator. This mechanical dissociation further disintegrates ECM to release loosely attached cells into the solution. Filter the contents to remove undigested tissue and ECM fragments and collect the filtrate containing various cells.
Resuspend the cells into a buffer containing polyvinylpyrrolidone or PVP-coated silica beads. The PVP beads selectively bind with fat-containing cells, effectively separating them from the rest of the cells.
Centrifuge at low speed to pellet the cell population; discard the supernatant containing debris and bead-bound fat cells. Resuspend the pellet and overlay the cell suspension onto a tube containing a density gradient medium to separate the cells based on their densities.
Centrifuge at low speed. The higher-density erythrocytes and granulocytes settle at the bottom, while lymphocytes appear at the interphase between two liquids.
Collect the cells from interphase and culture them in a selective medium for NK cell expansion.
Begin by identifying and sectioning the viable tissue areas using sterile surgical equipment to obtain lymphocytes. Then, place the sectioned tissues in 30 milliliters of HBSS without calcium or magnesium, and keep the tissue on ice until ready for isolation.
Working inside a biosafety cabinet, mince the tissue into less than 0.5-centimeter cubes with sterile razor blades and forceps. Place the minced tissue pieces — not more than 4 grams — in the tissue dissociator tubes, and add 10 milliliters of collagenase IV to the tissue pieces.
To mince the tissue thoroughly, treat the tissue dissociator tubes into a tissue dissociator at 37 degrees Celsius. After removing the tubes from the tissue dissociator, triturate the minced tissue through a 40-micron nylon cell strainer using the back end of a 5-milliliter syringe. Discard the large, undigested fragments.
Spin down the collected eluent at 400 g for 5 minutes at room temperature, and aspirate the supernatant before resuspending the cell pellets in 30% polyvinylpyrrolidone-coated silica to remove the fat cells. Spin down the cells as described, before resuspending the cell pellet in 9 milliliters of R-10 media.
To separate lymphocytes from red blood cells and polymorphonuclear cells, carefully layer the cell suspension over 4 milliliters of Ficoll or lymphocyte separation media. Separate the layers by centrifuging at 400 g for 23 minutes at room temperature with the acceleration and brakes off. Then, carefully decant the upper medium layer and harvest the interphase containing tissue-infiltrating lymphocytes.
Rinse the cells with 10 milliliters of media and proceed for analysis of primary Natural Killer, or NK, cells expansion protocol.
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