Natural Killer Cell Isolation from Liver Tissue for Selective Expansion
Transcrição
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.
