LAK cell generation in normal subjects: Ficoll-Hypaque vs. light spin purification

Research in adoptive immunotherapy such as lymphokine-activated killer cell (LAK) generation requires a large number of mononuclear cells (MNCs). The original procedure for generation of LAK cells used a Ficoll-Hypaque (FH) purification of the MNCs collected by a blood separator to remove granulocytes, platelets, and red blood cells. Since the Fenwal CS 3000 blood separator produces MNCs with reduced granulocyte contamination, it is possible that FH purification could be replaced by a light centrifuge spin (LS) purification. We report on a study comparing the MNC recovery, platelet and red cell removal, purification time, in vitro LAK cytotoxicity, and phenotypic characterization of the lymphocytes, collected by FH purification vs. LS purification by using the CS 3000. Six donors were leukapheresed with the CS 3000 using a shortened LAK Cell procedure. Following an LS (3 minutes at 1,000 rpm), the MNC collection was divided into two equal aliquots, one aliquot purified by FH and the other aliquot purified by LS. Both aliquots were then washed, cultured for 5 days in IL-2, and harvested with the CS 3000. Our findings suggest the LS purification resulted in improved 1) postpurification MNC recovery (89% vs. 60%), 2) purification time (54 minutes vs. 129 minutes), and 3) LAK cell generation by phenotype. LS purification was similar to FH purification in 1) platelet removal and 2) in vitro LAK cytotoxicity. We found the LS superior to FH when considering efficiency, economics, simplicity, and LAK cell generation and comparable in platelet removal and in vitro LAK cytotoxicity.