Simplified long term large scale production of highly active human LAK cells for therapy

Cells obtained by leukapheresis and separated without or with a Ficoll-Isopaque (FIP) gradient were cultured for 4 or 20 days to determine whether a more effective LAK cell population could be obtained compared to LAK cells generated by the commonly used method (FIP separated cells cultured for 4 days). After leukapheresis the cells were separated on a FIP gradient (FIP cells) or only washed to minimize platelet contamination (non-FIP cells). In some experiments, the cells were also pretreated with phenylalanine-methylester (PheOMe) to reduce monocytes. After culturing for 20 days in the presence of IL-2 (100 BRMP U ml-1) a significantly higher total number of CD3+ and CD56+ cells was noted in the non-FIP fraction compared to the FIP fraction. The total lytic activity of non-FIP cells (LU/tot. no. of cells) against Daudi targets after 4 days of culture was 11,820 +/- 624 versus 4770 +/- 550 of FIP cells (P less than 0.05). After 20 days of culture the non-FIP fraction exhibited a higher cytotoxic activity than the FIP cell fraction, 76,291 +/- 20,053 compared to 7169 +/- 1148 LU (P less than 0.05). Pretreatment of the cells with PheOMe induced a significantly more effective cytotoxic population when cultured for 4 days. However, at 20 days PheOMe did not enhance the LAK cell activity. Large scale production of LAK cells can be accomplished without FIP separation with an increased cell yield and cytotoxicity compared to FIP separated cells both in short and long time cultures.