EX-VIVO GENERATION OF DENDRITIC CELLS FROM CD34-PERMEABLE CONTAINERS UNDER SERUM-FREE CONDITIONS( CELLS IN GAS)

Dendritic cells (DC) are efficient and potent APCs that can be generat ed ex vivo. For them to be used clinically, however, a closed culture system using serum-free medium should be used. Our goal was to differe ntiate DC from human blood CD34+ cells in serum-free media in a new ga s-permeable culture container, PL2417, Apheresis products were collect ed from healthy G-CSF-mobilized donors, and CD34+ cells were selected using the Isolex(R) immunomagnetic cell selection system. Cells were c ultured in the presence of GM-CSF and tumor necrosis factor-alpha (TNF -alpha) in various serum-free media and compared with serum-containing medium in 4-well plates. One of the serum-free media was then selecte d and used in PL2417 containers and compared with serum-containing med ium in standard flasks. The cells were evaluated at days 0, 7, and 14 for the presence of DC, which were identified morphologically after Wr ight-Giemsa staining by cytoplasmic processes extending from the surfa ce of the cell. The cultures were evaluated phenotypically by flow cyt ometry and immunohistochemistry. The stimulatory capacity was examined in MLR, Overall, results from serum-free media and PL2417 containers were comparable results obtained under the other conditions. These dat a indicate that culture-deriving DC from CD34+ cells in PL2417 closed system containers using serum-free media is as effective as using stan dard flasks and serum-supplemented media.