Improved clinical scale culture conditions for expansion of cord blood

The use of allogeneic cord blood (CB) products, as a source of cellular support for patients undergoing high dose chemotherapy (HDC), had primarily been limited to smaller children due to the low numbers of cells in CB. Ex vivo expansion of CB cells has been proposed as a method to increase the number of cells. We have transplanted adult patients, undergoing HDC with CB cells expanded in static culture for 10 days. In these studies the CD34 selected cells were cultured for 10 days in 800 ml of defined media containing 100 ng/ml each of rhSCF, rhG-SCF and rhMGDF in 1L teflon bags. All patients achieved neutrophil engraftment in a median of 26 (range 15 to 45) days. In an attempt to hasten the time to neutrophil engraftment we have developed a 2-step culture system that results in increased expansion of total nucleated cells (TNC) and further maturation of neutrophil precursors. CD34+ cells selected from CB products were cultured for 7 days in 100 ml teflon culture bags containing 50 ml of DM plus SCF, G-CSF and MGDF (100 ng/ml of each). The cells were harvested from these bags and transferred to 1 liter telflon bags containing 1 liter of DM plus SCF, G-CSF and MGDF. After a second culture period of 7 days the cells were harvested washed and assayed for mature (GM-CFC) and primitive progenitor cells (HPP-CFC). The 2-step cultures resulted in a median expansion of TNC of 438 fold (range 286 to 952, N = 9) while the original 1-step cultures resulted in a median of 98 fold (range 59 to 350, N = 5). Equivalent expansion of committed progenitor cells (GM-CFC) and primitive progenitor cells (HPP-CFC) was obtained. The 2-step culture expanded cells contained more mature neutrophils similar to ex vivo expanded peripheral blood progenitor cells (PBPC). We propose that the availability of increased numbers of mature expanded cells may result in more rapid engraftment. These culture conditions are currently being incorporated into our clinical studies.