Increased expansion and differentiation of cord blood products using a two-step expansion culture

Objective. The use of allogeneic cord blood (CB) products as a source of ce llular support for patients receiving high-dose chemotherapy has been limit ed primarily to smaller children due to the low numbers of cells in a CB un it. Ex vi cro expansion of CB cells has been proposed as a method to increa se the number of cells available for transplantation. Following high-dose c hemotherapy administration, we transplanted adult patients with CB expanded in static culture for 10 days, in DM containing stem cell factor (SCF), gr anulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and d evelopment factor (MGDF). Patients achieved neutrophil engraftment in a med ian of 26 days (range 15 to 45). In an attempt to hasten the time to neutro phil engraftment, we developed a two-step culture system that results in in creased expansion of total nucleated cells and further maturation of neutro phil precursors. Materials and Methods. CD34(+) cells isolated from CB products were culture d for 7 days at 37 degrees C in 100-mL Teflon culture bags containing 50 mt of DR I containing SCF, G-CSF, and MGDF (100 ng/mL). The cells were harves ted from these bags after 7 days of incubation at 37 degrees C and transfer red to l-L Teflon bags containing 1-L of DM plus SCF, G-CSF, and MGDF. Afte r a second culture period of 7 days, the cells were harvested, washed, and assayed for mature (granulocyte-macrophage colony-forming cells [GM-CFC]) a nd primitive progenitor cells thigh proliferative potential colony-forming cells [HPP-CFC]). Results. The two-step cultures resulted in a median total nucleated cell ex pansion of 438-fold (range 286 to 952, N = II); the original one-step cultu res resulted in a median expansion of 98-fold (range 59 to 350, N = 5). Equ ivalent expansion of committed progenitor cells (GM-CFC) and primitive prog enitor cells (HPP-CFC) was obtained. CD34(+) cells were expanded a median o f 29-fold in the two-step cultures (N = 11). The two-step culture contained more mature neutrophil cells, by morphologic examination, than the one-ste p cultures, similar to ex vivo expanded peripheral blood progenitor cells ( PBPC). Conclusion. The two-step ex vivo expansion conditions described for CB resu lted in increased numbers of total nucleated cells, GM-CFC, HPP-CFC, and CD 34(+) cells and morphologically resembled ex vivo expanded PBPC, which have been shown to provide more rapid neutrophil engraftment than unexpanded PB PC. We propose that the availability of increased numbers of expanded CB ce lls may result in more rapid engraftment of neutrophils following infusion to transplant recipients. (C) 2000 International Society for Experimental H ematology. Published by Elsevier Science Inc.