AbstractConventional high-dose chemotherapy frequently leads to severe neutropenia, during which patients experience a high risk of infection. Although support care with donor’s neutrophils is possible this choice is largely hampered by the limited availability of matched donors. To overcome this problem, we explored a large-scale ex vivo production of neutrophils from hematopoietic stem cells (HSCs) using a four-stage culture approach in a roller-bottle production platform. We expanded CD34+ HSCs isolated from umbilical cord blood (UCB) using our in-house special medium supplemented with cytokine cocktails and achieved about 49000-fold expansion of cells, among which about 61% were differentiated mature neutrophils. Ex vivo differentiated neutrophils exhibited a chemotactic activity similar to those from healthy donors and were capable of killing E. coli in vitro. The expansion yield as reported herein was at least 5 times higher than any other methods reported in the literature. Moreover, the cost of our modified medium was only a small fraction (<1/60) of the StemSpan™ SFEM. Therefore, our ex vivo expansion platform, coupled with a low cost of stem cell culture due to the use of a modified medium, makes large-scale manufacturing neutrophils possible, which should be able to greatly ameliorate neutrophil shortage for transfusion in the clinic.