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Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O2) set for the incubator reflects the pericellular O2to which cells are being exposed. Studies have shown that low O2tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system. A combination of low ambient O2and dynamic culture conditions has been developed to increase the reconstituting capacity of human HSPCs. In this unit, the protocols for serum‐free expansion of HSPCs at 5% and 20% O2in static and dynamic nutrient flow mode are described. Finally, the impact of O2tension on HSPC expansion in vitroby flow cytometry and colony forming assays and in vivothrough engraftment using a murine model is assessed. © 2018 by John Wiley & Sons, Inc.

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell‐free matrices from bone marrow cells that mimic ‘human‐body‐niche‐like’ conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum‐free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering–based biomimetic scaffolds for HSPC expansion for clinical applications. © 2016 by John Wiley & Sons, Inc.