Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion

Abstract

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 (O ) set for the incubator reflects the pericellular O to which cells are being exposed. Studies have shown that low O tension 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 O and 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% O in static and dynamic nutrient flow mode are described. Finally, the impact of O tension on HSPC expansion in vitro by flow cytometry and colony forming assays and in vivo through engraftment using a murine model is assessed. © 2018 by John Wiley & Sons, Inc.

Effect of expansion of human umbilical cord blood CD34 + cells on neurotrophic and angiogenic factor expression and function

“…There has been considerable effort placed in identifying factors for use in the ex vivo expansion of CD34 + cells to promote proliferation and self-renewal. This includes the use of growth factors, inhibitors and small molecules (as reviewed by (Flores-Guzmán et al 2013;Xie and Zhang 2015;Zhang and Gao 2016)); the addition of cytokines including, interleukin 6 (IL-6), stem cell factor (SCF), thrombopoietin (TPO), interleukin 3 (IL-3) and FMS-like tyrosine kinase 3 (FLT-3) and to the culture media (Petzer et al 1996); and modulating oxygen levels under culture conditions (Tiwari et al 2018). More recently, attention has turned to the use of small molecules to modulate stem cell fate, with StemRegenin-1 (SR-1) inclusion to TPO, IL-6, FLT-3 and SCF-containing media shown to induce a 50-fold increase in the number of HSCs obtained (Boitano et al 2010).…”