Ex vivo expansion of hematopoietic stem cells in a proliferation chamber with external stirred conditioning tank: Sequential optimization of growth factors

The insufficient number of hematopoietic stem cells (HSCs) extracted from various cell sources creates the need for the use of growth factors in the culture systems. Numerous types and concentrations of growth factors have been reported in the literature. In this study, we investigated the effect of three important hematopoietic growth factors thrombopoietin (TPO), stem cell factor (SCF), and Fms-like tyrosine kinase-3 ligand (Flt-3) as well as cell-seeding density on ex vivo expansion of human HSCs using a factorial design. Sequential optimization was then followed by methods of steepest ascent and central composite design. The optimum concentrations of growth factors were 50, 90, and 34 ng/mL for SCF, TPO, and Flt-3, respectively, at an initial cell density of 2.5x10(5) cells/mL. Effective expansion factor value (EEF) of HSCs increased considerably and revealed almost similar results when the cells were cultured in a 24-well plate (EEF = 4.54 +/- 0.43) and a proliferation chamber with an external stirred conditioning tank (PC-ESCT; EEF = 5.1 +/- 0.35) at seeding density of 2.5x10(5) cells/mL after 7 days. The cells did not show considerable changes in proliferation when they were cultured in medium containing serum or in a commercial serum-free medium at the optimum concentrations of the growth factors. The present study demonstrated the optimum condition of hematopoietic growth factors as well as the potential of PC-ESCT for ex vivo expansion of HSCs.