The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, a serum-free, stroma-free and cytokine-containing culture system for CD34(+) and colony-forming cell (CFC) expansion was systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results show that the optimal compositions of the serum substitutes and the cytokine cocktail were BIT2 (1.5 g/l BSA, 4.39 mug/ml insulin, 60 mug/ml transferrin, and 25.94 muM 2-ME), and CC-S6 (8.46 ng/ml TPO, 4.09 ng/ml IL-3, 15 ng/ml SCF, 6.73 ng/ml FL, 0.78 ng/ml IL-6, 3.17 ng/ml G-CSF, and 1.30 ng/ml GM-CSF) in the Iscove's modified Dulbecco's medium, respectively. After one-week culture, the increases in the total number of white blood cells (WBC), CD34(+) cells and CFC were 64-, 27- and 22-fold, respectively. Its expansion ability of CD34(+) cells and CFC was comparable to that of X-vivo 20(TM), Stemline(TM), and Stemspan(TM) commercial media. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells. (C) 2003 Elsevier Inc. All rights reserved.