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Ex vivo expansion is an effective strategy to acquire cytokine-induced killer (CIK) cells needed for clinical trials. In this work, the effects of dynamic suspension culture, which was carried out by shake flasks on a shaker, on CIK cells were investigated by the analysis of expansion characteristics and physiological functions, with the objective to optimize the culture conditions for ex vivo expansion of CIK cells. The results showed that the expansion folds of total cells in dynamic cultures reached 69.36 ± 30.36 folds on day 14, which were significantly higher than those in static cultures (9.24 ± 1.12 folds, P0.05), however, the proportions of CD3

Stem cell factor (SCF) plays important roles in ex vivo expansion of hematopoietic stem cells (HSCs). In this study, the effects of dose and feeding time of SCF on ex vivo expansion of CD34 + cells were investigated in serum-free medium supplemented with a cytokine cocktail composed of SCF, thrombopoietin (TPO) and flt3-ligand (FL). Among the four tested doses (0, 5, 50 and 500 ng/mL), a SCF dose of 50 ng/mL was demonstrated to be most favorable for ex vivo expansion of CD34 + cells, which resulted in 34.22 ± 10.80 and 8.89 ± 1.25 folds of expansion regarding total cells and CD34 + cells, respectively. Meanwhile, the specific growth rate of cells, the consumption rate of SCF and the percentage of CD34 + c-kit + cells during the 21-day culture process were analyzed. The results indicated that initial 4-day period was a critical stage for SCF functioning on CD34 + cells during ex vivo expansion. Based on this, a modified SCF feeding regimen was proposed, in which SCF (50 ng/mL) was only supplemented on day 0 in the cytokine cocktail and cells were then fed with TPO and FL till the end of culture. It was found that this SCF feeding regimen could expand CD34 + cells efficiently, thus providing a cost–effect expansion protocol for HSCs.

Hypoxia favored the preservation of progenitor characteristics of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. This work aimed at studying the role of reactive oxygen species (ROS)-generating NADPH oxidase system regulated by hypoxia in ex vivo cultures of cord blood CD34+ cells. The results showed that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. Meanwhile the ROS generation was found to be inhibited by diphenyleneiodonium (the NADPH oxidase inhibitor), or N-acetylcysteine (the ROS scavenger). Accordingly NADPH oxidase mRNA and p67 protein levels decreased in hypoxia. The analysis of progenitor characteristics, including the proportion of cultured cells expressing the HSPCs marker CD34+CD38-, colony production ability of the colony-forming cells (CFCs), and the re-expansion capability of the cultured CD34+ cells, showed that either 5% pO(2) or reduced ROS favored preserving the characteristics of CD34+ progenitors, and promoted the expansion of CD34+CD38- cells as well. The above results demonstrated that hypoxia effectively maintained biological characteristics of CD34+ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase.

Oxygen tension regulates the biological characteristics of hematopoietic stem and progenitor cells (HSPCs) by modulating intracellular reactive oxygen species (ROS). To better understand oxygen tension mechanism on HSPCs culture, gene expression analysis of human CD34 + CD38 − HSPCs was performed using microarrays. The CD34 + CD38 − HSPCs cultured under normoxia, hypoxia, or with N-acetyl cysteine (NAC, an ROS scavenger) were isolated for transcriptional profilings. Compared to normoxia group, 1 gene was up-regulated and 22 genes were down-regulated in hypoxia group, while 1 gene was up-regulated and 29 genes were down-regulated in NAC group. These differently expressed genes were involved in cell surface markers, blood activation and differentiation. The common down-regulated genes related to dendritic cells (DCs) maturation (CD80, CD86, and JAG1) were confirmed by real-time RT-PCR. Furthermore, the analysis of the phenotypes of DCs, including the DC-characteristic surface molecule CD1a, the costimulatory molecules CD80 and CD86, and HLA-DR, associated with the capacity of DCs to stimulate allogeneic T cells, showed that hypoxia-mediating ROS inhibited the potential of CD34 + CD38 − HSPCs differentiating to mature DCs. All these results demonstrated that hypoxia-reducing ROS down-regulated the genes driving CD34 + CD38 − HSPCs differentiation, which provides an interesting molecular hint to direct their development to DCs during cultures.