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There are many important parameters when setting up and evaluating hematopoietic cultures. First is the cell source: hematopoietic cells from bone marrow, cord blood and mobilized peripheral blood may behave differently and result in different cell yields in culture. Most likely you would want to use CD34+ cells or even subsets of this population that are more highly enriched for stem cells and primitive progenitors (e.g., CD34+CD38- cells), but there may be applications in which non-purified cells or mononuclear fractions are sufficient. The cell concentration at the start of the culture is also important. At low cell concentrations (e.g. 10ˆ4 CD34+ cells per mL of culture medium) overall cell expansion (i.e., the number of cells generated per input CD34+ cell) is generally better than at high cell concentrations and there is less chance that the medium will be depleted due to overgrowth of cells in culture. However, this is very dependent on the quality and type of cells used, and on the culture conditions, specifically the properties of the medium and the types and concentrations of cytokines used. You also need to establish clear and measurable goals about the numbers and types of cells you want to generate in your cultures and what methods are needed to characterize these cells. I would monitor the cultures closely and do cell counts (of total and viable cells) regularly. You would want to use a medium that maintains high viability (> 90%) of the cells throughout the culture. StemSpan™ SFEM II is optimized for the culture of hematopoietic cells isolated from human bone marrow, cord blood and mobilized peripheral blood, and does not contain serum or cytokines, allowing you complete flexibility in defining your culture conditions. If your goal is to expand CD34+ cells you should monitor the cultures closely for changes in the % of CD34+ cells (or CD34 subsets) by flow cytometry. If it is important that the cultured hematopoietic cells retain their progenitor or even stem abilities they will need to be tested in appropriate functional assays, such as the colony-forming unit (CFU) assay, long-term culture-initiating cell (LTC-IC) assay or transplantation assays in immunodeficient mice. For more information on these assays, please read our mini-review on Hematopoietic Stem and Progenitor Cells.