Characterisation of dendritic cells generated using stemspan™ serum-free medium

Mature dendritic cells (DC) are often generated in serum-replete medium. One of the disadvantages of this system is the batch-to-batch variation in the cytokine content of serum-replete medium and the consequent variation in the functional properties of the DC generated. A serum-free medium, in which cytokine content is controlled is therefore preferable. Here we describe the use of StemSpan™ Serum-Free Medium for the generation of DC from a variety of sources. Lineage negative bone marrow cells (30-50% CD34+) were enriched using the StemSep™ immuno-magnetic negative selection procedure and cultured in StemSpan™ SFEM supplemented with GM-CSF and FLT3-ligand. After 14 days in culture, the cells lost expression of CD34, developed typical DC morphology, increased expression of costimulatory molecules and gained the ability to stimulate an MLR. In addition, these CD34-derived DC were able to present peptide to CD8+ T cells. Peripheral blood monocytes (CD14+) isolated using StemSep™ were cultured for 7days in StemSpan™ SFEM supplemented with GM-CSF (100ng/ml) and IL4 (20ng/ml). These cells developed typical DC morphology, lost expression of CD14, increased expression of CD86, CD40, CD1a and HLA-DR and increased their ability to stimulate an MLR. Peripheral blood DC (defined as CD4+ HLA− DR+lin−) were enriched using StemSep™ and cultured in StemSpan™ SFEM with GM-CSF (100ng/ml) and TNFa (10ng/ml). After 3 days, these cells developed a typical DC morphology, increased expression of costimulatory molecules and increased ability to stimulate an MLR. In summary, StemSpan™ SFEM, when supplemented with the appropriate cytokines, can be used to generate functionally competent DC from blood and bone marrow precursors.