Seven week culture of functional human mast cells from buffy coat preparations.

Functional, mature human mast cells have been generated by in vitro differentiation of CD133(+)/CD34(+) progenitor cells isolated from e.g. cord blood, peripheral blood, bone marrow or fetal liver. However, the protocols published so far require long term cultivation, i.e. up to 15 weeks for mast cell differentiation, which makes such approaches not only laborious but also costly. Here, we have developed a protocol for generating functional human mast cells from peripheral blood already within 7 weeks. Human CD133(+) progenitors were isolated from buffy coat preparations of peripheral blood and cultured in the presence of stem cell factor (SCF) and IL-6 for 7 weeks. IL-3 was added to the culture medium during the first 3 weeks, and fetal calf serum (FCS) added during the last week. In vitro differentiated CD133(+) cells exhibited multiple characteristics of mature mast cells. Thus, cells contained tryptase and expressed functional levels of FcepsilonRI. Anti-IgE stimulation induced significant release of histamine and PGD(2) and also of chemokines including MCP-1, IL-8, MIP-1alpha, and MIP-1beta. The fact that our in vitro differentiated mast cells are derived from a generally available source of progenitor cells makes this novel protocol widely applicable to any patient group, irrespective of age. Moreover, this progenitor source is more readily available than e.g. bone marrow or cord blood-derived progenitors. Consequently, our protocol has great potential in studies on mast cell biology and mast cell pathology, and e.g. on evaluation of drug effects.

Comparison of short term in vitro cultured human mast cells from different progenitors - Peripheral blood-derived progenitors generate highly mature and functional mast cells.

During the last two decades different scientific groups have investigated the phenotype and function of in vitro generated human mast cells (MC). The cells have been shown to display variable surface markers and functional characteristics. The phenotypic differences may reflect different culture conditions, protocols or the use of different progenitors. To investigate the significance of different progenitors, we have compared MC generated from CD133(+) progenitor cells from cord blood (CBMC) or peripheral blood (PBMC). The progenitors were cultured for 7 weeks in the presence of IL-6 and SCF, with addition of IL-3 the first 3 weeks, and FCS during week 7. The phenotype of the established MC was characterized by surface marker expression levels, metachromasia, histamine and tryptase contents and their function was evaluated by receptor-mediated release of histamine and PGD(2). The generated metachromatic (<99%) MC were 75% tryptase(+), regardless of the source of progenitor cell. Expression of c-kit/CD117, CD203c, and FcepsilonRI was comparable. The density of c-kit/CD117 receptors on CBMC was higher that of PBMC (p<0.001). The density of CD203c and FcepsilonRI was higher on PBMC (p<0.001). PBMC contained more histamine (p<0.001), expressed more FcepsilonRI (p<0.001) and released more histamine (p<0.001) and PGD(2) (p<0.001) upon ligation of FcepsilonRI, than CBMC. Culture with IL-4 increased expression of tryptase, FcepsilonRI, CD117 and CD203c, secretion of histamine and PGD(2) of PBMC, and histamine secretion of CBMC. Cord and peripheral blood may give rise to different types of MC. The question addressed should determine the progenitor cell and protocol to be used.