Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells

We describe a novel culture system for generating large numbers of murine skin-associated mast cells and distinguish their characteristics from bone marrow-derived cultured mast cells. Culture of day 16 fetal skin single cell suspensions in the presence of interleukin-3 and stem cell factor allowed expansion and maturation of mast cells in the presence of stromal cells. The average yield of mast cells after 2 wk was 7.3 million cells per fetus at a purity of 96%. These fetal skin-derived cultured mast cells increased their histamine content in a time-dependent manner to 3.6 pg per cell after 2 wk and 6.7 pg per cell after 4 wk. Phenotypic analyses revealed much greater expression of CD49b and CD81 and lesser expression of CD77 and CD102 on fetal skin-derived cultured mast cells as compared with bone marrow-derived cultured mast cells. These findings suggest a close similarity between fetal skin-derived cultured mast cells and freshly isolated cutaneous mast cells. Connective tissue mast cell characteristics of fetal skin-derived cultured mast cells were evidenced by: (1) their greater histamine content than bone marrow-derived cultured mast cells; (2) the presence of heparin; and (3) their degranulation in response to compound 48/80 and substance P. Importantly, fetal skin-derived cultured mast cells secreted greater amounts of interleukin-13 but much less MIP-1beta and interleukin-6 than bone marrow-derived cultured mast cells in response to ionomycin. Thus fetal skin-derived cultured mast cells have many characteristics distinct from bone marrow-derived cultured mast cells and can be used as a model of cutaneous mast cells to discern their functions.