Peripheral blood cultured mast cells: Phenotypic and functional outcomes of different culture protocols.

Abstract Background Mast cells (MCs) play a pivotal role in innate and adaptive immune responses. However, MCs are also involved in different pathologic conditions. Studies on the mechanisms that govern human MC functions are impeded by their limited and difficult recovery. Therefore, several research groups have developed protocols to culture human MCs from progenitor cells. These protocols vary with respect to culture duration and used maturation cytokines. How MCs obtained by different protocols differ in phenotype and functionality is currently unknown. Objective To compare different protocols for the generation of human MCs from peripheral blood progenitors. Methods Thirteen paired human MC cultures were investigated. MCs were cultured form CD34+ progenitors cells for 4 or 8 weeks and with or without the addition of IL-6. Phenotyping comprised staining for CD117, CD203c, FceRI, MRGPRX2, CD300a and CD32. Functional studies included measurements of the up-regulation of CD63 and CD203c after allergen-specific cross-linking of sIgE/FceRI complexes or ligation of MRGPRX2 with substance P and different drugs. Results Cell cultures for 4 weeks in the presence of IL-6 consistently yielded the highest numbers of MCs. MCs cultured for 8 weeks with IL-6 showed more autofluorescence significantly impeding correct analyses of FceRI and CD32. The density of FceRI and CD32 was comparable between the different culture conditions. MRGPRX2 expression was significantly higher in the 8 week cultures. The density of CD300a was increased in the cultures with IL-6. Cells cultured for 8 weeks were more responsive to MRGPRX2 activation. In contrast, the 4-weeks cultures with IL-6 showed significantly higher allergen-specific activation. Conclusion Four weeks of culture with IL-6 are sufficient to generate sizeable numbers of human mast cells from blood progenitors, thereby enabling simultaneous exploration of allergen-specific sIgE/FceRI cross-linking and non-specific activation via MRGPRX2.