Generation of Functionally Competent Human Mast Cells from Limited Blood Volumes

Mast cells (MCs) are innate immune cells that are derived from CD34⁺ hematopoietic stem/progenitor cells (HSPCs) and mature in peripheral tissues such as skin and mucosa. Mature human MCs can be generated from peripheral blood, but this process requires substantial blood volumes as HSPC frequencies are typically very low. The aim of this study was to validate a new in-house-developed protocol for the generation of MCs from less than 20 mL of peripheral blood. To this end, we used a magnetic bead-based procedure to isolate 'untouched' HSPCs from 14 to 16 mL peripheral blood (PB). In total, 12 cultures were set up with blood from seven healthy donors, wherein HSPCs were first expanded for 4 weeks, followed by another 8 weeks of culture in MC maturation-inducing medium. Flowcytometric analysis, histochemical staining, and degranulation assays were used to assess their phenotypic and functional features. Our data show comparable expression of cytoplasmic granules and cell-surface expression of MRGPRX2, FcεR1α, and CD117 in 8/12 blood-derived MCs (PB-MCs) and buffy coat-derived HSPCs (BC-MCs). PB-MCs responded to classic stimulating agents like IgE/anti-IgE and C48/80. Hence, our novel MC generation protocol yields functionally competent MCs with no compromise in their maturation or activation potential despite 12 weeks of in vitro culture.