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      A protocol for generating high numbers of mature and functional human mast cells from peripheral blood.

      Clinical and Experimental Allergy
      Blood Cells, drug effects, Cell Culture Techniques, methods, Cell Hypoxia, Cells, Cultured, Hematopoietic Stem Cells, Humans, Immunohistochemistry, Mast Cells, cytology, physiology, Stem Cell Factor, pharmacology

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          Mast cells (MCs) are multi-functional effector cells with an essential role in innate immunity and host defence, and under several pathological conditions, such as allergy. Here, we aimed at defining the culture conditions that would allow efficient generation of mature and functional human MCs from their progenitor cells. Human peripheral blood-derived CD34(+) progenitor cells were cultured in vitro under serum-free conditions with human stem cell factor for 9 weeks. Growth and differentiation of the cells into MCs were optimized by selected cytokines and a combination of hypoxic and normoxic conditions. MCs were phenotypically characterized by immunocytochemistry, their preformed mediators were quantified, and their functional ability to degranulate and release histamine was tested. On average, 20 x 10(6) mature MCs were generated from 0.5 x 10(6) progenitor cells during 9 weeks of culture, i.e. at least a 40-fold increase in cell number was achieved. The mature MCs had oval-shaped non-lobular nuclei, contained histamine, heparin, tryptase, chymase, and cathepsin G in their secretory granules, and strongly expressed c-kit (CD117) and Fc epsilon receptor I on their surface. Histamine release from the cells could be brought about by IgE-anti-IgE cross-linkage, compound 48/80, substance P, and anaphylatoxin C3a. The MCs remained functional for several weeks after their maturation. This study describes an efficient protocol for generating mature MCs from human peripheral blood with a functional phenotype of connective tissue-type MCs. Use of these cultured human MCs will increase our knowledge and understanding about human MC development and biology in human disease.

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