Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats

• Published in: Inflammation research Vol. 66; no. 1; pp. 25 - 37
• Main Authors: Tam, Issan Yee San, Ng, Chun Wai, Tam, See-Ying, Lau, Hang Yung Alaster
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 2017; Springer Nature B.V
• Subjects: Adult; Allergology; Anti-Inflammatory Agents - pharmacology; Antibodies - pharmacology; Antigens, CD34; Biomedical and Life Sciences; Biomedicine; Blood Buffy Coat - cytology; Cell Culture Techniques; Cell Movement; Cells, Cultured; Chymases - metabolism; Connective Tissue; Dermatology; Hematopoietic Stem Cells - cytology; Histamine Release; Humans; Immunoglobulin E - immunology; Immunology; Interleukin-8 - metabolism; Leukotrienes - metabolism; Mast Cells - drug effects; Mast Cells - metabolism; Mast Cells - physiology; Neurology; Original Research Paper; Oxygen; Pharmacology/Toxicology; Prostaglandin D2 - metabolism; Rheumatology; Tryptases - metabolism; Tumor Necrosis Factor-alpha - metabolism; Allergy; Inflammation; Culture protocol; Buffy coat; Human mast cell
• ISSN: 1023-3830; 1420-908X
• DOI: 10.1007/s00011-016-0989-z

Objective The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34 + hematopoietic stem cells which require less culturing time than previously reported methods. Methods CD34 + cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. Results We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MC TC type or connective tissue-type HMC. Discussion Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.