title: Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats.
abstract: 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.
  
  
  
  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.
  
  
  
  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 MCTCtype or connective tissue-type HMC.
  
  
  
  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.
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
authors.forename: Issan Yee San
authors.initials: IYS
authors.lastname: Tam
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
authors.forename: Chun Wai
authors.initials: CW
authors.lastname: Ng
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. stam@stanford.edu.
authors.forename: See-Ying
authors.initials: SY
authors.lastname: Tam
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. hyalau@cuhk.edu.hk.
authors.forename: Hang Yung Alaster
authors.initials: HYA
authors.lastname: Lau
mesh_terms: D000328:Adult; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D018952:Antigens, CD34; D058621:Blood Buffy Coat / Q000166:cytology*; D018929:Cell Culture Techniques; D002465:Cell Movement; D002478:Cells, Cultured; D053818:Chymases / Q000378:metabolism; D003238:Connective Tissue; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D016209:Interleukin-8 / Q000378:metabolism; D015289:Leukotrienes / Q000378:metabolism; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010100:Oxygen; D015230:Prostaglandin D2 / Q000378:metabolism; D053802:Tryptases / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D000906:Antibodies; D018952:Antigens, CD34; D016209:Interleukin-8; D015289:Leukotrienes; D014409:Tumor Necrosis Factor-alpha; D007073:Immunoglobulin E; D053818:Chymases; D053802:Tryptases; D015230:Prostaglandin D2; D010100:Oxygen
keywords: Allergy; Buffy coat; Culture protocol; Human mast cell; Inflammation