A protocol for generating high numbers of mature and functional human mast cells from peripheral blood
A protocol for generating high numbers of mature and functional human mast cells from peripheral blood
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DOI：
10.1111/j.1365-2222.2007.02778.x
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发表时间：
2007-09-01
影响因子：
6.1
通讯作者：
Kovanen, P. T.
中科院分区：
文献类型：
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作者：
Lappalainen, J.;Lindstedt, K. A.;Kovanen, P. T.
背景肥大细胞(Mast Cells，MC)是一种多功能效应细胞，在天然免疫和宿主防御中发挥重要作用，并在过敏等多种病理条件下发挥重要作用。本研究的目的是确定从人外周血来源的CD34(+)祖细胞在无血清条件下体外培养9周，以期确定从其祖细胞高效分化为成熟且具有功能的人巨噬细胞的培养条件。通过选择细胞因子以及低氧和常氧条件的组合，优化了细胞的生长和分化为MC。结果培养9周后，0.5×10(6)个祖细胞平均生成20×10(6)个成熟MC，细胞数量至少增加40倍。成熟的MC呈椭圆形非小叶状核，分泌颗粒中含有组胺、肝素、类胰蛋白酶、糜酶和组织蛋白酶G，表面表达c-kit(CD117)和Fc epsilon受体I。Ig E-抗Ig E交联剂、化合物48/80、P物质和过敏性毒素C3a可引起细胞释放组胺。结论本研究提供了一种从人外周血中分离出功能表型为结缔组织型的成熟MC的有效方法。使用这些培养的人类MC将增加我们对人类MC发育和生物学在人类疾病中的认识和理解。
Background 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.Methods 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.Results 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.Conclusion 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.