University of Antwerp, Faculty of Medicine and Health Sciences, Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Antwerp (Belgium) and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, Belgium.
University of Antwerp, Faculty of Medicine and Health Sciences, Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Antwerp (Belgium) and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, Belgium; Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent, Belgium.
J Immunol Methods. 2021 Aug;495:113061. doi: 10.1016/j.jim.2021.113061. Epub 2021 Apr 30.
Studies on the mechanisms that govern mast cell (MC) functions are hindered by the difficulties in isolating sufficient numbers of these tissue-resident cells. Therefore, many research groups use cultured human MCs obtained out of progenitor cells. However, these culture methods significantly differ regarding primary source material, culture durations and conditions. Consequently, the finally obtained cells are likely to exhibit morphological, phenotypical and/or functional heterogeneity.
To compare the phenotype and functionality of cells cultured from peripheral blood and bone marrow progenitor cells from patients with suspected clonal MC disease. These cells are designated as PBCMCs and BMCMCs, respectively.
Twenty paired PBCMCs and BMCMCs cultures starting from CD34 progenitor cells were compared. Cells were cultured for 4 weeks. Phenotyping included Giemsa and CD117 staining and flow cytometric staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a, CD32, CD63 and CD25. Functional assessment included measurement of the up-regulation of CD63 after cross-linking of the high affinity receptor for IgE (FcεRI) with anti-FcεRI and ligation of MRGPRX2 with substance P.
PBCMCs and BMCMCs are phenotypically comparable. Functionally, after activation with anti-FcεRI and substance P, PBCMCs and BMCMCs show similar up-regulation of the lysosomal degranulation marker CD63. However, the yield of PBCMCs is higher than BMCMs and peripheral blood cultures are purer than bone marrow cultures.
PBCMCs are an attractive alternative to the more difficult to obtain BMCMCs for the exploration of the complex mechanisms that govern IgE- and MRGPRX2-dependent MC activation and degranulation. Unlike BMCMCs, PBCMCs are easily accessible and enable repetitive analyses.
研究调控肥大细胞（MC）功能的机制受到难以分离足够数量的这些组织驻留细胞的阻碍。因此，许多研究小组使用从祖细胞中获得的培养的人 MC。然而，这些培养方法在原始材料、培养持续时间和条件方面有很大的不同。因此，最终获得的细胞可能表现出形态、表型和/或功能异质性。
比较从疑似克隆性 MC 疾病患者的外周血和骨髓祖细胞培养的细胞的表型和功能。这些细胞分别命名为 PBCMC 和 BMCM。
比较了 20 对源自 CD34 祖细胞的 PBCMC 和 BMCM 培养物。细胞培养 4 周。表型分析包括吉姆萨和 CD117 染色以及流式细胞术 CD117、CD203c、FcεRI、MRGPRX2、CD300a、CD32、CD63 和 CD25 的染色。功能评估包括通过交联高亲和力 IgE 受体（FcεRI）与抗 FcεRI 和与 P 物质结合的 MRGPRX2 来测量 CD63 上调。
PBCMC 和 BMCM 在表型上是可比的。功能上，用抗 FcεRI 和 P 物质激活后，PBCMC 和 BMCM 均显示出溶酶体脱颗粒标志物 CD63 的相似上调。然而，PBCMC 的产量高于 BMCM，外周血培养比骨髓培养更纯。
与更难获得的 BMCM 相比，PBCMC 是探索调控 IgE 和 MRGPRX2 依赖性 MC 激活和脱颗粒的复杂机制的有吸引力的替代方法。与 BMCM 不同，PBCMC 易于获得且可进行重复分析。
