Datasheet1_Functional human skin explants as tools for assessing mast cell activation and inhibition.docx

Mast cells are activated through a variety of different receptors to release preformed granules and mediators synthesized de novo. However, the physiology and function of mast cells are not fully understood. Traditional studies of mast cell activation in humans have utilized cultures of tissue-derived mast cells including CD34+ progenitor cells or well-characterized commercially available cell lines. One limitation of these methods is that mast cells are no longer in a natural state. Therefore, their applicability to human skin disorders may be limited. Human skin explant models have been utilized to investigate the short-term effects of cell mediators, drugs, and irritants on skin while avoiding the ethical concerns surrounding in vivo stimulation studies with non-approved agents. Nonetheless, few studies have utilized intact human tissue to study mast cell degranulation. This “Methods” paper describes the development and application of an intact skin explant model to study human mast cell activation. In this manuscript, we share our protocol for setting up ex vivo human skin explants and describe the results of stimulation experiments and techniques to minimize trauma-induced histamine release. Skin explants were generated using de-identified, full-thickness, non-diseased skin specimens from plastic and reconstructive surgeries. Results were reproducible and demonstrated FcɛRI- and MRGPRX2-induced mediator release which was inhibited with the use of a BTK inhibitor and QWF, respectively. Thus, this explant model provides a quick and accessible method of assessing human skin mast cell activation and inhibition.

肥大细胞（Mast cells）可通过多种不同受体激活，释放预形成的颗粒与从头合成的介质。然而目前学界对于肥大细胞的生理特性与功能尚未完全阐明。传统人类肥大细胞激活研究多采用组织源性肥大细胞培养体系，包括CD34+祖细胞（CD34+ progenitor cells）或经过充分鉴定的商业化细胞系。此类方法的局限性在于肥大细胞脱离了天然生存微环境，因此其在人类皮肤疾病研究中的适用性或受限制。人类皮肤外植体模型可用于探究细胞介质、药物及刺激物对皮肤的短期作用，同时规避了使用未获批试剂开展体内刺激研究的伦理争议。然而，目前鲜有研究利用完整人体组织探究肥大细胞脱颗粒过程。本“方法学”论文详述了用于研究人类肥大细胞激活的完整皮肤外植体模型的构建与应用。本文中，我们分享了离体人类皮肤外植体的构建方案，并详述了刺激实验结果及降低创伤诱导组胺释放的实验技术。本研究使用来源于整形与重建外科手术的去标识化全层正常皮肤标本构建皮肤外植体。实验结果具备可重复性，证实FcɛRI与MRGPRX2介导的介质释放可分别被布鲁顿酪氨酸激酶（BTK, Bruton's tyrosine kinase）抑制剂与QWF所抑制。综上，该外植体模型为评估人类皮肤肥大细胞的激活与抑制过程提供了一种便捷可行的研究方法。