Abstract:

Erythroblastic island （EBI） is the distinct microenvironment for mammalian erythroid terminal differentiation. Investigating EBI is essential for gaining a deep understanding of the cellular composition and phenotypic changes of hematopoietic microenvironment under both physiologic and pathologic conditions. The distribution of EBI in bone marrow is relatively scattered. Nowadays， existing enrichment methods exhibit unsatisfactory efficiency. Our research aimed to establish a novel and more efficient EBI enrichment method. Drawing on previous research， we introduced innovations and optimizations， developing a novel method centered on dual density gradient sedimentation combined with washing steps to remove non-EBI cell clusters. We compared the efficiency of our method with other existing methods using observing cell cluster structure and Wright-Giemsa staining and imaging flow cytometry （IFC）. Results showed that few single cells were found in the enriched product of Method 4 through direct microscopy and Wright-Giemsa staining， most of the products were typical EBI structures including a central macrophage and surrounding red blood cells. Using IFC technology to analysis F4/80， CD71 and CD11b in enriching products， about 63.7％ of the cell clusters enriched by our method were EBIs. This percentage was higher than the highest proportion obtained in previously reported Method 3， about 38.4％. As previously reported， EBI macrophages were CD11b^-. In some of the EBIs， there were CD11b⁺ cells other than erythrocytes and macrophages. These results showed that a significantly enhanced efficiency in our method， particularly in the removal of removing single cells and non-EBI clusters without causing significant destruction of EBI structures. Our modified method is suitable for research with high EBI purity requirements. Our research provided a more efficient method for researching hematopoietic microenvironment study.

Key words: erythroblastic island, red blood cell, enrichment method, imaging flow cytometry

摘要：

红系造血岛(erythroblastic island, EBI)是哺乳动物红细胞终末分化的独特微环境， EBI研究对于深入理解生理和病理条件下造血微环境的组成和变化至关重要。骨髓中EBI分布较为分散，现有富集方法的富集效果不尽如人意。研究旨在建立一种新的更高效的小鼠骨髓EBI富集方法。在前人研究的基础上进行了创新和优化，建立了以二次密度梯度沉降结合洗涤去除非EBI细胞团为核心的富集方法。通过细胞团形态观察、瑞氏吉姆萨染色、成像流式技术（imaging flow cytometry, IFC）等手段比较了该方法与现有富集方法的富集效果，在直接镜检和瑞氏吉姆萨染色中发现,方法4富集体系中少见单细胞，主要为以巨噬细胞为核心周围围绕红细胞的典型EBI结构。使用IFC技术对于F4/80、CD71以及CD11b进行分析，富集产物中约63.7％的细胞团为EBI，高出之前方法的最高标准（方法3），约38.4％。富集的EBI中发现巨噬细胞为CD11b^-,且除红细胞外，部分EBI中存在CD11b⁺细胞，与之前的研究相符。结果显示，该方法在没有造成EBI结构明显破坏的情况下，富集效果在去除单细胞以及非EBI细胞团等方面显著优于现有其他方法，适合对EBI纯度要求较高的研究，为造血微环境研究提供了有效的方法和手段。

关键词: 红系造血岛, 红细胞, 富集方法, 成像流式