An improved method for the isolation of endometrial epithelial and stromal cells from the total hysterectomy specimen

We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from hysterectomy specimens. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease-treatment and cell suspension conditions to dissociate single cells from the tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 x 10^6 of EMECs and 2.8 x 10^6 EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the epigenetic regulation underlying the dynamic changes of the endometrium during the menstrual cycle. Although protocols for the isolation of endometrial stromal and epithelial cells (EMSCs and EMECs) have been well established, the number of EMECs obtainable by the current protocols is relatively small. To improve the efficiency of isolating EMECs as well as EMSCs from endometrial tissues, we established protease-treatment and cell suspension conditions to efficiently dissociate single cells from endometrial tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. By conducting a microarray-based transcriptome analysis, we confirmed that the cells isolated by the modified protocol developed in this study maintain the transcriptomic properties of endometrial epithelial and stromal cells.

本研究旨在提升从子宫切除标本中分离子宫内膜上皮细胞（endometrial epithelial cells, EMECs）与子宫内膜基质细胞（endometrial stromal cells, EMSCs）的效率。通过免疫组织化学染色，我们发现胶原酶处理后残留的大块组织碎片（在常规实验流程中通常会在首次过滤后被丢弃）主要由腺上皮细胞与基质细胞构成。因此，我们建立了蛋白酶处理与细胞悬浮体系，以从该组织碎片中解离出单个细胞，并通过荧光激活细胞分选术分离得到上皮细胞（EPCAM阳性）与基质细胞（CD13阳性）。四项独立实验结果显示，每份子宫切除标本平均可分离得到1.2×10^6个EMECs与2.8×10^6个EMSCs。我们证实，本方法分离得到的细胞与常规流程获取的上皮细胞、基质细胞具有高度相似的转录组学特征。本优化后的实验流程可为后续研究月经周期中子宫内膜动态变化背后的表观遗传调控机制提供助力。
尽管目前已有成熟的子宫内膜基质细胞与上皮细胞分离流程，但现有方法可获取的EMECs数量相对较少。为提升从子宫内膜组织中分离EMECs与EMSCs的效率，我们建立了蛋白酶处理与细胞悬浮体系，以高效从子宫内膜组织碎片中解离单个细胞，并通过荧光激活细胞分选术分离得到上皮细胞（EPCAM阳性）与基质细胞（CD13阳性）。通过基于微阵列的转录组分析，我们证实本研究开发的优化流程所分离得到的细胞，仍保留子宫内膜上皮细胞与基质细胞的转录组学特性。