Development of in vitro model for implantation using the endometrial spheroid

子宫内膜是最复杂的组织之一，它会随着着床和妊娠过程的变化而发生动态变化。体外模型可能为阐明复杂的植入过程提供一种工具。然而，目前还没有合适的体外模型来研究子宫内膜功能。球体已被用于细胞生物学研究，因为它似乎模拟了活组织和器官中细胞的形态和生理，这与传统的单层培养不同。由正常成人细胞组成的多细胞球体可能为子宫内膜的研究提供一个更有用的模型。本研究的目的是建立由大鼠子宫内膜基质细胞(Res)组成的球体，作为分析子宫内膜功能的体外模型。用鲑鱼四元胶原(SAC)生成球体。将细胞接种在12孔SAC凝胶上(井本水三公司)。并在…中培养较多的培养液(DMEM/F12)中含有10%胎牛血清。当细胞在培养第7天达到融合时，胶原酶消化SAC凝胶以促进RES细胞膜的脱离。然后将漂浮的细胞片转移到琼脂糖包被的平板上，培养成球形。几天后，细胞片缩小，形成聚集的细胞团，最后形成圆形球体。细胞膜脱离后第5天，球体直径约为500μm，细胞活力的TUNEL检测显示，直到细胞片脱离后15天才出现细胞凋亡，20天出现TUNEL阳性细胞。此外，在增殖期的单层培养细胞中，未见增殖细胞核抗原阳性染色。明胶酶谱检测结果表明，在单层培养条件下均可产生基质金属蛋白酶-2和-9。然而，在细胞膜脱离后，两种MMPs的产生立即减少，直到15d才能检测到。体外，花生四烯酸(AA)诱导球体蜕膜化。RT-PCR分析显示，在AA处理2天后，蜕膜化的典型标志基因结蛋白和d/tPRP在球体中有表达，而在未处理的对照组中则没有表达。重要的是，现在的球体在结构和功能上都表现出类似子宫内膜的特征。该方法简便易行，为子宫内膜功能和着床的研究提供了新的视角。较少

The endometrium is one of the most complex tissues; it undergoes dynamic changes in response to implantation and pregnancy processes. An in vitro model may provide a tool for clarifying the complex implantation process. However, there is no suitable in vitro model for the investigation of endomotrial functions. The spheroid has been utilized in cell biology research because it appears to mimic morphology and physiology of cells in living tissues and organs, which is unlike conventional monolayer culture. Multicellular spheroids composed of normal adult cells may provide a more useful model for the study of endometrium. The purpose of the present study is to develop the spheroid composed of rat endometrial stromal cells(RES) as an in vitro model for analysis of the endometrial functions.The RES were prepared from rat endometrium at day 5 of pregnancy. Spheroids were generated using salmon aterocollagen(SAC). The cells were plated on 12 well SAC gels(Imoto Suisan Co.) and cultured in the … More culture medium(DMEM/F12) containing 10% foetal bovine serum. After the cells reached confluence at day 7 in culture, SAC gels were digested by collagenase to promote the detachment of RES cell sheets. Then the floating cell sheets were transferred to agarose-coated plates and cultured to form spheroids. The cell-sheet shrank and became an aggregated cell mass in a few days; it finally formed a round-shaped spheroid. Diameters of the spheroids were about 500μm at day 5 after detachment from SAC gels.TUNEL examination of cell viability in the spheroid suggested no cell was apoptotic until 15 days after cell sheet detachment, TUNEL-positive cells appeared at 20 days. Additionally, no positive staining of PCNA was observed in the spheroids in contrast to the strong staining in the proliferating monolayer cultured cells. The results of gelatin zymography showed that both MMP-2 and -9 were produced in monolayer culture. However, after the detachment of the cell sheet, the production of both MMPs decreased immediately and could not be detected until 15 days after detachment.In vitro decidualization of the spheroids were induced by arachidonic acid(AA) treatment. RT-PCR analysis showed that typical marker genes for the decidualization, desmin and d/tPRP, were expressed in the spheroids after 2 days of AA treatment, but not in the control groups without treatment.The results of the present study indicate that rat endometrial stromal cells are capable of being regenerated as a spheroid using SAC gels in vitro. Importantly, the present spheroid displays an endometrium-mimic feature in both the structural and functional similarities. The present method is simple and convenient, and therefore provides a new insight into the study of endometrial functions and implantation. Less