Development of the endometrial spheroid using tissue-engineering technique

本研究的目的是建立由大鼠子宫内膜间质细胞(Res)组成的球体，作为分析大鼠子宫内膜功能的体外模型。用鲑鱼四元胶原(SAC)生成球体。SAC的变性温度较低，需要进行交联才能作为支架使用。在本研究中，1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐或脱水热处理对SAC进行了交联化。似乎鱼胶原蛋白传播感染性疾病的风险较低。Res是在怀孕第5天从大鼠子宫内膜分离纯化的。Res在SAC上培养至融合状态。然后用胶原酶消化SAC。然后在琼脂糖包被的平板上培养RES细胞片，生成球体。经过胶原酶处理后，漂浮的细胞片在几天内缩小，成为聚集的细胞团，最终形成由RES组成的圆形异球形。用末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记法检测细胞存活率时，15天后球体内未见凋亡细胞。用增殖细胞核抗原(PCNA)抗体进行免疫荧光观察，球体内未见增殖细胞。结果表明，大鼠子宫内膜间质细胞可在体外用SAC再生为多细胞球体。由于目前的球体在结构和功能上表现出与子宫内膜相似的特征，因此它似乎是一种有用的大鼠子宫内膜的体外模型。该方法简便易行，为研究子宫内膜间质功能及植入提供了新的视角。

The purpose of the present study is to develop the spheroid composed of rat endometrial stromal cells (RES) as an in vitro model to analyze functions of rat endometrium. Spheroids were generated using salmon aterocollagen (SAC). SAC has a low denaturation temperature and need to be cross-linked before being used as a scaffold. In the present study, SAC was cross-linded by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride or dehydrothermal treatment. It seems that fish collagen has lower risk for transmission of infectious deseases.The RES were separated and purified from rat endometrium at day 5 of pregnancy. The RES were cultured up to the confluent state on SAC. Then digested SAC used collagenase. Then RES cell sheet cultured in agarose coated plate to generate a spheroid. After collagenase treatment, the floating cell-sheet shrank and became an aggregated cell mass in a few days ; it finally formed a round-shaped hetero-spheroid composed of RES. When cell viability was examined with TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling), apoptotic cells were not found in the spheroid for 15 days. Immunofluorescent observations used the proliferating cell nuclear antigen (PCNA) antibody showed that proliferating cells were not find in the spheroids. The production of proMMP-2 in the spheroid was similar to that produced by endometrial tissue in vivo.These results indicate that rat endometrial stromal cells are capable of being regenerated as a multicellular spheroid using SAC in vitro. As the present spheroid displays an endometrium-mimic feature in structural and functional similarities, it seems to be a useful in vitro model of rat endometrium. The present method being simple and convenient to form multicellular spheroid from RES, it provides a new sight in research of endometrial stromal functions and implantation.