Development of the endometrial spheroid using tissue-engineering technique

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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.