394 DEVELOPMENT OF A RAT ENDOMETRIAL SPHEROID AS A MODEL FOR THE ANALYSIS OF ENDOMETRIAL FUNCTIONS

Abstract

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 investigation of endometrial functions. The spheroid has been utilized in cell biology research because it appears to mimic the 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 the endometrium. The purpose of the present study was to develop a spheroid composed of rat endometrial stromal (RES) cells as an in vitro model for analysis of 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 DMEM/F12 containing 10% fetal 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 516.7 ± 28.9 ¼m at Day 5 after detachment from SAC gels. TUNEL examination of cell viability in the spheroid suggested that no cell was apoptotic until 15 days after cell sheet detachment; TUNEL-positive cells appeared at 20 days. Additionally, no positive staining of proliferating cell nuclear antigen (PCNA) was observed in the spheroids in contrast to strong staining in proliferating monolayer cultured cells. The results of gelatin zymography showed that both matrix metalloproteinases (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 was induced by arachidonic acid (AA) treatment. RT-PCR analysis showed that typical marker genes for the decidualization, desmin and decidual/trophoblast prolactin-related protein (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-mimicking feature in both 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.

This research was supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Kiban-kenkyu C 18580282).