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FORMATION OF MULTICELLULAR SPHEROID AS A MODEL OF BOVINE ENDOMETRIUM.
Yamauchi, Nobuhiko1,2, Yamada, Osamu1,2, Takahashi, Toru1, Takezawa, Toshiaki1, Hashizume, Kazuyoshi1, 1 2
ABSTRACT- The purpose of the present study is to develop the multicellular spheroid composed of bovine endometrial stromal cells (SC) and epithelial cells (EC) as an in vitro model to analyze functions of bovine endometrium. Spheroids were generated using the stimulating effect of ascorbic acid on collagen synthesis of SC. The original SC and EC were separated and purified from bovine endometrium at day 1-4 of estrus cycle. Firstly, SC were plated and cultured on 48 well culture plate up to a confluent state, and then EC were plated onto the SC monolayer. L-Ascorbic acid phosphate magnesium salt n-hydrate (Asc) was added to the culture medium. The multicellular spheroid was prepared by detaching EC-attached SC monolayer (cell sheet) and culturing it on non-adhesive substratum. When cells were cultured in the medium containing Asc more than 10 uM, cell sheets were detached easily from culture plate by agitating with pipette in EDTA/PBS solution and then floated as a wavy sheet. The floating cell sheets shrank and condensed into aggregate in a few days and finally formed a spheroid with oval-shape in 5 days after detachment. Histological observations showed that spheroids were covered with cuboidal cells on outer layer and contained rich extracellular matrices (ECM) inside. In immuno-cytochemical observations, the cells on the outer layer and ECM inside the spheroid showed positive staining with anti cytokeratin and anti type-I collagen antibody, respectively. These results demonstrate that bovine endometrial SC produce a plenty of ECM response to Asc addition, and formed spheroid efficiently. EC in the spheroid were relocated to the outer layer and cover the SC and ECM inside. The present method being simple and convenient to form multicellular spheroid from bovine endometrial SC and EC, it provides a new sight in research of endometrial functions and implantation. This research was supported by grants from BRAIN, Japan.
KEY WORDS: spheroid, bovine, endometrium, in vitro