A three-dimensional cell culture model for bovine endometrium: Regeneration of a multicellular spheroid using ascorbate

N. Yamauchi, O. Yamada, T. Takahashi, K. Imai, T. Sato, T. Ito, K. Hashizume

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46 Citations (Scopus)


The development of a multicellular spheroid comprising bovine endometrial epithelial cells (BEE) and bovine endometrial stromal cells (BES) is described in this study. The BES were cultured to confluence in medium with L-ascorbic acid phosphate magnesium salt n-hydrate (AsA-P) which stimulates collagen synthesis in BES. The BEE were co-cultured on a BES cell-sheet for 24 h before detachment of the cell-sheet to generate a hetero-spheroid. After EDTA treatment and agitating with pipette, 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 BES and BEE. Histological examination found that hetero-spheroids were covered with BEE on the outer laver. When cell viability was examined with TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling), no positive signal was detected in the spheroid for up to 10 days. Immunofluorescence observations showed that spheroids contained abundant extracellular matrices, including type-I, -III, -IV collagen, fibronectin, and laminin. PGF produced by hetero-spheroids in response to oxytocin was significantly higher than those produced by monolayer cultured BEE (P<0.05). MMPs were not detected in media from spheroids cultured for 5 days after detachment of the cell sheet. These results indicate that bovine endometrial cells have the capacity to regenerate as a multicellular spheroid after treatment with ascorbate in vitro. The spheroid displays an endometrium-mimic feature. Thus, we conclude that spheroids formed by BES and BEE are a useful in vitro model of bovine endometrium.

Original languageEnglish
Pages (from-to)258-269
Number of pages12
Issue number2-3
Publication statusPublished - 2003
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Reproductive Medicine
  • Obstetrics and Gynaecology
  • Developmental Biology


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