A three-dimensional cell culture model for bovine endometrium: regeneration of a multicellular spheroid using ascorbate.
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 24h 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 layer. 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(2alpha) 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.
Yamauchi
N
N
Laboratory of Reproductive Biology and Technology, Department of Developmental Biology, National Institute of Agrobiological Sciences, Ikenodai 2, Kukizaki, Inashiki-gun, Ibaraki 305-8602, Japan.
Yamada
O
O
Takahashi
T
T
Imai
K
K
Sato
T
T
Ito
A
A
Hashizume
K
K
D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002417:Cattle; D002449:Cell Aggregation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D004717:Endometrium / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008954:Models, Biological; D009924:Organ Culture Techniques / Q000379:methods; D010121:Oxytocin / Q000494:pharmacology; D011453:Prostaglandins / Q000378:metabolism; D012038:Regeneration; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D023822:Tissue Engineering / Q000379:methods
D011453:Prostaglandins; C011669:ascorbate-2-phosphate; D010121:Oxytocin; D020782:Matrix Metalloproteinases; D013755:Tetradecanoylphorbol Acetate; D001205:Ascorbic Acid