The objective of this investigation was to establish a three-dimensionally cultured human endometrium which could be used as a tissue model for the mechanism study of implantation in vitro. By using human endometrial stromal (ES) and epithelial cells (EE) from hysterectomy specimens, reconstruction of endometrium in culture was established by first layering a collagen gel containing ES cells, then overlaying with the Matrigel containing endometrial epithelial (EE) cells. Ultrastructural examination of the 48 h-endometrial cell culture revealed monolayered columnar EE cells with microvilli on the collagen layer containing ES cells and appearance of the tight junctions and desmosomes between EE cells, a cell layer closely resembling the native endometrium. Immunohistochemical characterization of the reconstructed endometrium showed a strong immunoreactivity for cytokeratin, integrin alpha1, alpha4 and beta3 subunits, cyclooxygenases-1 and -2, matrix metalloproteinases-1, -2, -3 and -9, and tissue inhibitor of metalloproteinases-1 and -2 in the EE cells comparable to the native endometrial epithelium. ES cells also showed stronger immunoreactivity for cyclooxygenases, integrins and MMPs, but less for cytokeratin. Gelatin zymographic analyses of the media obtained from the reconstructed endometrium model showed gelatinase activity bands at 57, 60, 72, 92 and 97 kDa molecular weight, respectively. The present study provides a possibility that our three-dimensionally cultured endometrium model could mimic the morphological and functional characteristics of the native endometrium. The model could be used to clarify the roles of various molecules involved in the human implantation.
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