177 ISOLATION AND CULTURE OF MOUSE ENDOMETRIAL CELLS IN A THREE-DIMENSIONAL CULTURE SYSTEM

Abstract

Implantation of mammalian embryos occurs only during a restricted narrow window. The endometrium becomes highly receptive for the embryos during this period of time. The objective of this study was to establish an in vitro culture system for pre- and post-implantation mouse embryos. In Experiment 1, mouse uterine horns were excised at Day 3.5 post-coitus. After being washed with Dulbecco's phosphate-buffered solution (DPBS), the uterine horns were cut open and incubated with 0.05% trypsin in DPBS at 4°C for 2 h. After trypsinization, the tissues were incubated at 37°C for an additional 30 min. The solution containing epithelial cell suspension was recovered after 30 s of vortexing. The trypsinized uterine horns were then cut into 1-mm³ pieces and digested with collagenase (type I, 1 mg mL^-1 in DPBS) at 37°C for 3 h with vigorous shaking. At the end of digestion, the solution was filtered through 40-µm nylon mesh and the flowthrough containing stromal cells was collected. The isolated epithelial and stromal cells were characterized by their morphology and immunocytochemistry. Both types of cells showed positive immunocytochemical reaction with desmin antibody. The cultured epithelial cells formed polyhedral shapes, and more than 95% expressed epithelium-specific protein, cytokeratin-18. On the other hand, most of the spindle-like stromal cells had no signal for cytokeratin-18 expression, although a few scattered cells were positively labeled. In Experiment 2, for construction of the 3-dimensional culture system, epithelial cells obtained by the method described in Experiment 1 were seeded on an artificial basal membrane (ECMatrix^TM; Millipore/Upstate/Chemicon, Temecula, CA, USA) with underlying stromal cells embedded in the type I collagen matrix. The whole system was settled in a Millicell^® (Millipore) hanging in a 24-well culture plate, and immersed in DMEM medium supplemented with 10% fetal bovine serum, 20 ng mL^-1 epidermal growth factor, 63.5 nmol progesterone, and 7.14 nmol 17β-estradiol. The morphology of epithelial cells on the matrix became cuboidal after 48 h of culture. Additionally, the columnar cells with a basal nucleus were observed on the paraffin wax sections. In Experiment 3, mouse E3.5 embryos were recovered and cultured in this established culture system. Normal hatching and/or attachment of the blastocysts were observed after 2 days of culture. In conclusion, our results showed that epithelial cells formed morphologically columnar monolayers and apparently interacted with blastocyst embryos. Successful construction of this model system would facilitate the study of early embryo development through the implantation stage.