Establishing a three-dimensional in vitro model of the mouse endometrium

One of the many scientific fields relying heavily on animal research is the investigation of the implantation process leading to pregnancy. During implantation, the blastocyst attaches to the endometrium by invading through the epithelial cells into the stromal tissue. This triggers the decidualization process in stromal cells, a crucial part of successful implantation. To replace the animals used in the research of these procedures, a murine three-dimensional in vitro implantation model is being developed. An important part of that project is the endometrium model, which consists of isolated primary cells from murine endometria. Before their implementation into the implantation model, a characterization concerning their general behaviour in cell culture and their ability to decidualize is crucial. During this master thesis, the isolated primary endometrial stromal cells were investigated with different fetal calf serums as media components through a proliferation assay, immunofluorescence microscopy and gene expression. Furthermore, formation of the isolated cells into three-dimensional organoids was attempted using agarose forms and an orbital shaker. The isolated cell material was shown to express marker proteins specific for stromal and epithelial cells. The primary stromal cells were able to propagate under cell culture conditions in medium containing non-stripped fetal calf serum and medium with charcoal-stripped fetal calf serum, but the proliferation with charcoal-stripped serum was more consistent. Through morphological assessments and gene expression analysis, the decidual ability of the stromal cells could be shown, although the results varied depending on the analysed gene. However, replication of these results in immunocytochemical analysis was not possible. The threedimensional organoid formation was successful, displaying both a stromal compartment and an epithelial monolayer as confirmed by confocal laser scanning microscopy. These characterization steps are a fundamental part in the successful establishment of the in vitro murine implantation model, which could be used in studying human pathologies like implantation failure and preeclampsia. Furthermore, such a model could be used to replace uterotrophic assays using mice for toxicological studies, or to examine chemical products for adverse consequences on implantation before releasing them to the market.