Bovine Trophoblast Cells: Establishment of Cell Culture System and Comparison to In Vivo Qualities

Abstract

Cell culture offers a specifically good understanding of single factors during complex signal transduction pathways, and cell–cell interactions can be examined in small scale. In the bovine synepitheliochorial placenta the peculiarity of ‘restricted’ trophoblast invasion is found as non‐polarized trophoblast giant cells migrating into the uterine epithelium. Specific expression patterns of integrin receptors in trophoblast cells have already been shown in relation to the differentiation of the haemochorial placenta of humans and rodents. For evaluation of integrin function during the precisely controlled trophoblast invasion in cow, it is important to develop a model system, where primary cell cultures from cotyledons are compared to in vivo trophoblast cells. Uteri of pregnant cows were gathered during routine slaughtering. In parallel, placentomes were snap‐frozen for the production of cryosections, and other placentomes were manually separated into fetal cotyledon and maternal caruncle. Dissociation of the cotyledonary cells was accomplished with collagenase I, and cells were cultivated in Quantum 286 (PAA Laboratories GmbH, Linz), a special medium for epithelial cells, containing 10% fetal calf serum. A population of mainly epitheloid cells was achieved by controlled trypsin/EDTA treatment, because of different adhesive forces of fibroblasts and epithelial cells to the substrate. Both, confluent growing cells and tissue sections, were investigated by indirect immunofluorescence with antibodies against cytokeratin, integrin subunits α6, β1, α and β, and the extracellular matrix (ECM) components laminin and fibronectin. The epithelial character of the propagated cells was proven by their distinct immunostaining for cytokeratin. In vivo expressed integrin subunits α6 and β1 were found colocalized along cell‐cell borders and towards the flask ground. The corresponding ECM ligand laminin occurred in both, cells and tissue. In tissue sections fibronectin and subunits α and β3 were not detected in trophoblast cells, however, in vitro these cells displayed a weak immunoreactivity. Summarizing, a method for the cultivation of bovine trophoblast cells was successfully established. The comparison of in vivo and in vitro expressed integrins and ECM showed that the cells in part maintained their in vivo attributes, and therefore are suitable for the study of cell–cell interactions.Funded by German Research Foundation.