Morpho-functional characterization of equine endometrial epithelial cells in vitro – preliminary results

Abstract

Influences of different culture media and growth surfaces on morpho-functional characteristics of equine endometrial epithelial cells (EEC) in vitro were investigated in order to optimize culture conditions. Therefore, the results were compared to the endometrium in situ. Equine EEC were cultured using different media with varying serum components and additives: foetal bovine serum plus additives (MFBS+A), foetal bovine serum (MFBS), horse serum 1 (MHS1), horse serum 2 (MHS2) and horse serum 3 (MHS3). Uncovered and Matrigel™-covered cell culture inserts, as well as cell culture flasks were used. Cultured cells were examined cytomorphologically (haemalaun-eosin staining, giemsa staining), cytochemically (alcian blue staining) and immunocytochemically (oestrogen receptor (ER) α, progesterone receptor, proliferating cell nuclear antigen, inhibin-α, cytokeratin 8, 18, 19, vimentin, α-actin, desmin, transforming growth factor (TGF)-α,-β1,-β2,-β3). Further, tissue samples of the uteri from which the cells were isolated were examined histopathologically (haemalaun-eosin staining), histochemically (alcian blue staining) and immunohistochemically, using the above-mentioned antibodies. Regardless of the used media and the state of the endometrial cycle at point of isolation, two morphologically different cell types and three growth patterns were found. None of these facts led to apparent differences concerning immunolabelling. Whereas growth surfaces, duration of culture and state of endometrial cycle at point of isolation did not cause any obvious influences on the immunolabelling of EEC in vitro concerning the utilized antibodies, the culture media did. Especially cells grown in MFBS+A exhibited characteristics comparable to uterine glandular epithelia in situ concerning cytokeratin 8, 18, 19, TGF-β1,-β3 and ERα.

Establishment and characterization of a coculture system of equine endometrial epithelial and stromal cells

“…In accordance with findings of Böttcher et al. (), cytokeratin 19 was expressed in ECs and absent in SCs.…”

“…While cells that appeared morphologically as epithelial could only be observed in a minor fraction of cocultured ECs, the immunocytochemical detection of specific antigens (cytokeratin 19, UG, CAL and scattered expression of ERα and PR) in this cell population supports the conservation of an epithelial phenotype during coculture, in principle. Similar findings have been described in monoculture studies (Böttcher et al., ). However, the preservation of functional characteristics was only partially given under the applied culture conditions.…”

“…These circumstances may be explained by the restrictions imposed by the coculture system. A shortage in the supply of the cocultured cells by the use of a culture medium optimized for monoculture studies (Böttcher et al., ; Theuß et al., ) may have led to an increase in cellular dedifferentiation. It is known that environmental conditions directly influence cellular differentiation (Freshney, ).…”

“…For cell culture purposes, biopsy material as well as tissue samples collected from freshly deceased mares are most frequently used (Böttcher et al., ; Theuß, Böttcher, Kappe, Schoon, & Ellenberger, ). Monocultures of equine endometrial epithelial and stromal cells derived from these sources have already been established and characterized (Böttcher et al., ; Szóstek et al., ; Theuß et al., ). Because a monoculture approach can only rudimentarily reproduce the in situ situation, several studies in human and veterinary medicine focus on the establishment of coculture systems.…”

Establishment of a new method for isolation and culture of equine endometrial epithelial and stromal cells

“…However, for the first three passages, epithelial cells had to be seeded at higher density to achieve satisfactory results. As aforementioned, non-cultured cells were embedded in paraplast for immunocytochemistry (Böttcher et al 2010), as well.…”