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For exploring the processes leading to successful reproduction, differentiated long-term in vitro systems modelling the mammalian oviduct are needed. Therefore, in the present study culture conditions for primary porcine oviductal epithelial cells were optimized with regard to morphological differentiation and usability for extended cultivation periods. To evaluate different growth media for the primary cells, we used morphological criteria as well as real-time impedance measurement. After an initial media testing, the cells were grown on hanging membranes and the culture settings (conventionally cultured, serum gradient over the membrane and air-liquid interface) were assessed by histology and electron microscopy. We proved long-term expression of an oviduct specific marker (oviductal glycoprotein 1) and showed a hormone responsiveness of the culture system by means of quantitative reverse transcription-PCR. Differentiated epithelial cells could reproducibly be cultured up to 6 weeks in an air-liquid interface. After 3 weeks of culturing, the cells were clearly polarized and exhibited cilia. The model maintains physiological properties such as morphological features (mixed cell population of ciliated and secretory cells, apical cell-cell contacts typical for columnar epithelial cells) and oviduct-specific markers showing hormone responsiveness. We established a polarized long-term in vitro-system of the porcine oviductal epithelium preserving detailed features of the porcine oviduct. Therefore, we provide a useful tool to elucidate unsolved scientific questions concerning reproductive physiology.