Fachbereich Veterinärmedizin



    Establishment of in vitro cultures of porcine reproductive epithelia (2014)

    Mießen, Katrin (WE 3)
    Berlin: Mensch und Buch Verlag, 2014 — IV, 73 Seiten
    ISBN: 978-3-86387-509-1
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000097164
    Institut für Veterinär-Biochemie

    Oertzenweg 19 b
    14163 Berlin
    Tel.+49 30 838 62225 Fax.+49 30 838-62584

    Abstract / Zusammenfassung

    The molecular pathways in the morphologically and functionally different tissue of the reproductive tract of mammals are still incompletely elucidated. For exploring the processes leading to successful reproduction, differentiated in vitro systems modelling the mammalian reproductive tract are needed.
    The pig serves as a valuable resource for adequate material for several reasons. Since anatomy of the organs, as well as physiological and pathophysiological responses have been found to be very similar to those in humans, the pig is a more valid model for human research than rodent species and, therefore, an important alternative as a non-rodent animal species. This is especially true for the early processes in reproduction. Furthermore, due to the industrial porc production, porcine material for cell culture is easily available (slaughterhouse). Biological variations between the different individuals concerning age, cycle stage (mostly pre-pubertal animals) and breed (hybrids of German Edelschwein, German Landrace and Pietrain) are reduced. This leads to a low biological variation.
    In the present studies, culture conditions for primary porcine epithelial cells derived from the oviduct and the cervix uteri 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 realtime 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 (for the oviductal cell culture only) and air-liquid interface) were assessed by histology and electron microscopy (only oviductal cells).
    For the oviductal cell culture, we proved long-term expression of an oviduct specific marker (oviductal glycoprotein 1) and showed a speci_c hormone responsiveness of the culture system by means of quantitative reverse transcription PCR (qPCR). Dfferentiated epithelial cells could reproducibly be cultured up to six weeks in an air-liquid interface. After three weeks of culturing, the cells were clearly polarized and exhibited motile 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 could reproducibly culture pure epithelial cells out-growing from fresh tissue explants of the porcine cervix uteri. The optimised growth medium was conditioned Ham's F-12, containing 10 % FCS1, EGF2 and insulin. When growing cells in an air-liquid interface for three weeks, the cells showed a multilayered phenotype. The cells were of epithelial origin and showed beta catenin expression as well as production of mucopolysaccharides.
    Finally, polarized in vitro-systems of the porcine cervical and oviductal epithelium preserving detailed features of the native tissue were successfully established.
    Especially the oviductal cell culture model is very promising and will further promote subsequent research projects.