Freie Universität Berlin, Institute of Veterinary Biochemistry, Berlin, Germany.
Theriogenology. 2011 Sep 15;76(5):900-10. doi: 10.1016/j.theriogenology.2011.04.021. Epub 2011 Jun 30.
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.
为了探索导致成功繁殖的过程，需要建立能够长期分化的哺乳动物输卵管体外模型。因此，本研究旨在优化原代猪输卵管上皮细胞的培养条件，使其具有形态分化能力，并能延长培养时间。为了评估原代细胞的不同生长培养基，我们使用了形态学标准和实时阻抗测量。在初始培养基测试后，将细胞种植在悬膜上，并通过组织学和电子显微镜评估培养条件（常规培养、膜上血清浓度梯度和气液界面）。我们证明了输卵管特异性标记物（输卵管糖蛋白 1）的长期表达，并通过定量逆转录 PCR 显示了培养系统的激素反应性。分化的上皮细胞可在气液界面长期培养至 6 周。培养 3 周后，细胞明显极化，并出现纤毛。该模型保持了生理特性，如形态特征（纤毛细胞和分泌细胞的混合细胞群，柱状上皮细胞的典型顶端细胞-细胞接触）和对激素反应的输卵管特异性标记物。我们建立了一种长期体外极化猪输卵管上皮细胞系统，保留了猪输卵管的详细特征。因此，我们提供了一种有用的工具，可以阐明生殖生理学方面尚未解决的科学问题。
