Wakabayashi Yoshiyuki, Chua Jennifer, Larkin Janet M, Lippincott-Schwartz Jennifer, Arias Irwin M
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Histochem Cell Biol. 2007 May;127(5):463-72. doi: 10.1007/s00418-007-0274-x. Epub 2007 Feb 17.
Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.
了解上皮细胞如何产生并维持极性和功能需要进行活细胞成像。为了使细胞完全极化，有必要将它们培养在可渗透的膜滤器上；然而，半透明的滤器会阻碍定量活细胞成像所需的显微镜光路。或者，可以切除膜滤器，但这会消除对顶面和底面的选择性接触。相反，直接在玻璃上培养的上皮细胞表现出与在滤器上生长的细胞不同的表型和功能。在这里，我们描述了一种在Transwell滤器插入物上培养极化上皮细胞的新方法，该方法能够实现具有时空图像分辨率的卓越活细胞成像，这是使用传统方法无法实现的。细胞在滤器支架的下侧培养。以这种倒置配置生长的上皮细胞呈现出完全极化的结构，包括存在功能性紧密连接。这种新的培养系统允许对内涵体和高尔基体动力学进行四维（随时间的三个空间维度）成像，并允许对顶面和底面进行选择性操作。这项新技术在极化上皮细胞的可视化和操作方面具有广泛的适用性。
