Abstract
Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single‐cell and subcellular levels, and can be extended to other cell types with minor modifications.
Highlights
Tissues such as dissociated frog skin were used to investigate epithelial physiology (Ussing and Zerahn 1951)
Epithelial cultures grown on porous membranes develop a basolateral and apical polarization corresponding to the interstitium and lumen, respectively, of ducts of many organs
We performed several experiments to test the functionality of our disk membrane culture system
Summary
Tissues such as dissociated frog skin were used to investigate epithelial physiology (Ussing and Zerahn 1951). Preparation of epithelial tissue is technically challenging and requires sacrificing multiple animals. Sample quality is paramount for data quality. These factors often result in inefficient collection and variability of data. Dissected monolayers are not optimal for microscopic analysis, since structural stability is compromised in the dissection process. To circumvent these issues, investigators have developed cell lines from animal and human tissues (Simmons 1982; Widdicombe 1990; Bens and Vandewalle 2008). Epithelial cultures grown on porous membranes develop a basolateral and apical polarization corresponding to the interstitium and lumen, respectively, of ducts of many organs. A common approach for these studies involves the use of compartmentalized culture systems which enable separate control of the apical (luminal) and basolateral (serosal) media
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