Epithelial monolayer culture system for real-time single-cell analyses

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.

In Pursuit of Authenticity: Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for Clinical Applications

“…These iPSC‐RPE had not been identified previously as outliers by structural analysis or gene expression suggesting that evaluation of cellular function is a potential tool for discerning the quality of iPSC‐RPE. The intactness of the epithelial sheet and the electrical activity of the RPE monolayer are essential for its function; thus, iPSC‐RPE are more fully characterized by assessing the responses of confluent monolayers rather than individual cells [49]. Therefore, we expanded our functional analysis of the intact monolayer to several components of the ATP‐mediated purinergic signaling pathway, critical elements in controlling the chemical composition and volume of the SRS [19, 22, 23, 50, 51].…”

Airway Surface Liquid pH Regulation in Airway Epithelium Current Understandings and Gaps in Knowledge

“…The main relevant studies are summarized in Table 1 , Table 2 , Table 3 and Table 4 . In vitro studies have shown that ASL pH is decreased in the context of CFTR mutations, as compared to epithelia carrying WT CFTR [ 8 , 22 , 42 , 43 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 ]. The majority of studies focused on the most frequent CFTR mutation, p. Phe508delCFTR (F508del).…”