Cell-based high-throughput odorant screening system through visualization on a microwell array

Abstract

The development of a cell-based high-throughput screening system has attracted much attention from researchers who study drug screening mechanisms and characterization of G-protein coupled receptors (GPCRs). Although olfactory receptors (ORs) constitute the largest group of GPCRs that play a critical role recognizing and discriminating odorants, only a few ORs have been characterized, and most remain orphan. The conventional cell-based assay system for characterizing GPCRs, including ORs, is very laborious, time consuming, and requires an expensive assay system. In this study, we developed a simple, low-cost miniaturized odorant screening method by combining Micro-Electro-Mechanical system (MEMs) technique and visualization technique for detecting an odorant response. We fabricated PEG microwell from a photocrosslinkable polyethylene glycol diacrylate (PEGDA) solution and applied it to cell culture and a reverse transfection platform for cell-based high-throughput screening. For the first time, the olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptor genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a CAMP response element (CRE) reporter assay. We tested this platform using four de-orphaned ORs. This new cell-based screening method not only reduced numerous time-consuming steps but also allowed for simple, efficient, and quantitative screening and patterning of large numbers of GPCRs including ORs, which can help to visualize the OR response to odorants on a microwell. (C) 2013 Elsevier B.V. All rights reserved.