Can flow cytometry play a part in cell based high‐content screening?

Abstract

In parallel to gene therapy and cellular therapy it is clear that the more traditional pharmacological approaches still have a major role to play in the treatment of most diseases. Two drug-discovery strategies are widely used in the pharmaceutical industry: the so-called rational drug design and the most recent screening-based strategy. Rational drug design requires usually an excellent knowledge of the biomolecular target: molecular mechanisms of action, three-dimensional structure, etc. It is scientifically sound and often efficient but rather time consuming. To cope with an exponentially increasing number of potentially interesting targets derived from genomic and proteomic studies, the industry has developed the screening-based approach since the 1990s. It has proven to be very efficient to afford original hit compounds that may open the way to drug development. During the last decades there has been a rapid development of assays and screening methods to identify promising drug candidates. Using cell-based disease models will improve the predictive value of drug discovery (1). When compared with a classical drug design approach (based on a single target protein involved in a given biological event) the use of a cell-based essay is dramatically efficient for drug discovery and development. Pace has been increased via combinatorial chemistry, genomics knowledge, proteomics technologies, new target validation, methodologies, miniaturization, and high-throughput screening as some of these issues were recently profiled by Smith et al. for the cytometry community (2). Once you are convinced that understanding how drugs affect cellular processes and cellular pathways in situ is the most biologically reliable route to discover effective new drugs; high-content screening (HCS) will be your technology of choice. HCS is based on multiparameter analysis of intact cells by coupling the power of fluorescent trackers to bioinformatics and robotics. Typically the system is based on epifluorescence microscopy and combines automated cell field focusing and multichannel image capture with sophisticated proprietary algorithms to extract high biological content information from fluorescently labeled cells. Recent technological breakthroughs in automated microcapillary cytometry allowed avoiding expensive and time-consuming image analysis apparatus and replace them in most cases by faster FACS analyses (FCS, SCS, and today up to four fluorescence channels). For example, microcapillary cytometry HCS includes the following: receptor internalization, multiparameter apoptosis, mitotic index, cell cycle, mitochondrial activity and of course cell viability, cell tracking, and real-time cellular concentration. At present, a substantial variety of automated single-cell measurements can be assessed, such as the metabolic state of viable cells by flow cytometry or laser scanning cytometry (3, 4). Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. Even though there is no exact definition of HCS, it typically refers to a cytometric technique based on automated fluorescence microscopy. By connecting the imaging of cells in microtiter plates with powerful image analysis algorithms and data visualization software one can acquire knowledge on multiple biochemical or morphological pathways at the single-cell level (5). A recent review by Paul Lang et al. describes how cellular imaging technologies contribute to the drug discovery process (6). They concluded that indeed several factors limit the extensive use of cell