Screening methods cell-based

Daugherty PS, Iverson BL, Georgiou G. 2000. Flow cytometric screening of cell-based libraries. J Immunol Methods 243 211-227. 

A variety of different assay chemistries have been developed for screening using cell-based assays. Making the most logical choice among the different methods will depend on what you want to measure at the end of the experiment. The number of viable cells, the number of cells that died as a result of treatment, and investigating whether cell death occurred via apoptosis or necrosis can all be determined with homogeneous assays that have adequate sensitivity for use in 1536-well formats. 

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Two main factors have guided the need for optimization of the early screening techniques on one hand the use of simple, quick and high-capacity cell monolayer methods, e.g., Caco-2 cell and MDCK and on the other hand the increased synthesis of more lipophilic, insoluble compounds from combinatorial libraries. This has created a vast number of different variants of cell-based assays and has resulted in variability among the data obtained. A need for optimization of as many as possible of the different parameters in order to increase the predictivity and throughput of the model has been suggested in the literature [98-100]. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

Dissimilarity and clustering methods only describe the compounds that are in the input set voids in diversity space are not obvious, and if compounds are added then the set must be re-analyzed. Cell-based partitioning methods address these problems by dividing descriptor space into cells, and then populating those cells with compounds [67, 68]. The library is chosen to contain representatives from each cell. The use of a partition-based method with BCUT descriptors [69] to design an NMR screening library has recently been described [70]. 

The selections of compounds are made using a variety of methods, such as dissimilarity selection (16), optiverse library selection (17), Jarvis-Park clustering (18), and cell-based methods (19). All these methods attempt to choose a set of compounds that represent the molecular diversity of the available compounds as efficiently as possible. A consequence of this is that only a few compounds around any given molecular scaffold may be present in a HTS screening... 

In our study we compare two diversity-driven design methods (uniform cell coverage and clustering), two analysis methods motivated by similarity (cell-based analysis and cluster-classification), and two descriptor sets (BCUT and constitutional). Thus, our study addresses some of the many questions arising in a sequential screen how to choose the initial screen, how to analyze the structure-activity data, and what molecular descriptor set to use. The study is limited to one assay and thus cannot be definitive, but it at least provides preliminary insights and reveals some trends. 

Giuliano, K.A., Johnston, P.A., Gough, A. and Taylor, D.L. (2006) Systems cell biology based on high-content screening. Methods in Enzymology, 414, 601-619. 

---