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This paper reports the chemical identity and mechanism of action and formation of a cell growth inhibitory compound leached from some single-use Erlenmeyer polycarbonate shaker flasks under routine cell culture conditions. Single-use cell culture vessels have been increasingly used for the production of biopharmaceuticals; however, they often suffer from issues associated with leachables that may interfere with cell growth and protein stability. Here, high-performance liquid-chromatography preparations and cell proliferation assays led to identification of a compound from the water extracts of some polycarbonate flasks, which exhibited subline- and seeding density-dependent growth inhibition of CHO cells in suspension culture. Mass spectroscopy, nuclear magnetic resonance spectroscopy, and chemical synthesis confirmed that this compound is 3,5-dinitro-bisphenol A. Cell cycle analysis suggests that 3,5-dinitro-bisphenol A arrests CHO-S cells at the G1/Go phase. Dynamic mass redistribution assays showed tha...

This article describes a solvent treatment process for transforming standard flat polystyrene surfaces into textured microenvironments for three-dimensional (3D) cell culture. The process can be used with off-the-shelf microplates having 6, 12, 96, or even 384-well formats and completed in a few steps. The surface microtexture resembles tightly packed, non-interconnected micropores having a median pore size of either 115 or 19 μm depending on the strength of the solvent mixture. Hansen solubility parameter analysis was used to map out the polymer–solvent interaction conditions necessary for creating the surface texture. Primary human hepatocytes cultured on two-dimensional control surfaces attached with a flat monolayer morphology while those cultured on surfaces having 115 μm-sized pores exhibited a round 3D morphology. The cells also showed more in-vivo like behavior on the porous surface by exhibiting significantly higher basal enzymatic activity and expressing higher levels of several drug metabolism-related genes. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40181.

The increased number of drug targets and compounds demands novel high-throughput screening technologies that could be used for parallel analysis of many genes and proteins. Protein microarrays are evolving promising technologies for the parallel analysis of many proteins with respect to their abundance, location, modifications, and interactions with other biological and chemical molecules. This chapter specifically describes the fabrication of G protein-coupled receptor (GPCR) microarrays, a unique subset of protein microarrays, using contact-pin printing technology. The bioassays and potential applications of GPCR microarrays for the determination of compound affinities and potencies are also included.

The possibility of using microarray technology for mechanistic understanding of drug toxicity has opened up a new research field in Toxicology. In an attempt to build knowledge in the field, we have designed a 1C-gene array composed of 85 known human genes with toxicological interests and 15 control genes. HepG2 cells were treated with ethanol and two anticancer drugs, mitomycin C and doxorubicin. RNA were isolated and labeled by fluorescent dyes, then hybridized to the 1C-gene array. Our results showed that a number of cytochrome P450 genes, such as CYP4F2/3, CYP3A3, CYP24, and CYP51, were consistently responsive to the toxicant treatment. However, different genes response to different toxicants. For example, CYP24 and CYP51 were up regulated by the ethanol treatment but remained unresponsive to the other two drugs. The anticancer drugs, but not ethanol differentially regulated several other genes including CYP3A3, TNFRSF6 and CHES1, implying that the two drugs might function through a similar mechanism, which differs from that of ethanol. The reproducibility of our results suggests that microarray- based expression analysis may offer a rapid and efficient means of assessing drug toxicity.

This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.