(423g) Development of a 3-D Whole Cell Gfp-Based Assay System with High Sensitivity and Accuracy for Cytotoxicity Studies

Live cell fluorescent kinetic assays are always desired by the pharmaceutical industry, safety evaluation agencies and even basic science research because they are suitable for automatic high-throughput design without requirement for manual sample preparation and enable characterization of rapid cellular events. However, fluorescent signals generated in such assays are always too weak to be sensitive enough for in situ measurements. Three-dimensional (3-D) cell culture systems are better than 2-D cultures for efficacy analysis, because 2-D culture models are inherently prone to error lacking proper in vivo tissue functions. We have designed, built and tested a simple 3-D whole cell GFP-based assay combining 3-D culture system into live cell fluorescent kinetic assay, which can increase the signal to noise radio at least one order of magnitude compared to standard 2-D cultures, and thus enables much more applicability for parallel, automated, long-term (more than one week) assays. This system has potential for the development of 3-D high-throughput biosensors, scale-down system of large scale bioreactors, and assays for laboratorial routine uses such as cytotoxicity assays and the whole-process monitoring of embryonic stem cell differentiation. In this 3-D system, embryonic stem cells with stable enhanced green fluorescent protein (GFP) expression were cultured in pre-treated nonwoven polyethylene terephpthalate (PET) fibrous scaffolds. The whole culture system was carried out in modified 96-well microplates stacking on a lab shaker in a cell culture incubator. A general fluorometer was used to quantify fluorescence intensity in real time. Effects of fetal bovine serum and fibronectin coated on 3-D ES cell culture were evaluated in the system. Embryocytotoxicity assays of Dexamethasone(DM), Diphenylhydantoin (DPH), Penicillin G, 5-fluorouracil(5-FU) and assays to monitor acute cell responses of detergent demonstrated the appropriation to biosensor technology: enhancement of signal to noise ratio, elimination of labor intensive manual sample preparations, improvement of accuracy from 3-D culture superiority and minimization of errors due to biological system changes caused by cell activities. In addition, with the introduction of live cell kinetic assays, instead of fixed endpoint assays, cytotoxicity can now be readily expressed upon a constant standard with more accurate and real-time monitoring. The results in this new 3-D sytem also showed significant differences in drug cytotoxicity responses as compared with those from the conventional 2-D cell culture assays. Applications and advantages of this new high-throughput system in drug discovery will be discussed in this paper.