Cell assays for high-throughput screening (HTS) of potential drug candidates are important tools in the process of drug discovery. Most cellular assays are currently based on 2-D monolayer cell cultures, but 3-D cell cultures could better mimic the in vivo characteristics of actual organism tissues. Unfortunately, assays using 3-D culture models usually require significant manual manipulation and are therefore not suitable for HTS. Research under Dr. Shang-Tian Yang has resulted in a functioning system for high-throughput 3-D cellular assays using engineered cells to express enhanced green fluorescence protein (EGFP) quantifiable through fluorometry. System improvement to allow rapid assessment of cellular events, such as specific gene expression or cell cycle progress is limited by the long persistence of the current reporter protein in the cells.
In this study a new fluorescence reporting cell line was established using a destabilized EGFP (d4EGFP) expressed in Chinese hamster ovary (CHO) cells. Correlating fluorescence with cell number for the d4EGFP cell line in 2-D assays indicated that d4EGFP expression may be too low for use in high-throughput cell number reporting. The fluorescence and cell number correlation in 3-D assays indicated better performance could be achieved in 3-D but the fluorescence was sensitive to duration between sampling, possibly due to oxygen transfer limitation, hindering reliable use for cell number reporting. Response to such factors could still serve a purpose for culture condition monitoring, and could be applied in culture development and optimization.