Abstract 5498: Microenvironmental control eliminates edge effect from 96-well plate-based in vitro assays

Abstract The most widespread format for in vitro drug discovery is the 96-well-plate, but edge effect causes inconsistent results in edge wells due to variations in plate environment. Researchers often avoid using the outermost edge wells, reducing plate capacity by 37 percent, requiring more plates, and increasing assay costs and time. Using the entire plate could save an estimated USD 119M in research costs annually and reduce waste. Finding methods to counteract edge effect is crucial for efficiency, sustainability, and reproducibility of in vitro drug discovery. Previous studies showed that constant thermal control of plating conditions reduces variability in cell distribution in edge wells. Here, we ask the question: can evaporation be eliminated as a difference between edge wells and the other wells of a plate? We plated water in traditional 96-well plates or plates with a water-filled moat around the edges. The plates were covered with either traditional plastic lids that allow gas exchange at the plate edges or an adhesive film that allowed gas exchange across the whole plate. Triplicate plates were incubated for 3 days at 95% relative humidity (RH) in the Xvivo System, a closed system which controls temperature, humidity, and gas levels for cell incubation and handling spaces. Crystal violet dye was added to each well and 0.1ml transferred from each well to a fresh plate. Absorption was read on a plate reader to assess dilution of the dye by the volume of water remaining in each well after incubation (triplicate plates each experiment, n=3 separate experiments). The wells were grouped as four concentric rings, with edge wells as Ring 1, and the center 8 wells as Ring 4. One-way ANOVA with Brown-Forsythe test was used for comparisons. Even at 95 percent RH, traditional plates/lids had edge wells volumes slightly lower than the central rings of wells. There was more overall evaporation from traditional plates covered with film than from the other plate/cover combinations, but there was no difference between the variance in edge wells and other wells. With a moat plate and regular lid, there was no difference between concentric rings on the plate, and there was less overall loss of water across the whole plate. Studies with A549 human lung carcinoma cells also confirmed that the edge effect can be eliminated when temperature and humidity are controlled at all times in a closed cell handling system. We concluded that the proper control of the plate microenvironment in a fully controlled cell handling and incubation environment can allow usage of edge wells for in vitro drug discovery, improving efficiency, sustainability, and reproducibility. Citation Format: Shannon Darou, Graham Jackson, Yan He, Alicia Henn. Microenvironmental control eliminates edge effect from 96-well plate-based in vitro assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5498.