Abstract 2438: Reducing plate edge effect by controlling cell handling conditions for in vitro tumor hypoxia assays

Abstract

Abstract To reduce the impact of high variability in the edge wells of 96-well plates, the "edge effect", researchers often fill edge wells with buffer to try to insulate the rest of the plate. This removes over a third of the wells from the assay, adding cost, handling time, and space problems to in vitro assays. This is of particular concern for researchers working with cells in low oxygen, as handling more plates increases the time that cells are out of optimum conditions, risking HIF-1a modulation. For this project, we tested the idea that controlling the entire cell handling space to conditions that match the incubator, including the working space floor temperature and gas levels, may help reduce the edge effect seen in 96-well plates. The null hypothesis was that cell handling conditions would make no difference in the variability of cell density in 96-well plates loaded in traditional room air conditions compared to plates loaded in controlled conditions matching the incubator. To address cell loading specifically, A549 cells were harvested from cell culture flasks in traditional cell handling conditions (room temp, 20% O2, 0% CO2), then split. Half of the cells were transferred to an Xvivo System for controlled cell handling conditions. The cells in both conditions were transferred to media pre-conditioned to hypoxic incubator conditions and then plated in 96-well plates. Temperature mapping was performed with 16 thermal probes simultaneously in replicate plates loaded with medium only. The cells were incubated for 24 hours, then stained for cell density, and read on a plate reader. Cell viability was greater than 90% (n=4 plates/condition/trial x 4 trials). We found that edge well temperatures were more stable in plates handled in unbroken optimal conditions and edge effect on cell density was greatly reduced. In room temperature conditions, columns 1 and 12 cooled most rapidly. We concluded that full-time control of environmental conditions allowed for use of the whole plate with reduced edge effect. This helps reduce cost, time, number of plates and the risks to cells during cell handling. Citation Format: Alicia Henn, Shannon Darou, Randy Yerden. Reducing plate edge effect by controlling cell handling conditions for in vitro tumor hypoxia assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2438.