Controlled temp for cell plating eliminates room temp cell settling in 96-well plates, reducing Edge Effect and improving cell conditions

Abstract

Abstract The “edge effect” has always limited the use of edge wells of widely-used 96-well plates, reduced useable assay space by 37%, increased materials, time, and costs for these assays. Methods reported to reduce edge effect include leaving the plates at room temperature (RT) for extended cell settling times (30–120 min). We previously reported that controlling conditions to 37° C during cell plating prevents thermal currents and non-random distribution of cells seen during cell settling in edge wells. Here we report studies with human A549 lung carcinoma cells, used for in vitro viral assays, and human bone marrow mesenchymal stromal cells (MSC). Our null hypothesis is that there would be no difference in cell density or viability between cells plated/settled at constant 37° C and extended cell settling times at RT conditions. We plated A549 or MSC under controlled conditions in modular cell handling and incubation chambers in the Xvivo System. We pre-incubated plates for 30, 60, 90, and 120 min at RT and compared these with cells handled at constant 37 degrees. We used the HoloMonitor M4 (PHI, Inc) to monitor cell settling and assessed cell density at 24 and 48 hrs later using crystal violet. We found that cell settling at RT resulted in delayed cell attachment, delayed time to first division, and different cell densities as compared with cell handling at 37° C. Cell agitation and thermal variation was still seen between center and edge wells during cell settling with medium warmed to 37 degrees before traditional cell plating at RT. We concluded that full-time control of 37° C during cell plating is necessary to prevent cell density and cell viability edge effect that occurs during routine handling of cells under RT conditions.