BioSpherix & Phase Holographic Imaging in printed media

Abstract 2157: Eliminating Edge Effect in 96-Well Plates by Controlling Thermal Conditions during Cell Plating

DOI: 10.1158/1538-7445.SABCS18-2157 Published July 2019

 

Abstract

The traditional approach to avoiding the increased variability in 96-well plate edge wells has been to avoid using them. 

This results in a 37% loss in usable assay space, increasing time and materials needed for cell-based assays. 

Approaches to reducing this “edge effect” have included reducing plate evaporation during long-term incubation. 

However, we have preliminary data that plating cells at a constant 37 degrees C dramatically reduces edge effect. 

We have previously showed that in plates filled at room temp (RT), the cells in edge and corner wells experienced the most directional cell rolling during cell settling, preventing random cell deposition. 

In contrast, cells plated at 37 degrees C settled randomly in edge wells with less or no directional rolling. 

Here, we extend these findings with studies of well-specific cell settling patterns associated with plate-wide thermal changes after cell plating. 

Our null hypothesis was that intra-well thermal currents as plates warm to 37 degrees C from RT do not disrupt random cell deposition during settling. 

A549 human lung carcinoma cells were plated in warmed medium in either traditional uncontrolled RT conditions or in an Xvivo System under completely controllable conditions. 

In this closed chamber, everything (chamber floor, atmosphere, the pipettor, tips, fluids, gloves, and plate) was at the same temp as the incubator, where cells typically settle in the wells after plating.

We used the PHI HoloMonitor M4 microscope to record holographic time-lapse images of cells settling and adhering to the well floor. 

Crystal violet staining was used to assess cell deposition patterns. We found that not only did constant temp conditions change well-specific cell settling patterns, but when plate-wide thermal changes were inverted, by plating cells at 37 degrees C and letting them settle at RT, we were able to alter well-specific cell rolling and cell settling patterns. 

We concluded that it is thermal changes that drive edge well variability and that constant temperature control during cell plating can reduce or eliminate edge effect. 

Plating cells under full-time control of conditions could have a tremendous impact on cell-based drug discovery and pre-clinical testing by allowing full use of 96-well plates. 

This could reduce assay time and materials as well as improve assay reproducibility.

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