The edge effect: A global problem. The trouble with culturing cells in 96-well plates.

Background: The use of 96-well plates is ubiquitous in preclinical studies. Corner and edge wells have been observed to be more prone to evaporation compared to interior wells.
Methods: Mammalian cells were cultured in 96-well plates over a period of 72 h. VWR and Greiner plates were tested. MTS reagent was added, and metabolic activity was determined after 2 h.
Results: When using VWR plates, cells showed a highly heterogeneous pattern of cell growth. The outer wells showed 35% lower metabolic activity than the central wells. Cells grown in rows two and three also grew sub-optimally (25% and 10% reduction compared to central wells). Greiner plates showed better homogeneity. Cells grown in the outer wells showed 16% lower metabolic activity while cells in rows two and three showed reductions of 7 and 1%, respectively. This edge effect was partially mitigated by storing the plates in loosely sealed wrapping during incubation. Placing a buffer between the wells of the plate further improved homogeneity for the Greiner plates.
Conclusion: Different brands of 96-well plates show different levels of the edge effect. Some clearly are inappropriate for such studies.
General Significance: Each laboratory needs to determine their own optimum conditions for culturing cells empirically before continuing to use multiwell plates. Otherwise, large artifacts may arise, affecting the quality of data, with the potential of introducing type I or type II errors.
Competing Interests: This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(© 2021 The Authors. Published by Elsevier B.V.)