Related Experiment Video
Updated: Nov 9, 2025
Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates
Published on: March 7, 2017
The edge effect: A global problem. The trouble with culturing cells in 96-well plates
Morva Mansoury1, Maya Hamed1, Rashid Karmustaji1
1RCSI-Bahrain, PO Box 15503, Adliya, Bahrain.
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.
Insights
The edge effect in 96-well plates causes significant cell growth variability, with outer wells showing lower metabolic activity. Different plate brands exhibit varying degrees of this effect, impacting preclinical study data.
Area of Science:
- Cell biology
- Preclinical research
- Assay development
Background:
- 96-well plates are standard in preclinical studies.
- Evaporation is more pronounced in corner and edge wells compared to interior wells.
Purpose of the Study:
- To investigate the impact of the edge effect on mammalian cell culture in 96-well plates.
- To compare the homogeneity of cell growth between different brands of 96-well plates.
Main Methods:
- Mammalian cells were cultured for 72 hours in VWR and Greiner 96-well plates.
- Metabolic activity was assessed using MTS reagent after 2 hours.
- Experimental conditions included loosely sealed wrapping and buffer placement to mitigate evaporation.
Main Results:
- VWR plates exhibited significant heterogeneity; outer wells had 35% lower metabolic activity than central wells.
- Greiner plates demonstrated better homogeneity, with outer wells showing 16% lower metabolic activity.
- Mitigation strategies like loose sealing and buffer placement improved homogeneity, especially in Greiner plates.
Conclusions:
- Different 96-well plate brands display varying susceptibility to the edge effect.
- The edge effect can introduce significant artifacts, potentially leading to Type I or Type II errors in preclinical data.
- Laboratories must empirically determine optimal cell culture conditions for multiwell plates to ensure data integrity.
