Beating the edge effect

There are several different factors which can increase both noise and variability of cell-based assays for high throughput screening. In particular, the edge effect causes a significant increase in plate rejection rates - and it needs to be fully understood in order to beat it.

The microplate is an important element of cell-based assay protocols and is designed so that multiple tests can run in parallel. The plates are easily incorporated into high-throughput workflows, to increase screening efficiency. However, the occurrence of edge effect poses a major limitation in obtaining a streamlined, high throughput protocol. During culture, water and media commonly evaporate from the wells that are closest to the perimeter of the plate, with the outer 36 and corner wells being the most affected. The result is a variation in cell growth across the plate, while any media components, such as salt, can become concentrated to the point where they are harmful to the cells. A volume loss as small as 10% can concentrate media components and metabolites enough to alter cell physiology, consequently impacting on the viability of downstream data, causing heterogeneous or biased results to occur. As a result, the trend has been to not use the outermost wells to avoid this variability. Not using all 96 wells means that throughput and therefore efficiency, is substantially reduced.

In order to keep the time and cost involved in drug screening and the identification of lead compounds to a minimum, it is critical that the methods used are optimised to produce the best results.

[caption id="attachment_50076" align="alignnone" width="550"] Figure 1: Comparison of cell count per field in the Edge plates with either a filled or empty moat. The graphs show the cell count per field distribution across the entire plate (96 wells)[/caption]

Cell culture incubation is a vital and dynamic process in which there are a large number of variables to consider so that the cells are protected and thriving. In order to reduce evaporation, it is therefore important that culture plates are incubated in an environment with at least 95% humidity. Evaporation is almost four times higher at 80% than 90% humidity, demonstrating the significant difference that humidity levels can make on resulting viability. By ensuring that the water gradient is as small as possible, water loss during culture will be less critical to the cellular mass in the long-term, thus reducing plate rejection rates due to evaporation.

Another critical factor affecting evaporation is the frequency with which plates are removed from the incubator for microscopic examination. Furthermore, as is often the case, incubators can be shared by multiple users, making frequent door openings another issue. Exposure to external conditions decreases humidity control and temperature within the incubator, thus increasing the rate of evaporation. However, in order to maintain accurate and consistent media concentrations across the microplate, and prevent cells from drying out, media levels may need to be topped off throughout the incubation period – another process that requires removal from the incubation environment. This puts further emphasis on the importance of maintaining evaporation levels as low as possible to prevent wells from running dry.

With a significant cost, both in terms of resource and money being invested in the identification of lead compounds in the drug screening process, it is pivotal that researchers have a highly efficient, reliable, and often automated, method of culturing for various cellular assays. Since efficiency is key, researchers ideally need to be able to culture cells in all 96 wells of a microplate, without any worry of encountering significant evaporation and thus, edge effect. Although employing a CO2 incubator with advanced humidity control and limiting exposure to external conditions can significantly reduce evaporation, novel new plate formats can further reduce its occurrence, while removing the time-consuming need to regularly replace lost culture media.

[caption id="attachment_50078" align="alignnone" width="550"] Figure 2: A comparison of overall plate evaporation after 4 and 7 days’ incubation across a range of plates.[/caption]

By incorporating a large perimeter buffer zone – essentially a moat surrounding the outer wells –  microplates such as the Thermo Scientific Nunc Edge plate can eliminate well-to-well variability, while dramatically reducing overall plate evaporation to <7% after seven days’ incubation. This moat can be filled with sterile water or, to eliminate spillage, 0.5% agarose, to provide a solid, jelly-like material. As a result, the plate can be incorporated into a fully-automated workflow, thus increasing screening efficiency.

In order to assess the effectiveness of this evaporation buffer, two plate types (one with a moat filled with sterile water and one without a moat) were used to culture HeLa cells in an incubator at 37°C and 5% CO2. After seven days of culture, the cells were fixed with formaldehyde and stained with fluorescent dyes for visualisation. The plates were then imaged with a high content screening reader using a 10x objective and cell count data was collected. Data shown in figure 1 demonstrate that well-to-well results were more consistent in a microplate with a filled buffer zone, than one without.

In order to assess evaporation rates, six plate types were filled with 100µL of water and incubated at 37°C for four days in a humidified atmosphere of 5% CO2 in air. In order to simulate common laboratory conditions, the incubator door was opened for 15 seconds, seven times each day. To compensate for favourable versus non-favourable positioning within the incubator, three plates of each type were placed at different positions and a mean value calculated. The protocol was repeated over an incubation period of seven days using 200µL of water. Figure 2 demonstrates that the presence of the evaporation buffer zone dramatically reduces overall plate evaporation to less than 1% after 4 days’ incubation, or 2% after seven days’ incubation.

An evaporation buffer zone fill consisting of 0.5% agarose provides the same low plate evaporation as water, while providing the solidifying effect required to prevent spillage during automation. Both remain below 2% after seven days’ incubation, in comparison to over 8% observed with a standard 96-well plate.

Evaporation across cell culture microplates becomes increasingly problematic the longer the culture period. However, the edge effect may cause critical volume loss in the periphery and corner wells much earlier. As such, there are several steps that researchers can take to reduce the occurrence of evaporation:

• Use an optimally performing humidity control of at least 95%
•  Limit the number of inspections that occur external to the incubator
•  Do not open the incubator door unnecessarily
• Reduce the need to replace lost media by employing novel plate formats that incorporate a large evaporation buffer zone in their perimeter

In order to obtain a streamlined, high throughput method of assay based cell culture for the screening of potential drug compounds, it is vital that an effective and reliable method is obtained. One issue that has been commonly encountered in cell culture laboratories is the phenomena known as edge effect. Evaporation naturally occurs along a gradient, which can result in concentration of media components, such as salt, which can be harmful to the cells. Over long-term incubation, this will occur across the plate in its entirety; however this can be amplified in the outer and corner wells of microplates where a larger water loss can occur over a much shorter time frame. In order to combat this effect and maintain the integrity of resulting data, while keeping all 96-wells in use, the incubation process and the plate itself need to be as efficient as possible. Users need to adhere to best practice measures, ensuring that a consistent incubation environment is maintained throughout the culture period, with minimal exposure to the external environment. In addition, plate formats such as that of the Thermo Scientific Nunc Edge plate, incorporate an evaporation buffer which surrounds the periphery wells. As a result, well-to-well consistency is maintained across the entire plate, and edge effect is essentially eliminated. Cell cultures can therefore occur in a highly efficient manner, with reliable and reproducible results for subsequent lead compound analysis.

Contact: www.thermoscientific.com/edgeplate