Hello Erica Weber

The reason being a different environmental condition. The environment surrounding the cells in the flask is different than that in a 96-well plate. The major problem in a 96-well plate is the “edge effect”. The 96-well plates are designed with a high surface area to volume ratio within each well. This is a key feature that makes them useful for various assays, particularly those involving cell culture in small volumes. But they are prone to evaporation from the edges. Evaporation can lead to many things that can affect cell growth and viability such as increased concentration of medium components, changes in solute concentration can lead to osmotic stress, impacting cell function; evaporation can also affect the pH of the medium, disrupting cellular processes and causing stress; uneven evaporation can also result in temperature differences across the plate, creating inconsistent growth conditions.

You can overcome these problems by incorporating the following in your protocol.

1. Pre-incubate the plate for a short period at room temperature after seeding as this can help cells adhere more evenly before incubation.

2. Add sterile water or media to the wells surrounding the experimental wells which can help reduce evaporation and maintain more consistent humidity and temperature.

3. Adjust the cell density and incubation conditions to help mitigate the impact of edge effect.

4. Ensure even cell distribution by gently pipetting against the well walls and thoroughly resuspending cells before plating.

5. Closely monitor cells and environmental parameters like pH and nutrient levels and adjust accordingly.

6. Use different 96-well plate because some 96-well plates are manufactured with different surface treatments or materials.

So, whenever you culture cells in 96-well plate, the differences in geometry, surface area, and potential for evaporation and temperature gradients in 96-well plates compared to flasks should be looked into, and careful consideration of plate choice is necessary to ensure optimal cell health and reproducible results.

Best,

If your adherent cells appear stressed after overnight incubation in a 96-well plate, but remain healthy in a T75 flask under the same conditions, the issue likely stems from microenvironmental differences inherent to smaller wells. Factors such as evaporation, especially in edge wells, can lead to osmotic and pH stress, while low seeding density, reduced medium volume, or non-treated plastic surfaces may impair attachment and nutrient availability. Unlike flasks, 96-well plates require careful control of humidity, cell density, and media volume (ideally ≥150 µL), and may benefit from surface coatings or using treated plates to support optimal adhesion and morphology. Addressing these aspects usually improves cell health during longer incubations.