Hello everyone,
I am culturing MDA-MB-231 cell line in Leibovitz's l-15 medium supplemented with 10% FBS and 1% Pen/Strep in an incubator at 37°C without humidifying pan. I observed that some of the culture medium vaporizing during culturing and water droplets on the upper surface of the flask. How can I solve the evaporation from the culture medium without disturbing the cells?
Sorry for the inconvenience.
Thanks in advance,
Mervenur
Hello Mervenur Yavuz
To avoid significant evaporation from culture flask, the flask is placed in an incubator with humidity control aimed at 100% relative humidity. However, the highest obtainable humidity in practice would normally be about 95%. A small water vapor gradient is therefore still operating causing water loss which may become critical during long term culture. One reason for the inability to keep the humidity at 100% is that the incubator is normally opened frequently during incubation.
The effect of culture medium vaporizing may cause problems to the cultured cells since the medium components, especially the salts, may reach concentrations which may be harmful to the cells. So, you need to place the humidifying pan in the incubator and you should fill the humidifying pan with sterile distilled water to ½ inch from the top or fill only to the “max” indicator if available. Keeping the humidifying pan full with water is important for good, consistent humidity and best growth conditions for the cells.
Best.
Dear Mervenur Yavuz,
the media and culture conditions are all suited for the growth of your cell line. However, it is equally critical to ensure that your CO2 incubator maintains just the right level of humidity. Due to differences in the humidity levels across the tissue culture flasks, you are witnessing evaporation of your culture media. Therefore, you must add sterile water inside a stainless steel tray and place it inside the CO2 incubator. This will maintain a stable relative humidity (at equilibrium), thereby preventing any evaporation from your culture media. Make sure that at all times that the water level in the tray is at least 1/4th of its capacity and change it regularly (every week) to prevent any microbial contamination.
Good luck!
Dear Mervenur Yavuz,
Humidity is so important of protecting cultures from desiccation. Humidity pans are commonly used in small to medium-sized incubators to generate humidity through evaporation. Humidity pans produce relative humidity levels ranging from 95 to 98 percent. You can provide adequate humidity by placing ultrapure water on the stainless steel tray inside the incubator. While this prevents evaporation of the media in the cells, it also prevents the compositional changes that may occur in the cell media content as a result of evaporation. Regardless of whether antimicrobial reagents are used, the water in the humidity tray should be closely monitored to avoid undetected contamination growth or the accumulation of harmful substances. Instead of just refilling the water, it is best to completely replace it on a regular basis and clean the humidity tray (e.g., with 70% Ethanol) before adding fresh water. You should replace the water in the CO2 incubator once a week, according to general recommendation.
Best wishes,
Zeyno
Thank you for all your answers, Zeyno Nuhoğlu Öztürk Jatin Chadha Upasana Kachroo
Malcolm NobreI add a humidifying stainless steel tray with autoclaved distilled water, and 2 days later the evaporation problem was solved! Thank you so much.
Mervenur Yavuz Good to know that your problem was solved. Good luck with your experiments! :)
Kindly maintain humidity inside incubator and if it has water pan.. then keep it upto 70% full always to avoid media evaporation
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