Hello!

I'm new to cell culture, but I need to cultivate the Mle-12 cell line. For three months, I haven't been able to get more than 5-6 passages. With each passage, the number of dead cells increases. This is very frustrating, and unfortunately, I don't have any colleagues who can help me. Could you please tell me what's critical for this cell line and for lung epithelial cells in general? Are they more susceptible to overexposure to trypsin or mechanical stress?

My protocol:

1. Pour off the old culture medium.

2. Wash with PBS (3 ml).

3. Add 1.5 ml of trypsin and wait 1 minute. While I wait, I tap the sides of the culture medium, otherwise the cells do not detach.

4. Pour in 3 ml of culture medium and wash the walls of the culture medium several times with a pipette until most of the cells detach.

5. Pour into a test tube and centrifuge for 5 minutes at 13,000.

6. Pour off the liquid and add 2 ml of new medium.

7. Pour 4 ml of new medium and 1 ml of cells into the new culture flask.

8. Mix the cells vigorously with a pipette about 20 times (if I don’t do this, they will remain stuck together).

The first photo shows cells from passage 2-3. I do the passage every day at about the same time, but after a few days, the number of dead floating cells increases. And the second photograph is cells after 6 pass

I will be very glad to receive any advice and will be happy to answer all questions.

On 1/12/2026 at 5:39 AM, Clavicula said:

I don't have any colleagues who can help me.

Isn't anyone else doing cell cultivation? One thing I would check is to make sure that the culture medium is still fine (and has all required additives) and that the incubator is up to spec (especially check the that the CO2 is steady and at the correct level).

Based on your description I also think that you might be treating the cells too harshly. While I haven't got MLE 12 specifically in my lab, lung cells have a propensity to clump up if agitated and they do not like it if you mix them too much. They are not as stable as, say, HeLa. If detachment is difficult, it is better to incubate a bit longer at 37C and wait for detachment. You will have to be a bit more patient with lung cells.

You have not provided trypsin concentration (or flask volume), but instead of PBS washing (which could be too harsh, if not done carefully), I would just rinse the cell with trypsin/EDTA solution to get rid of inhibitors, before adding additional trypsin/EDTA solution. PBS is also frequently used, but I generally like to ensure a fixed concentration of trypsin as the detachment kinetics is a bit more controllable in my hands.

Detachment happens usually within 5 mins (so observing it is important). But if it takes longer, instead of agitating rather place them in 37C in give them another 5-10 mins or so before (and again, observe the process so that you get an idea how well the protocol works). Once larger detachment happens, I interrupt the trypsinization by adding about 2x volume of the complete medium.

Also, I do note generally recommend centrifuging lung cells but if you do, use low g. 13,000 (regardless whether you meant RPM or g, even if it is really a tiny rotor) would be too high.

If they grow to confluence within a day or two (which seems to be the case?) I would increase the passage ratio a bit.