hi,

There are some non-enzymatic cell dissociation buffers you can buy.

You may also try EDTA solution (can't remember the concentration but should be easy to find).

Good luck.

Thank you. But I have tried using EDTA 0.02%, it didnt lift the cells. I did a quick trial and I had to wait for 3 hours and it hardly did anything!

Try subculturing when they are about 75% confluent. After trypsin is added place on warming tray and watch them carefully (view each 30 seconds using phase) after adding the trypsin and agitate (tap culture on countertop while holding top in place to prevent splashes) to speed up the detachment process. Inactivate trypsin the second most of the cells have released (with complete media containing fetal calf serum) or you can kill them easily. Don't try to release all the cells from attachment. Replate with fresh media and incubate quickly.

Hi, this discussion is missing details about the cells used. Are these primary cells or lines? There are several established lines. See for instance:

https://www.dsmz.de/nc/search.html?id=329&tx_mnogosearch_pi1%5Bq%5D=endometrium&tx_mnogosearch_pi1%5Bsubmit%5D=Search

There you will find suggested routines for such cell lines. If the link should fail, go to www.dsmz.de and enter "endometrium" as search term.

Thank you very much for your help.

Wolfgang Asche: They are primary cells

Hi, this means that your cells do not show any adaptation to cell culture so far. You will have a limited number of division cycles If the gentle trypsin treatment (as suggested by Betty) does not work, you might try to add some dispase as well for the detachment. But watch the culture during the process. Inactivate the proteases as suggested by Betty.

Make sure your definition of confluent is not "overgrown". If this is not the case then try subculturing a bit earlier as suggested buy others. Make sure that you do not overtrypsinize and perhaps check the concentration of trypsin as I think for these cells it is much lower than for other cell types. Also, I read that for human cells you need to split into 2 as otherwise cells will not survive at lower concentrations.

Stromal cells should grow reasonably well and be able to be passaged with trypsin/edta. Epithelial cells generally require a matrix (matrigel) to proliferate and survive passage - these can be passaged 1-3 times. Make sure you are killing the trypsin activity with serum-containing media immediately after removal from the plate.

When trypsinising the endometrial cells, I use 4 ml of trypsin/EDTA for T25 flask. I usually wait for about 5-7minutes and I always monitor the flask under the microscope. Once the cells have lifted off, I use DMEM containing 10% FBS to inhibit the enzyme, then centrifuge the cells at1000xg for 5 minutes.

Hey Osman, I've worked with human endometrial stromal cells before, but not mouse so I dont know if this will work the same. Here's how I went about it: I waited until the cells were about 75-80% confluent, aspirated the media, added around 2-4 ml of 0.25% trypsin in EDTA and left in the incubator for 5-7 minutes, occasionally taking the flask out and giving it a gentle tap on the bottom (like Betty suggested) or gently rocking it back and forth for a few seconds to speed up detachment. Then once most of the cells have been confirmed as detached under microscopy, pipette an aliqout containing the cells (I think it was around 2-4ml but Im not sure) to a fresh flask and add 20ml of fresh media (I found you didnt need to centrifuge them). This worked pretty well and I managed to continue passaging them this way for ages. Anyway, hope this helps and I'll see you soon (then I'll be aksing you all the questions!). All the best, Matt

Well Matthew I guess we have cracked it!!!!

The new medium we have tried (SensiCell DEMEM/F12) worked quite well. We have managed so far to do 2 passages.