So, I have been culturing MRC-5 cells for a while now. I cultured them at passage 18, managed to reach confluence in T75 flasks in a few weeks and subcultured them. My problem is, this subculturing happened a month ago, and most of my flasks seem to not be confluent yet and I would risk saying that there haven't been significant changes in growth within the last week. I would just like to ask if this is normal or if there is any advice anyone could give me. The culture medium I am using is DMEM with 10%FBS and 1%antibiotic/antimicotic and when changing medium I only remove 50% and replace with fresh medium so it's 50%old/50%new.
Thank you!
I'm not familiar with the particular cell line but for your observations I would guess you cells are becoming senescent. Do you have information from your lab of how many times can those cells be passaged?
Other possible explanations can be that your culture medium is old and it has lost glutamine or that your culturing techniques might be stressing cells and reducing their expansion potential. Check out if your medium changes are as often as they should (2 to 3 changes per week depending on the cell line and how confluent it is), if cells become too confluent before subculturing or that trypsinization of cells is too aggressive and cells suffer damage. For example for trypsinization of mesenchymal cells (that are similar to fibroblast) I use 5 minutes of trypsin 0.05% at 37º C.
Hope it helps
Have you ever successfully cultured fibroblasts to passage 18 before? Unless they are immortalized, fibroblasts don't grow indefinitely. .
Your cells seem senescent especially if as you said, they reached confluence in a few weeks. Healthy fibroblasts will reach confluence, when subcultured, in less than a week most of the time. You may have hit the Hayflick limit. MRC5 cells are not immortalized cells and will become senescent after 42 to 46 populations doublings as stated on ATCC website. You may have to buy or find new ones.
Good luck
Thank you very much for all your replies! As for changing medium I did it as often as they should and the medium was fresh. As for culturing until passage 18 before, I have personally not done it but people that did before me had done problem. For now I will try to use new cells and see if the problem persists.
It might be patronizing, but hopefully you realize that passage number and population doublings are not the same thing. Their correlation can vary dramatically depending on the seeding density of each passage, so the number of passages before senescence may differ greatly.
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