Last year, we got HUVEC (RFP) and we were having trouble since Day 1 (it has proven to be a lot more work than I expected compared to other cells.
After we gotten the cells, we thawed them at 38C and seed them in flasks. Normally, what we would do is to switch out the media 24 hours later, but 70% of the cells did not adhere and it took between 48-72 hours for them to adhere.
Assuming they are p3, they seem to adhere and proliferate properly until p5, when we thought, we had enough and will freeze them and keep a few for our experiments. We were able to at least store >100,000 cells / cryogenic vial x7. After a few experiments, we wanted to thaw the p5 (which is about a month later) and do more experiments. However, we are having some problems. The p5 stop adhering to the plate! None of the p5 worked, we have tried up to 5 vials now and not willing to try more. We are not sure where we have gone wrong.
Do you think using Trypsin 0.25% is one of the factors? I have also been using cell recovery freezing medium which I have used for other cell types (C28, MSC, Osteoblast). We are using the Nunclon Tissue Culture plate (we did not coat the flask with anything). Their current morphology is floating in the flask and rounded.
Please help
Hi Anne!
All what you see is normally for HUVEC. It isn't stable cell line, p6 is limit of its seeds independently of freezing. 30% adhesion if good result, we have 5-10%, that reduce seds to p3.
I also had significant trouble culturing HUVEC cells. I should point out that there are normal HUVEC cells and a HUVEC cell line. I worked with the cell line. It did not adhere well, and got worse with passage - consistent with what you have seen.
One possible explanation is an old finding known as contact inhibition. Normal adherent cells must be passaged before they reach confluence. If they reach confluence, they stop dividing and are very difficult - if not impossible - to revive. Passage should occur at 30 - 50% confluence.
Another factor may be the concentration of trypsin you are using to detach the cells and the amount of time they are in trypsin, because if the trypsin digestion goes too far, the cells can be damaged too badly to recover.
O.25% trypsin is pretty standard, but I do remember that when I used fibroblast feeder cells, I was told to use 0.05% trypsin, and to observe the cells with an inverted microscope. I would watch for the cells to round up, and then gently smack the flask to release the cells and add medium with serum to stop the digestion. If I waited for the cells to detach and float, I did not get reattachment and formation of a monolayer.
Hope this helps!
Multiple factors are likely to contribute to the observations you shared. However, the most likely one is that your primary cell population of HUVECs had reached the number of cell doubling limit (10-16 doublings) before their freezing. Therefore, the frozen cells are not likely to give a cell culture population (due to limited ability or inability to attach and proliferated).
Hello all, thank you very much for your replies! Really appreciate it and glad that I am not the only one having trouble (I hope we all get answers!)
Does anyone know how do we revive the cells that do not adhere anymore? (aka p5, p11?)
We have leftover p8 cells (from the frozen vials) that have been attaching and proliferating properly.
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