I am working with bovine aortic endothelial cells and after thawing I split them at 1:3 ratio. The cells didn't grow, so I came across an article saying that BAEC's divide in the presence of fgf. I added fgf at a concentration of 5ng/ml but still I don't see a considerable growth in cell number. Can anyone suggest what could be the problem?
My colleagues use BAEC very often and seem to have no problem. How did you get your BAEC? Was the transport appropriate? Were the cells stored in liquid N2 in freezing medium? One new tech put wrong freezing medium into the cells in our group and nobody was able to culture the cells after that. BAEC grow happily in DMEM with 5-10% FCS. I believe you have experience before so I do not assume you are doing the culture first time. Just some of my thoughts.
Thanks for your response Jeng. I grow my cells in bovine calf serum. I tried growing them in FCS but they didn't grow very well. Cells were growing absolutely fine earlier but all of a sudden they have stopped dividing. I has once split them at 1:10 ratio and that is when they stopped dividing so I split them at 1:3 but still I see no growth. One unusual behavior that I have observed is that the cells increase in size considerably and remain in that shape.
What is the passage number. We routinely culture pulmonary artery endothelial cells from fetal sheep. When the passage number reach 6-8 we start to see morphological changes and cells grow slow. We even see some batches change into spindle shape like fibroblast or smooth muscle cell but we never got a chance to check it. Occasionally we have cells stop growing even at 4th passage but cells from the same sheep from another tube grow very well. It is always a mystery to me why some batch just do not grow. Also, if the cells increase in size then you should consider some other cell type is taking over (macrophage?). I will suggest you just change to another batch of BAEC if possible.
The 1:10 split was the problem. Endothelial cells like cell-cell contact. When you go higher than 1:3 or 1:4, the cells have a tendency to stop growing. The increase in size is a sign of senescence. Once that has happened, I've never been able to jump start them again. Start a fresh vial (that was not split 1:10 previously), and the culture conditions you used previously should work.
BAEC are pretty easy to culture and to spread. Follow Wayne's recommendation with a new vial and do not use these cells above P16.
Su-Li Cheng
I have worked with bovine AEC from Lonza in the past 2 years. I usually maintain the bAEC culture in the EGM-MV Bullet kit medium (containing brain extracts, glucocorticoid, gentamycin, EGF, and 5% serum) as recommended by Lonza. For experiments, I split the cells into DMEM growth medium. The cells do not grow as well in DMEM growth medium as in the EGM-MV medium and tend to show some endothelial-mesenchymal transition (i.e. increased expression of SM22alpha and decreased expression of claudin 5, etc) in DMEM growth medium. 1:3 split should be fine. I hope this information help you.
Hi Su-Li,
We got the cells from some other university, I am using the recommended conditions. As mentioned earlier I seed them in 10% calf serum and DMEM. I am still facing the problem of slow growth and I see that the cell cytoplasm enlarging. I cannot deduce as to what the problem could be- I certainly see a change in morphology. Have you ever seen such a morphology in endothelial cells. If yes, what could be the reason for such change. Also, do you seed cells in plastic cell culture dish or use a different cell culture dish ? I would really appreciate suggestion as I am unable to figure out such unusual behavior of these cells.
Thanks
Sorry the attachment sent was how the cells were looking earlier, the second attachment shows the changed morphology of the cells
These cells are definely senescent. In my lab, we seed these cells in tissue culture dishes that have been coated with gelatin. You can prepare a 1% stock solution of gelatin, filter it with a 0.45um filter and store it at 4 degrees (stable for 3 months). Incubate your dish with .05% gelatin in PBS for 30 minutes at 37 degrees then wash with PBS and sterilise under UV, to be sure. We use 10 cm dishes.
Thank you Nicolas !
One quick question - Should I thaw a new vial of cells or would these senescent cells get back into the cell cycle if grown on gelatin treated tissue culture plates ?
You should definitely thaw a new batch of cells. Also, if you still have the same problem with the new vial, you should even buy a new vial from a renowned company. Your time is more precious than a vial of cells. Also, never use these cells after P18. Good luck with your experiments!
Thanks again Nicolas- I was wondering that since its a cell line, shouldn't it continue to proliferate without reaching senescence.
BAEC are not an "immortal" cell line, they are primary cells, just like HUVEC. Anyway, it is never a good idea to use cell lines above a certain number of passages, even HeLA that are "immortal" for example. The difference is in the maximum number of passages you can do with one specific cell line: P7 for HUVEC, P18 for BAEC, P100 or more for HeLA. Hope this helps you.
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