We've been culturing Human Brain Endothelial Cells (HBEC-5i, Cerebral Microvascular Endothelium) for about 5 months now. Usually our cells appear as shown below in figure 1. However, on three separate occaisions, our cells seemed to adopt the morphology and confluency seen in figure 2. On this occaision we began observing this phenomenon around P14. Previously we've cultured HBEC cells until ~P20 before thawing a fresh stock, without seeing this happen at all.
For those of you with experience or know much about working with HBEC-5i cells, have you observed this before? Have they differentiated? Is it normal/to be expected?
Dear Scott,
I have to start by saying that i am not familiar with this specific type of cell, but I am familiar with endothelial and epithelial cells.
Usually after many passages and especially if they are kept in exhausted medium they start de-differentiating. This is the case if your cells present a spindle-like shape. I can observe some of them in your second picture. Moreover, confluence of cells may be another reason for their different shape. Indeed especially endothelial cells tend to form a layer when the confluence is getting high. This may change their shape too.
I hope this can be useful.
Regards
Hi Scott, I have cultured many endos, though not HBEC-5i.
First of all, the top figures appear to be copied from ATCC.
https://atcc.org/~/media/Attachments/5/0/5/7/CRL-3245-comparison.ashx
Secondly, the culture in the lower figure is way overconfluent. They should have been split long before this picture was taken.
When primary endos are this confluent they become senescent. I know from working with HUVECs, BAEs, etc that after passage 6-7, the cells start behaving differently.
You appear to have some giant cells in there but these endos are transfected with SV-40 so who knows what's going on.
Dear Scott,
Endothelial cells can show spindle-like as well as cubble-stone morphological structures, this often also depends on media compositions and differentiation grade. You could check the effects of e.g. hydrocortisone or dexamethasone on their morphology and should be able to observe these changes, especially with primary cells. However, normally the morphology should not change over time, if it is not intended by your experimental set-up. Therefore, changes in morphology gives you a good hint to be careful with your results. Why did not you show regular images of your cells without the mentioned changes instead of copying ATCC images. This would be more helpful for the comparisons. Sometimes, cells just do not look like the images provided by ATCC. Best wishes, Winfried
Hi all, thanks for your responses and feedback.
In response to your queries about why I've used an ATCC image as opposed to one of our own, we hadn't taken pictures of our cells prior to them changing morphology as we had no reason to. Thus, I used the ATCC picture as this was the exact morphology we were observing.
I do absolutely realise the cultures are "overconfluent" however, in normal situations when the cultures are confluent, they do not look like this. They should be much more like picture on the right in the first figure. The cells we are seeing are much smaller, do not represent a similar morphology and form a far denser monolayer.
Giulio, we followed your advice and split the cells at a much lower density than usual on Friday. When we observed them today, they had grown far quicker than usual and were once again exhibiting morphology and confluency seen in figure 1. Today we will split again at an even lower density than previous to see if we see a difference and we will of course keep on top of media changes.
Steingrimur and Winfried, thank you for your advice, however I must point out that these are an immortalised cell line, not primary cells. Winfried, we could potentially see if hydrocortisone or dexamethasone influences them anyway, and of course, for now we are holding fire on any of our experiments involving these cells.
Hi Scott, what do your sub-confluent or confluent cells look like vs overconfluent cells?
If you are only seeing weird cells when they are overconfluent, there is a simple solution.
Split them before they become overconfluent.
Hi Steingrimur.
Firstly, you'll note that the cells depicted in figure 2 are not "overconfluent." By definition cells become overconfluent when they have occupied all available space; there is still space for them to grow into as you can see from the image. The image was representative of the culture dish as a whole, thus there were plenty of gaps for the cells to occupy. So at that point the cells were 90-95% confluent and ripe for splitting. The only issue was that since the last time we observed the cells, they adopted this abnormal morphology.
Since I last commented, we rigorously split the cells numerous times to see if they would adopot their previous morpholgy with plenty more space to grow. Time and time again we've observed the cells again, still with plenty of space for them to grow, continuing to adopt the same morphology. Therefore, we've deduced that this is not an issue with confluency. Although we havent tried the hydrocortisone/dexamethasone test, we believe that it's probably an issue with passage. Thus we are thawing new vials soon. If we encounter this issue again, I may revisit this thread.
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