Primary endothelial cells went differentiated - Medium, serum or other factors? (Aug/18/2006 )
I have experienced some problems with bovine aorta endothelial cell culture. The cells looked fine when I harvested, purified, and grew them up from 96well->24 well->6well plates->100 mm dishes. I started to notice large (giant) sized cells appeared to be differentiated. I have since reduced split ratio from ~ 1:4 to 1:2, and I have tested other factors and found that cells are happier in opti-MEM+10% FBS, instead of M199+10% FBS. Could any one with first hand experience on this cell type please advice me where did I do wrong and is it possible to get rid of these giant cells? How did you culture BEAC? Thanks in advance.
After how many passages do you start to see the 'large' cells? What do they look like? Do you have a picture? Have you tried staining with pecam, ve-cadherin, or other marker to check the endothelial-ness of your culture?
If we plate our mouse endothelial cells too sparse, they do tend to spread out more and not be as 'happy'.
Way back when I was culturing BAEC, we did so in DMEM with 10% calf (not fetal - we were cheap) serum, with pen/strep, and glutamine. They seemed to do well in that, but we weren't isolating our own.
Hope this helps
15 years ago we used to isolate primary BAEC's but used them after the first passage. We did this because the Prostaglandin profile we were interested in at the time changed dramatically over just a couple of passages. Later when we were interested in Nitric oxide, again the NO profile changed over time. Thus we were getting fresh Aorta's on a regular basis.
The morphology also changed over the passages from the classic "cobblestone" to a more fibroblastic appearance. We used Factor VIII staining for characterising our endothelial cells.
Our growth media was DMEM (1000mg/litre) + 10% FCS, 2mM L-Glutamine and Pen/strep.
I am suprised that you start them in 96 well plates as you should get loads of cells from the Aorta.
Do you go and get the tissue or do you use a company ? I only ask because when we tried to use a company, the abattoir workers usually handle the tissue very roughly. This means that there is detachment of the cells from the aorta before it even arrives in the lab. I used to go to the abattoir and personally supervise the excision of the aorta. As a rough guide, a portion of the thoracic aorta should provide enough cells to seed at least 3 or 4 T25cm TC flasks.
Other factors affecting the cells :
Grade of FCS i.e. use the best serum you can get like New Zealand sourced FCS.
The TC plastic will affect the growth and morphology....try from different suppliers.
The collagenase you use for isolation i.e. the batch and Type and the length of time you use it.
Seeding density : endothelial cells require a high seeding density and low split ratio.
jmdp, and Rhombus: thanks for your help. I think most likely is due to cell density that was a bit low during my 24 to 6 well expansion step. We did harvest ourselves. We used mechanical scraping method, rather than enzymatic digestion to get cell off the tissue, therefore cells may not be 100% BAEC. We picked up colonies that are typical of endothelial cells and expanded from these colonies stepwise. Now cells are roughly in P6. As these gaint cells do not divide, they get diluted away. I am pretty happy with what we have at this point.
We tried scraping because it is alot easier and cheaper to do. But cell viability and the possibility of cross contamination from other cell types i.e.fibroblast/SMC was more probable. Collagenasing although it takes longer should give you a far cleaner and more viable cell harvest, no need to pick colonies that look like endothelial cells.
The aorta is very thick and very hard. I have no ideal how do I seal it into a compartment, so I can fill it with collagenase solution and keep it from leaking. Do you have a clever way to do it right that you can share with us? Thanks in advance.
There are little vessels that can be tied off with cotton or clamped with spensor wells. Again both ends of the aorta can be clamped in the same way but with much larger spensors.Again if you go to the abattoir and explain to the meat inspector how you want your aorta, they will generally oblige and thus leave these intercostal vessels more or less intact. We went away from bovine to Porcine generally because of the ease of ligating the intercostal vessels and the increased number of abattoirs that do pig as opposed to cow.
Hope this helps, it brings back bad memories as this is all I did for about 4-5 years.