Dear Sushmita,

I don't have an experience with lung endothelial cells but I have a long experience with human umbilical vein endothelial cells, and I can say that 2 min is very long period you can try to wash your cells with PBS twice then trypsinize  for about 30 seconds and as soon as they get rounded under the microscope, you remove the trypsin and tap the plate on the table few times then add your medium and then pipette the medium up and wash the plate withe medium several times till you collect all the cells. In this way you will not affect the viability of your cells by the long trypsinization process.

I hope that will help

Good luck

Dear Sherif, thanks a lot for your reply..i do exactly the same for HUVECs but the mouse cells don't come off in 30 seconds..i have carefully titrated the time and found that it requires a minimum of 2 mins.

even if you washed them with PBS before you start?

yes..i always wash the cells with PBS to remove the medium because serum in the medium slows down the trypsinization.

Dear Sushmita,

The thing you are describing is what usually happens with mouse lung endothelial cells. They are more vulnerable than HUVEC to passaging, so it takes longer for them to recover and they grow as colonies, as you have observed. The only tips I can give you is: don't divide them more than 1:2 or 1:3, be patient with them and only use them until passage 4. This cells are intended to be isolated and immediately used in the experiment you have planned, not to culture them for long time because they can't bear it. Hope this helps.

Thanks a lot Noelia! I actually was passaging them only 1:2. The reason for me to passage them (i usually only work with unpassaged primary cells)  in the first place was to have them more in number because when I isolated them using CD31 coated MACS beads, i got a lot of unwanted fibroblasts as well...and then had to wait for a month to get large endothelial colonies, but these are mixed with a lot of fibroblasts. Therefore, I was passaging it just once atleast with a hope to enrich for my endothelial colonies. Alas, every time the fibroblasts take over due to their super high proliferative index!

Yes, fibroblast are a usual problem in this type of primary cultures. What I do is to do a first "negative selection" with anti-podoplanin (other protocols suggests anti-CD16/CD32)  to remove non-endothelial cells, then a second "positive selection" with anti-CD31 to pick the endothelial cells out of the mix. Then I culture them for a couple of days until they grow and repeat the positive selection to further purifiy the culture. You will never have a 100% endothelial cell culture, but these are the majority in the culture. It is OK that you passage them as you have said to get higher number of cells, just keep in mind that you shouldn't go further than P4.

I totally agree with Noelia and have similar experience, we always prefer to do a negative selection first, followed by positive selection with CD31 twice. As Noelia said, even after doing this EC population is not 100% pure but I can say about 90-95% pure. Mouse lung ECs are more sensitive and we prefer to use 0.05% trypsin.

Thanks a lot Noelia and Abha. I do positive selections exactly like you said, once during the isolation and later on during further passages ( and i don't passage more than P3....but i didn't start off with the negative selection due to lack of those antibodies you mentioned. But maybe I will do it for the next time. Was nice to know your experiences in this regard and it definitely helps. Thanks a lot for your replies!!

I donot know what is exactly your protocol of isolation .but I think if you got a lot of fibroblasts ,maybe you can try another protocol to isolate lung endothelial cells .if the primary endothelial cells are cultured too long in the medium ,it is difficult to trypsin . 

http://www.ncbi.nlm.nih.gov/pubmed/19223532

thanks a lot Guangxiang Zang