Is it early or late EPC you refer to? I used to work with early EPC in past, and as far as I can remmeber we seeded them in most of the cases right away into experimental dishes after isolation, to avoid passaging afterwards.

Especially for FACS, I would recommend using Accutase instead of Trypsin, as trypsin might also cut some proteins from the cell surface, and that will compromise your FACS analysis!

And a very obvious thing, but still: make sure you wash them well (at least twice) with PBS without Ca/Mg ions before applying trypsin, to remove any residual FCS (contains trypsin inhibitors) and weaken integrins/cadherins (by depriving of ions they need to function).

Good luck!

Hi Maulasri,

Passaging EPC (or ECFC) with trypsin won't work as trypsin is toxic to EPC. Toxicity will start as low as 0.02% of trypsin. Better use accutase (PAA labs), like Monika suggests, or dyspase instead. These reagents are more gentle to the EPC. Passaging should work afterwards.

Best wishes,

Guido

There are also specialistic culture vessels, that supports cell adhesion only in 37 C, while when put on RT - cells will detach. However I don't remember the vendor, but You should look for such solutions.

Regards

Hi Maulasri,

I have been working with ECFC (EPC) for a while now and trypsin is absolutely fine for them. Wich protocol are you using to isolate the EPC from mouse BM? Do the isolated cells proliferate? Do they form colonies with a cobble stone appearance? For easy isolation and expanion you could see our publication in Blood 2009 or you could watch my video protocol on JOVE.com.

I hope this will help.

Good luck,

Nicole

@Monika I want to use early EPCs (10 day old EPC culture) for FACS. After FACS sorting, I want to re-plate CD34+ cells or use them for transplantation.

Thank you everyone for the suggestions. Also, I want to ask one little thing that what is the optimal fibronectin concentration should be used for the EPC culture? I use 2 ug/cm square.

@Nicole I am following Ashara et al protocol for the BM-derived EPC culture and yes they form beautiful colonies. But, I am unable to passage them with trypsin, even if I wash three times with Ca/Mg free PBS. Could you please send me the link of your paper?

I also have the same problem with detach EPCs isolated from mouse bone marrow. Maybe you could try HyQtase, it may work.

Hi Maulasri,

You can try TrypLE Express, which is a chemical dissociator. It's not so aggressive for surface receptors and with trypsin our cells tend to "go bad" in a few passages. Most of our isolations maintain their phenotype for quite a long time if passaged with TrypLE. It might take slightly longer to detach them this way, but you don't run the risk of damaging them by incubating them for too long, like you do with trypsin. I've never tried Accutase with ECFCs but I would guess it would work too since it's safe to use with stem cells. Also, we use collagen coating instead of fibronectin, but from what I gather, it should work both ways (there's a recent article by Colombo et al. comparing colagen and fibronectin for ECFCs, if you're interested).

Best,

Tiago

Hi Guys,

Here is the method we published in 2009. I hope it will help.

Best,

Nicole

And this is the video protocol we published after.

http://www.jove.com/video/1524/isolation-large-scale-expansion-adult-human-endothelial-colony

The problem in using Asaharas protocol is, as has been published by David Ingram, Mervin Yoder and also Eva Rohde, that you generate a mixed population of endothelial cells, macrophages and mesenchymal cells. True ECFC, which are CD45 negative (!), CD31 positive and CD90 negative, do not need a coating on cell culture treated plastic surfaces to grow. I have used both trypsin and TrypLE Express and both work very well without harming the cells. Please don't hesitate to ask if you have any questiones, I hope I will be able to help.

Best,

Nicole

Hi, I used PBS-EDTA 1% (for 2-5 minutes) for EPC passage.

Hey Nicole,

Your paper demonstrates human peripheral blood growing on non-coated plates. Do you think it would be the same with mouse bone marrow cells or is your protocol specific for human blood sample?

And also, if you are using direct plating technique then there are more chances of getting platelets, macrophages and mesenchymal cells because you are eliminating them by using gradient method, right?

Another option in culture would be to grow them on coverslips and move the coverslips over to a new plate.

Hey Maulasri,

I have heard that this techniqu is also aplicable for pig PB, I suppose it should work for mouse BM. Plateletes and macrophages are in this system not such a problem. However, MSPCs could be challend´ging, since they love EGM-2 as well. Morphologically you will be able to seperate them immediately. If you have MSPCs in the culture they will overgrow all other cells. But density centrifugation will unfortunately not solve the problem, since the MSPCs will also stay in the MN cell layer. I wuld try it out. The safest was is to use umbilical cord blood or umbilical cord, which is tricy in mice I suppose....

Hey Rajesh,

In my experience ECFCs (EPCs) do not grow an glass.

Not glass. plastic coverslips coated with fibronectin.

Oh, that might work. But removing the medium does the same trick...

Dear colleagues,

Be aware that the identification of ture vascular precursors has been a matter of debate for the past 15 years since the publication of the study Asahara (Science 1997) who described putative endothelial progenitors. Subsequently, diverse cell subpopulations have been studied as colony forming units-Hill (Hill NEJM 2003), circulating angiogenic cells or CAC (e.g. described by Dimmeler group, Vasa Circulation 2001) and endothelial colony-forming cells as those studied by Yoder (Yoder Blood 2007) that varied in terms of phenotype, contribution to vascular homeostasis and purity. In addition, cell isolation and culture techniques are different for each one subpopulation.

If it helps, we have recently published an extensive revision regarding this strong controversy (Roura J Vas Res 2013).