Isolation of rat endothelial cells - (Nov/04/2011 )
Hey everybody... hope that anyone can help...
i am trying to isolate endothelial cells from large rat vessels... now that that isn´t really succesfull, i read some paper to get ideas, what other tissue i could use.. i often read about isolating microvascular cells from adipose tissue... i think i will try this, because our rats are reeally fat can anyone tell me, whats the advantage of this tissue? are there maybe less fibroblasts or something like that? would be great if you guys could help...
Many years ago I tried to isolate endothelial cells from Rabbit aorta, the vessels being alot larger that the equivalent in rat. It was impossible to grow enough cells to do experiments because by the time the cells I did isolate had grown, they were pharmacologically different from in vivo cells.
What we did was isolate PORCINE aortic endothelial cells. We did this because we could obtain large numbers of fresh vessels from abattoirs. The number of cells we isolated was in the tens of millions, easily enough to perform experiments with very low passage cells that were similar pharmacologically with in vivo endothelial cells. Due to the large numbers of cells we could easily perform immunohistochemical staining to characterise them as "endothelial".
The experiments were in the 1980's when primary cells could not be purchased from companies like Promocell, who sell a large variety of primary cells. Again the disadvantage with using these companies is that the cells are expensive and have been grown in "specilaised low serum media" that you have to use from that particular company....again being alot more expensive than DMEM/RPMI.
We were always criticized by groups who suggested the lack of relevance of our work because we were using porcine and not human cells. I think our work speaks for itself as "very relevant" as we had 3 papers publsihed using these porcine cells...the references are below:-
Nature Vol.320. No.6061 pp. 454-456, 3rd April 1986. "Superoxide anion is involved in the breakdown of endothelial- derived vascular relaxing factor". S. Moncada et al.
Nature Vol.327 pp. 524-526 11th June 1987. " Nitric Oxide release accounts for the biological activity of endothelium-derived relaxing factor" S. Moncada et al.
Nature Vol. 333 pp. 664-666 16th June 1988. " Vascular endothelial cells synthesize nitric oxide from L-Arginine" S. Moncada et al.
Thus my advice would be to use larger animal vessels so that all the problems associated with using minute vessels are circumvented.
I do hope this is useful.
hey uncle rhombus
thanks for your answer! the problem is, i have to use rat cells... my boss wants this. I read something about using the microvessel endothelial cells from the lungs or the liver... just mince it, than MACS sorting. sounds easy... i think we will try this.. another group here uses porcine vessels.. last week i just watched them isolating...sooooooo much easier!!!
i also think, the number of cells is so low, and by the time we have enough, there might be some receptors or integrines or something like that got lost, so that we might have endothelial cells, but cant show it with staining... hmmm.. so i am thankful fpr ideas