I am studying barrier function in HMEC1 cells. However, before continuing to use them, I'd like to know how these cells differ from HUVECs or HDMEC's in terms of the the following:
Differences in barrier properties
Differing expression levels of Adherens Junction and Tight Junction
proteins or barrier signaling molecules
Are some more polarized than others?
Is one cell line a more preferred, accurate model of endothelial cells?
Any other information about endothelial cells and barrier function is also appreciated.
Thanks
HMEC-1 were immortalized using transfection with PBR-322-based plasmid containing the coding region for virus 40 A gene product, large T antigen, as described by Ades et al 1992 Article HMEC-1: Establishment of an Immortalized Human Microvascular...
HUVECs are not immortal. HUVEcs are derived from fresh umbilical veins and those cell lines can last about 5-6 passages. So HUVECs are closer to in vivo real situation than HMEC1, but HUVECs batches are heterogeneous as they are derived from different human beings. Your data from HUVEcs might differ from batch to batch. You have to repeat experiments about 5-6 times to get good statistics. HUVECs were tested quite well for permeability, but there are no published data about comparison of the permeability between HUVEC and HMEC1. You might need to do it by yourselves.
It is logical to use HUVEC as they are considered as very close to in vivo situation and they are not immortalized. HUVECs can form surrogate blood vessel ( It is called tube fomation assay :Article Sphingosine-1-phosphate receptor 1 transmits estrogens' effe...
)The good paper about HUVEc permeability is here
Article HSPA12B inhibits lipopolysaccharide-induced inflammatory res...
Thanks!
What plating density( cells/cm^2) would you recommend for HMEC1s, and how long do they need to grow on transwell filters before they form a proper barrier?
experiments should be completed in either a 24-well polystyrene plate (catalogue number 08-772-1; Fisher, Pittsburgh, PA - or similar plates) seeded at a concentration of 40,000 cells per well, or on 6.5-mm-diameter polyethylene terephthalate inserts (100,000 cells per insert) with a pore size of 0.4 μm (catalogue number 351,181; BD Falcon, San Jose, CA or similar filters). You have to check cells in the microscope to make sure that cells are confluent -there are not visible gaps/spaces between cells. When cell monolayer is confluent - cells are ready for testing.
It might take a day or even overnight for cells to get confluent. It is hard to predict for your cell condition. You have to test this by yourselves.
Cells should be cultured under standard conditions (21% O2, 5% CO2, and balance N2), passaged 1:4, and used between Passages 8–20 for all experiments. HMECs-1 should be also checked daily (use microscope) for changes in appearance of morphology, and before experiments. The ideal checking includes flow cytometry. Some laboratories check HMECs-1 for dil-labeled low-density lipoprotein uptake by flow cytometry to verify an endothelial cell phenotype. Cells should look flat, sperad, and have uneven edges. You can take a look at my images of endothelial cells here:
Article Sphingosine-1-phosphate receptor 1 transmits estrogens' effe...
(I have got also full text available for this articel)
All the best for your experiments!
Hey, I'd like to image HMEC1 cell to cell contacts using ZO-1, VE-Cadherin, and Cl-5. However I'll be growing the HMEC1s, for some experiments, on solid substrates, not transwells, so I'll need a way to be sure of polarity. Do you have a recommendation for apical/basolateral markers I could use for HMEC1s? Thanks
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