Growing a monolayer on transwells - (Jul/16/2008 )

Hi,

I want to do a migration assay using Jurkat T-cells through endothelium.
I seed endothelium on a gelatin-coated polycarbonate membrane, but - despite the number of cells (I tried a range of 10^4 - 2X10^5) - do not reach the <5% permeability that I want even after four days.
Am I doing something wrong technically?
I've tried growing the monolayer with medium in the lower well (then they migrate to the bottom well), without medium in the bottom well, and with PBS in the bottom well.
I also notice that after a two-three days the insert becomes leaky - meaning that if I add medium to the upper well before I add medium to the lower well, the medium drips down to the lower well.

I would really appreciate some advice.

Thanks,

What kind of endothelium are you growing? I grow Caco2 cells for 21 days and use them for drug transport studies. If your endothelial cells couldn't form a monolayer on transwell membrane, you might want to try using Caco2 as a preliminary model, and you could try your migration assay before 21-day when they form mature tight junctions, say 3, 5 or 7 days. May not be the relevant model you want, but at least you could start screening and testing your migration inhibitors.

Wow, 21 days, really?

I'm using brain microvascular endothelium, not a commercial cell line. People from my lab who have worked with them previously have never grown them for more than 3-4 days.

The problem is - one time I was able to form a monolayer, but not since.

When you grow the monolayer for 21 days, how often do you change the medium? Do you also put medium in the bottom well? Have you experienced a "leaky" transwell?

Thanks