co-culture of two adherent cells - (Jan/01/2005 )

Hi,

I am trying to establish a co-culture of NIH/3T3 fibroblasts and monocyte/macrophages. As it is known fibroblasts keep on proliferating as co-culture time extends. This makes it difficult to analyze monocytes as their ratio changes. I want to keep all cells adherent to be sure that they are cross-talking. So what is the best way to study this co-culture; to irradiate fibroblasts?, to treat them with mitomycin? or to fix them in paraformaldehyde? and then add the monocytes... ?

But how long fixed or irradiated fibroblasts can be maintained effectively (surface protein expression must be OK) in culture?

And how can i be sure that there wont be any trace of mitomycin or paraformaldehyde to affect monocytes badly? even if i wash these adherent fibroblasts many times...

Does anyone have an experience on co-culture of two adherent cell types? Any idea?

Thanx by now...

Gunes.

Hi. I cultured mouse embryonic stem cells on fibroblast. This fibroblast were treated with 10 ug/ml mitomicyn C (Sigma, M0503) in culture medium during 3 hours. After this the monocape was cleaned 3 times with PBS. & hours after they can be used as feeder layer o subcultured to other flask in the adecuated density. I used them during one week after inactivation with mitomicyn.
I hope that my experience help you.

Do you want the cells to grow together on the same surface? If that's not critical, you could try tissue culture inserts: grow the fibroblasts to confluence in the well and then "co-culture" the monocytes by seeding them into a tissue culture insert and then analyze what you want. I'm not sure whether you would need to irradiate the fibroblasts or add mitomycin. If you do, it would be wise to see whether protein expression is still the same. I doubt it would be. You could analyze protein expression with a protein cytokine array with and without treatment. Definitely don't fix the cells with paraformaldehyde - I'm pretty sure you won't get any protein expression if you do.

You may also want to consider growing the cells in collagen (type I) gels (2 adjacent, separate layers). Growth in collagen gels would be more similar to in vivo, but its usefulness depends on what you want to test. For RNA expression, it works well; I'm not so sure it would be so easy to test for protein expression, yet it should be possible if you first treat with collagenase, type I to degrade the collagen gel.

irradiated ur fibroblast when it becoms confluent, seed ur macrophage cells later. iradiated fibroblast keep ur fibroblasts still alive while still not proliferating!

Hi
We routinely co-culture monocytes and fibroblasts in our lab. We find that using PET inserts with 0.4micron allows the cells to be in enough contact to "talk" to each other. The protocol for culturing the two cells (one type on each side of the insert) is relatively simple you would need a sterile tip box (place some double sided tape in the bottom prior to autoclaving). Take the inserts (coat them if required i.e. gelatin/albumin) then invert them in the tip box and take your cell suspension and pipette onto the underside of the insert (just enough to cover surface) and incubate for between 30mins and 1 hour to allow cells to settle then simple pop them in a plate add medium and then seed other cell type onto the inside of the insert.
Hope this helps