My study needs to test the adipose tissue derived stem cells' effect on the fibroblasts. I planned to perform a indirect co-culture of these two kinds of cell through seeding ADSCs in the 0.4 um transwell and seeding fibroblasts in 6-well plate. The question appears because ADSC grows slower than fibroblast, so I need to wait ADSC until it reach 80% confluence. While, during ADSC culture in transwell, I found that culture medium leaked to the well under the transwell. So should I add medium in the well also? And if the medium didn't leak into the well, will the secreted cytokine or exosomes from ADSC transport to the medium when co-culture with fibroblasts?
In this case, you should add medium in transwell only. The leaked medium (and everything secreted in it or added in it) from upper transwell will leak down and support the growth of fibroblasts. Let me know if you agree
It looks like you are investigating the paracrine effect of the ADSCs on fibroblasts. Maybe you can try to culture the cells separately and collect the ADSC-conditioned medium. There are many commercially available concentrators for this procedure. Under this scenario, you can starve fibroblasts to eliminate the autocrine effects and titrate the conditioned medium to optimize your study.
Subhash C. Juneja Thanks for your reply. I found that the membrane of transwell seems to be unidirectional because once the medium leaked, the upper chambers were nearly dry. This affected a lot to the ADSCs in transwells. Maybe I should change the transwell to other brand's.