Filtration of primary cell culture, how?? - (Feb/21/2005 )

Hi all (sorry long post)

I am new to the field of cell culture. I'm trying to growth fibroblast from adipose tissue. In brief, I digest the tissue with collagenase, filter it through 250micron nylon mesh, centrifuge and wash 3 time with dmem/mcdb131 (Gibco) +10%FBS , then platte in 24 wells plate.

There is just a little bit of fibroblast that growth on the side of the well and there is a lot of dying or dead cell in the middle of the well. I thought the dead cell were blocking the growth of the fibroblast, so I trypsinized the cell to detach all cell from well and was hoping that only fibroblast would re attach and that dead cell would be wash in the next media changing. It partially work. Fibroblast growth more but there is still too much dead cell (Less then before though)

So I wanted to filter the cell throught 40micron nylon mesh before centrifuge/wash step. But the problem is that the media with the cell dont passively pass throught the 40micron mesh. How can I filter throuth that small mesh? What kind of device would be able to hold tightly my filter (I have a sheet of nylon mesh that I cut to the desire size) so I could apply vaccum ???

any suggestion??

Thanks
Christian

hi
40 micron filter would retain all the fibroblast so i don't think that's a great idea.
i think it would be good to try a trypsinisation (if you think it's a hard treatment try a 1/5 dilution of trypsin in PBS) before platting cells...
You can also try growth in 15% FBS.

hope tha helps you.
Keep us informed.

Hi

Fibroblasts are pretty fast growing cells, I would suggest just digesting the adipose tissue, or just chopping it finely (see Freshney: Cell Culture text) and plating resulting bits onto 6 well plates , and leaving for a few days. After this you should be able to see outgrowth of fibroblasts from the adipose tissue. At this point trypsinise the wells so that the fibroblasts lift off, but not the rest of the tissue lumps and proceed from there. After a few passages (2-3) you should have reasonably pure cultures of fibroblasts.

Good luck
Bob