Dear Marcia,

to my mind, there are 2 cell populations in these pictures: epithelial (pavimentous) and fibroblasts-like (elongated). I usually work with skin explant culture where I have alredy seen such phenomenon. But after the first passage, epithelial cells do not survive in DMEM.

Regards,

Gwen.

From their appearance in your picture I would say they are smooth muscle cells (SMC). Since the Oviduct is surrounded by a thick muscular layer (tunica muscularis) it is common to get SMC as a contamination. You could use alpha-smooth muscle (alpha-sm) actin as a marker to verify it.

As for a protocol it would be good to know beforehand what you are doing right now to isolate them and what you have already tried to get rid of the contamination.

Thank you.

To isolate the fibroblasts I am incubating the oviducts in Trypsin-EDTA 0.1% for 90 minutes, than I squeeze it to remove epithelial cells, open the oviduct, wash to remove remaining epithelial cells. And proceed in 2 different manners:

1. squeeze it harder with a blader and collect the cells

2. cut pieces smaller than 1 mm3, digest using collagenase type I for 30 min and filter to remove non-digested tissue.

In both ways of isolating the cells, I have a huge epithelial cell contamination, some fibroblasts and this other kind of cell.

I've tryed different trypsin treatment times, to try to pass the fibroblasts (are supposed to deattach faster), but I still have epithelial cells on it.

I also tryed to culture small pieces of oviduct, but again I just have few fibroblasts.

You will most likely always get just a small portion of fibroblast, since there content in the tissue is rather low.

I would have suggested trying a sequence of detaching methods, but you already have done this with trypsin. Have you tried detachment with just PBS/5mM EDTA (5-10min, 37°C)? It works to get rid of some fibroblast-contamination in primary epithelial cells cultures of bovine placental cells. The fibroblasts will detach, while the epithelial cells will not.

Sometimes it is also possible to use the attachment rate (which is different depended cell-typ and substrate (plastic as standard, but you can also try fibronectin- or collagen-coat)) directly after isolation. Therefore, put the cell-suspension in a culture flask and incubated it for 30min-1h (incubator), take it out, shake it gently and transfer the not-yet attached cells in the new flask. Then wait again and repeat the whole process over and over again. Fibroblasts should be the first to attach.

Are you using any marker to be sure, that you have at least some fibroblast in your culture directly after isolation? SMCs tend to be quite difficult in culture, since they change their morphology depending on cell density and age. When they dedifferentiate in culture and they often look like fibroblasts.

What medium are you using? Fibroblast are sometimes susceptible to high glucose (it can arrest growth) so be sure not to use high glucose medium. (My bovine fibroblast growth fine in DMEM/HamsF12)

Thank you very much Nina,

I am using DMEM/F12 10% FBS

I haven't stainned these cells yet. I thought that I could define it by morphology, and then I had this third kind of cells...

I will try the PBS-EDTA and the attachement rate, and also save some to immunostainning. Thanks for the tips :)

Have you tried to cut the tube into small segments place the segments luminal side up on coated dishes and left the fibroblast grow out? It takes about a week.

I've tried small pieces, but luminal side down and non coated dish. I will try it, thank you!

Hi there,

I can definitely see fibroblasts in the pictures but am not sure about the nature of the other cells. As regards a protocol, I would suggest to chop the tissue into as smaller pieces as possible, put the pieces on the bottom of dry plastic flasks, incubate it for 3-4 minutes and then pour in DMEM+FBS+PSF. In low glucose DMEM usually after 2-3 passages you should not see epithelial cells. You can also look for "fibroblast-selective media" if there is any available. You can test for the fibroblastic nature of your cells by measuring their alkalkine phosphatase levels and/or doing a pcr for fibromoduline as a marker for fibroblast.