fiborblast contamination in primary culture - (Nov/27/2006 )

Hai Friends,

I am inolved with primary culture of epithelial cells of tumor origin. Now i am facing a major threat of fibroblast overgrowth in my culture. Initially i was using DMEM+Serum free keratinocyte medium in that both epithelial as well as fibroblast growth were minimal . But now when i Use DMEM+Hams F12 medium, epithelial cells seems to grow but fibroblast outgrowth is maximal and with an aggressive mode. I tried differental trypsinisation methosd and i see again fibroblast growing in culture .can any one suggest a good method to get a culture devoid of fibroblasts

Thank you
Anand

Do you do gravity separation to separate the epithelials and fibroblasts into different layers before culturing them...if u dont u might wanna try that..it works well for my labmate!

you might have to purify epithelial cells by cloning ring/trypsin treament method.

follow older discussions such as

http://www.protocol-online.org/biology-forums/posts/3572.html 1://http://www.protocol-online.org/biol...sts/3572.html 1://http://www.protocol-online.org/biol...sts/3572.html 1://http://www.protocol-online.org/biol...sts/3572.html 1

Hai,

I am happy to get the responses from those who working in the same area. But i am unaware of how to do gravity seperation as well ring/trypsin cloning method.Can you brief me the protocol as still i am facing the problem of fibroblast contamination in my culture

thanks once again for the quick response
Regards
Anand

I think gravity separation refers to differential adhesion separation (or I can be wrong?), meaning you seed the cell suspension on to a plate, let it set for 10-20 min or shorter, then transfer to a fresh one. Fibroblasts tend to stick to the plate faster than other cell types. You repeat this several times, you may be able to remove some of them. Cloning ring is a plastic or glass cyrinder (you can get it from vendors such as VWR or Fischer, youcan also made yourself by cutting off a 1 ml pipette tip) you spread some grease at one end, push and seal it to the plate. This can give you a small well to allow you to add trypsin-EDTA solution to lift cells. If you seed cells at a low density, cells can form colonies without other contamination nearby and you can isolate colonies this way. Afterwards, you expand from your isolations and grow them up. Be careful some cells need certain cell density to replicate. You can use both methods together.

Hai,

Thank you for the quick response. I have a population of cells which is very much similar in epithelial cells like morphology, but upon trypsinisation and seeding, its changing its morphology to a clear cut fibroblastic pattern of growth. I dont know whether i can attach photo in this forum. can you please give me your personal ID so that i can send the pictures so that you can comment on that.

Regards
Anand