Tips on growing fibroblasts - semi-urgent! - (Oct/24/2006 )

I hope someone can help me! I've just started a new job last week and have been put in charge of a cell line which is patient derived fibroblasts. They have none frozen down and apparently this is the only source of DNA for this patient....so no pressure, eh!? I've also been informed by my boss that another patient derived fibroblast cell line is coming for me to look after in the next couple of days. They don't do much cell work and the cell facilities are quite disorganised so I am trying to sort those. I also have no experience growing these types of cells. My background is virology and we were using typical veros, bhk, hep-2 etc.

Does anyone have good tips on growing these cells? I also need to freeze them down and was thinking of using 5-10% DMSO plus 10% FBS in BME (this is what they are currently being grown in). Any suggestions welcome. They don't have the big book of all there is to know about cells here so I am at a bit of a loss, to say the least.

Thanks much,

Rachelle

Thanks for your suggestion, that sounds like a good place to start. Boss is not a cell biologist so he doesn't know!

Rachelle,
I'd say up the FCS % in your freezing medium to 50% with 40% complete medium and 10%DMSO. Would making a lymphoblastoid cell line by immortalising B-cells from the patients with Epstein-Barr virus be a better source of patient DNA? The fibroblasts will become senescent (30+ passages) eventually so it's worth freezing down alot of early passage cells. I think people using primary fibroblast for experimental purposes use them at early passages to be sure that they're not measuring artifacts.

Ceri

I cultured fibroblast like synoviocytes from patients.
I only used them between passage 3 and 10. early passages : cells were contaminated with other cell types, and later passages were no more used, because they could start to be senescent.
So I froze as many cells as possible at passage 4 to 6, to be sure to have enough stock.
The cells were frozen in culture medium containing 10% FBS plus 10% DMSO, in an isopropanol box at -80°C for 24 hours and transfered in liquid nitrogen.

Thank you all for your suggestions. The immortilization may be something to think about. I am not 100% sure what they intend to do with these cells since it is not my project and I've only been working here 2 weeks now. Last week I attempted to concentrate some of the flasks of cells because they had been split v v v v v thinly by my colleague. They were not growing and did not have enough neighbouring cells to form cell-cell contacts. Unfortunately since then I think they've become contaminated (see my post above!) so I'm not sure what to do with them.

Something else I had forgotten about was immortalisation of fibroblasts using a retrovirus expressing hTert (telomerase). A group in the department I was in used this technique to immortalise some fibroblasts.

Ceri