I am trying to culture fibroblasts from the skin of Black 6 cross mice. I have tried releasing cells with collagenase, dispase, or just culturing explanted fragments (cut with scalpel or scissors) with or without a cover slip over them. I have tried DMEM (low and high glucose) and DMEM/F12, with FCS or with heat-inactivated FCS, and serum concentrations of 10%, 15%, 20%, 30%. I have tried naked plastic, or plastic coated with gelatin or Type-I collagen. I have tried plastic from several manufacturers. In all cases I get a few cells which look OK, in some cases go through a number of divisions forming small (a few 100 cell) 'colonies', but then slow, flatten and 'senesce', and die. Many of these combinations have worked fine with human skin and muscle explants in the past, giving healthy cells that grow for 40 - 50 doublings. Any protocols, and especially 'don't do' lists which might point to what is going wrong, would really help. Thanks.
Many thanks again for your help. I have got mouse cells to grow. I used explants as above, without cover slips, I did not worry if they were the right way up (could not tell!), did not use enzymes to dissociate the tissue, I just let the cells migrate out. Trypsin did release more cells, but they did not seem viable (may have to work on that). I tried various processes for handling the tissue before culture - shaving hair off (good idea), mincing it (bad idea), cutting into small pieces with a scalpel (best). Optimal was putting the fragments in a 60mm dish with ~0.5ml of medium. More allowed them to float around (bad), less was prone to dry out. Top them up with another ml of medium after 3 - 4 days. As Prof Torday said, the lot of FBS was critical - one of three worked well, one poorly, one not at all. Also coating the plates with collagen - I used 3ug/ml rat tail collagen in 0.2% acetic acid, leave on plate for 1-2 hours at 37oC, take off surplus and air dry. Once established the cells grow OK on this, very poorly on 'bare' plastic. (I have not tried gelatin yet). I tried 10%, 20% and 30% serum in the initial culture, that did not make an obvious difference - serum batch was much more important. Hypoxia is not essential - I used 5% CO2 and normal air, but they are looking a bit stressed and senescent so this may be the next step. So far I have split them 1:10 four times and they are going strong. I do notice that they need medium changing more often than the human fibroblasts, turning high-glucose DMEM very acid (yellow) if left more than 2 days.
So - thanks again, gentlemen. Best wishes, William
Hi,
In my experience, you got better results with the explant method. I added FGF-2 (4ng/mL) to DMEM/F12 with 20% FCS. Once I passage the cells I switched to 10% FCS. I cultured them on home-made gelatin coated Petri dishes. Use explants of at least 2mm diameter whenever possible, I know it depends on the anatomical site of sampling.
Good luck and tell us if you succeded.
Hi William,
in our experience, we use the explant method with good results. We make small pieces of the tissue (around 1mm3) in Petri dishes and then we use colagenase type I (about 30-40min) and the trypsin (about 15 min). After that, we put DMEM (with 20%FBS), just sufficient to cover the bottom of the dishe. It is important to let the pieces attach and in the next day remove those do did not attach to the plate. Also, try to put the fragments in the correct position (dermis up).
Hope it help you.
Best regards,
Hi William,
I had the same experience as you, recently while culturing mouse dermal fibroblasts. The real issue with senescence is because of the oxygenic conditions rather than the media or plastic. The protocols to culture mouse dermal fibroblasts (specifically) recommend hypoxic conditions i.e. 3% oxygen rather than 20% oxygen ( atmospheric oxygen in a usual cell culture incubator). This resolved my issue and I was able to passage the cells at least 5 times. I had availability of incubator at 4% oxygen levels that worked for me.
Hope this helps,
Good luck.
In my 50 yr experience culturing lung fibroblasts, trypsinization is the best method, and the amount of fetuin in the FBS is critically important for differential adherence to tissue culture plastic (it should only take 30 min to an hour for the fibroblasts to differentially adhere to the plastic surface). The 'adherence variable' is critically important and is a function of the 'lot' of FBS you purchase from the vendor. The independent variable is the price of veal! which I have assumed affects how young the calves are when they are slaughtered..... Just as an aside and fun fact. That aside, my practice was to request several different lot samples of FBS from the vendor and determine empirically which lot is best and then buy as much as you can and freeze it!
Many thanks to Benjamin, Carlos, Rochan and John - all really useful tips. I had heard the issue of FBS 'lot' before, but not realised that it made such a difference.
For trivial financial and logistical reasons the project has been on hold for a couple of months, but when I return to it I will record here what I found. Again, thanks for your input.
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