Greetings
I have plated 50,000 fibroblast cell/ml in a 60 mm dish & around 5000 cell/ml in a 12 well plate, since about 5 days.
I'm monitoring them & they still didn't attain a monolayer yet. They are growing slowly.
I'm not sure if the seeding density was initially low. Can anybody suggest a solution for this?
And for how long can i wait for them to attain the monolayer before they approach senescence???
Thanks in advance
More info would be helpful. What is the source of fibroblasts, what is the composition of your growth medium (and added volume), and what sort of doubling times are you in fact seeing, approximately? For future expts, you might want initially to titrate the density in a smaller format (e.g. 96-well plate) and see how that goes before scaling up - also remembering that keeping the volume of added medium on the low side will allow helpful growth factors to build up.
Hello Jane
My fibroblasts were purchased from the ATCC, it is derived from the foreskin of a newborn male. I cultured them in low-glucose DMEM, supplemented with 10% FBS and 1% streptomycin, penicillin, fungizone mix.
Thanks for your help
Well... just looked that up on ATCC and they use quite a few supplements (e.g. insulin) for these cells, although I was guessing at exactly which type you purchased (see "Culture conditions" for your cells - I looked at http://www.atcc.org/products/all/PCS-201-010.aspx#cultureconditions). Especially if you put them straight from frozen into your medium, they may be in the process of adapting to the change, or they may just not do so well in it. Considering these primary cells have limited potential for doubling anyway, you might be better off going with what is recommended if you can. Also, fungizone is pretty toxic so unless you have good reason (hopefully not!) for adding this you might exclude it.
Good luck!
I culture Foreskin fibroblasts and I would seed 200,000 cells into a well of a 12 well plate and the next day they would be about 80% confluent. To give you an idea about how long it takes for fibroblast to divide I would split a confluent flask of fibroblasts into 2 flasks and it would take 1-2 days to be confluent again. Fibroblasts don't like to be too thin in their monolayer so I wouldn't split them more than 1:3.
The numbers I use for general planning.
12% confluency is 8-11K cells/cm2 and would be the minimal I seed at and will require splitting in 5 days.
100 % confluent is 60-80K cells/cm2. 100% confluency is hard to see with fibroblasts because at around 80-90% the cells start to form 3-d structures.
Hope this helps.
Is it necessary to reseed fibroblasts when they form monolayer or I just can change the growth medium that they will stay alive? I want to avoid unnecessary division.
I recommend you to ressed or to freeze.
Since I had have only changed the medium, after complete confluence was got, I became to see many cellular debris nas some breaks in my bottles.
Thank you very much! Would it be okay if I will use mixture of 8%Dmso, 15%fbs and 77% DMEM growth medium for cells freezing?
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