6 replies to this topic

[yjp]

my MRC-5,normail human fibroblast cell were detached from the flask suddenly after it almost confluent,I can exclude the reason of culture medium and trypsin,because at the same time we culture other two flasks cell,they grow normal.

[franck]

the  basis of cell could be dead then there is no fixation point, it's probably a reason

[Sean Peel]

In my experience with a variety of connective tissue cell types, once cells reach confluence it is not uncommon for certain cell lines or primary cell types (I have no experience with your particular cell line) to detach from the dish bottom and for the cell layer to roll up.

If you were to leave the flask the detached cell layer will have cell outgrowths. (This is the best way to confirm that the cells are not dead.) It has even happened to me that the entire cell layer has rolled up into a ball !
However, once again if you let the ball settle you end up with a cellular out-growth.

I cannot say what it does to the phenotype of your cells, but my best advice is to subculture at between 80 and 90% confluence to prevent this from happening.

[yjp]

i think it is better for me to describe the details of what happen to my cell.the cell grew well after I subcultured it ,it almost confluented on the third day,but at that time ,I saw some spots dispersed on in the flask,meanwhile,the spots looked like 'flowers',then dispersed 'flowers' began to detached from the flash,at last,all the cells detached.
 P.S. i still don't know whether the cells still live or not.because the culture medium became yellow,and I can't see any difference of the cells under microscope.maybe just the materials sent out by the cell after it died that tured the medium yellow.

[Sean Peel]

Its hard to say what is happening based on your description. Some possibilities include:

1) an infection - possibly bacterial (generally the medium will become cloudy if this is the case) or mycroplasma (you can't see these and often they don't kill cells just make them behave abnormally). To test for mycroplasma you can get a staining kit from Sigma or a PCR kit from ATCC.

2) The cells were not feed for a prolonged period

3) ??? its just one of those cell culture things that happens sometimes.

Sorry I don't have any better ideas.

[yjp]

hello,sean
 thank you for your help.I am a new comer in cell culture,so there are so many difficulties i meet.I still have one question,do you have any experience in synchronizing fibroblast cell in each cell cycle?

[Sean Peel]

I have no experience with cell synchronization personally. However, I believe that one way is to serum starve the cells for a period (24 hrs ?) the cells all gather in g1/g0 and then can be returned to the cell cycle by restoring serum to the medium. Please check the details before doing this.

I also remember that you can collect some cell types in the M phase because they round up and are poorly attached to the substratum. You shake the medium to dislodge them and then recover them from the medium by centrifugation. I don't know how good this is though.

If you have any general questions on cell culture you are welcome to e-mail me at seanpeel@yahoo.com. I have 12+ years experience with cell culture, mostly primary bone, cartilage and ligament fibroblasts but also with various connective tissue cell lines.