giant cells? - (Oct/15/2008 )

Hi all,

Im growing some HeLa cells in 10cm dishes and T-25 flasks. I usually split them 1:10 or 1:5 from 70-80% confluent plate but i noticed many giant cells in my culture. I know giant cells may appear once a while but there are just too many to be normal.

Can anyone enlighten me on what have i been doing wrong?

I culture my cells in DMEM (Gibco) with 10% fcs and supplement with L-glut.

Thanks.

Jing

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When a monocyte enters damaged tissue through the endothelium of a blood vessel (a process known as the leukocyte extravasation), it undergoes a series of changes to become a macrophage. Monocytes are attracted to a damaged site by chemical substances through chemotaxis, triggered by a range of stimuli including damaged cells, pathogens and cytokines released by macrophages already at the site.
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thanks for your input but im not using macrophasgs or monocytes

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Dear Jing,

Large cells normally appear when they are in trouble. This could be due to a number of factors:

i) Some cells have defined passage ranges and will deteriorate over time. You need therefore to go back to your masterbank of cells and initiate a new vial. I have to say that Hela's should have a longer passage range than most cells.

ii) The dreaded contamination: this could be just a subtle, low level contamination, either bacterial or fungal. If you use antibiotics then this can be disguised. Mycoplasma contamination will NOT affect the media colour or be visible to the naked eye.....test cells regularly.

iii) Media variations/Serum variations: This is relatively uncommon as media is produced by companies with good records for GMP. HOWEVER, low quality serum will also affect the cells greatly.

iv). Over Trypsinisation: this is common as well. If you leave the trypsin on too long...or...use the wrong concentration, the cells will suffer.........the large cells you see are cells that are just hanging in there and are probably apoptosing before your very eyes.


These are just a few ideas, hope this is more useful to you than the previous response.

Kindest regards.

Rhombus

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Thanks for the advice. I actually got these cells from a colleague and his cells are all good. We use the same lot of media and serum. I think it's something that i have done, maybe over trypsination. I usually trypsinize them between 2-5 mins at 37 deg.

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Dear Jing,

I think that's where you maybe going wrong. I have grown hela's in the past. I use 0.25% Trypsin/0.05% Versene at Room Temperature for 30-60 seconds. If you use trypsin for too long, it will be internalised and therefore will degrade intracellular proteins....and definetely cause apoptosis.

Regards.

Rhombus

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Usually, large cells indicate senescence, and will frequently go on to apoptosis, though not always. There isn't much you can do about it when the cells have senesced, the rest of the cells in the culture should be fine, though in theory you should discard the culture as you may have just selected out a sub-population of cells.

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