Keratinocyte Cuture conditions - (Jun/30/2008 )

Hi,
I am an immunologist working with a dermatologist and know little about skin cell biology. We are treating keratinocytes derived from foreskin in tissue culture conditions with a purified protein. We do not use serum in the media. The keratinocytes change morphologically with this treatment, but without the background, I'm not able to guess at what the morphological change means. We have induced apoptosis and that looks distinctly different. With this protein treatment we get elongation, lack of proliferation, lack of migration, but the cells remain adherent and tend to clump. Any ideas as to what we might be observing?
Thanks for you help.

I was working with keratinocytes a few years ago - not easy. I remember that they were fussy when it came to serum, either they do or don't like it (can't remember which one) and they hate when you use the other condition so stick with the one that works. Sounds like it's the serum they like, if background is an issue can you do a negative control?

The morphology change is differentiation and cell death. This is what they are programmed to do and it's one of the issues with keratinocytes. If you think about their role in the body (to form skin), they start off as normal cells in the basal layer and their destiny is to move up to form the skin layers and eventually become the hard dead elongated flat squamous cells needed to form the protective outer layer of the skin (this all happens within 14 days i think). So the elongation you see is that progression from round keratinocytes to flattened skin cell layers. The lack of migration is because they don't need to migrate as much as they get to the surface of the skin. And the lack of proliferation is because they are destined to die as they differentiate (apoptosis), so they don't want to proliferate. All this makes keeping keratinocytes in that keratinocyte state difficult.

With regards to the clumping, this is how they grow. They grow next to each other presumeably because they feed growth factors to one another (not sure what biological purpose this serves). They certainly depend on one another to grow well and don't like it when they are too far apart. For this reason, probably the best advice i can give to you is that when you split them, split them lightly. That way, when they are single cells they are never to far away from a neighbour and re-establish a lot better and keep their morphology (that pointy star-like shape) and growth characteristics a lot better. Sometimes i would trypsinise them and literally put the whole lot back into the same flask, just to make sure they were close to one another.

They also get contact inhibited though so don't let them get any more than 70% confluent because that will stop them growing completely. They will differentiate in the middle of those clumps, which is that death/flattening program in action, so watch out for that because you want to keep them in the keratinocyte state.

So the best way to keep them going as long as you can is to split them frequently (every 3-4 days) to prevent contact inhibition and split them lightly to keep them close to one another for growth. I used to change the media every two days as well to keep them happy. They are fussy so you need to do that. The idea is that you want to keep them 30% confluent once you've split them to keep them close to each other for growth but split them once they get close to 70% (and not a day longer!) to prevent contact inhibition and differentiation. You'll never prevent differentiation completely (some accumulation each passage) but you can maintain the poulation of keratinocytes well and pleasingly keep them growing for maybe 10 to 15 passages for primary cells (they are destined to die so you will only get this many passages). Be warned though! One slack day where you can't be bothered looking after them is enough to pay the price and say good bye! Unfortunately, that means at least one day in on the weekends but if you look after them they will look after you.

Bet you weren't expecting that reponse! Hope it goes well.

Good luck, Rob

Forget your experiment, publish this somewhere!

Thanks for the replies. Because I am trying to assay the effect of a particular protein on keratinocytes, I do have a non-treated control that seems to like the culture conditions in which we are growing them. The treated cells are the ones elongating and appear to be flattening. Can you suggest any molecular markers I might be able use to clearly define that what I am seeing is normal terminal differentiation?
Thanks again!

Did you coat your plate with fibronectin, collagen and albumin? If you didn't the cells will not spread out.