No experience with keratinocytes - (Jan/19/2006 )

Dear all,

First of all, excuse me for my bad english (I am from the netherlands). At the moment I am having my workplacement periode at a technical university. Part of my research is to discover how urine causes skin damage. Before I will use skin equivalent EpiDerm, I need to ' practise' with a monolayer keratinocytes. I have no experience with cell culturing at all. Do you know which medium I have to use? I have read something about calcium free medium to prevent differentiation. But to be really honest, I don't know if I want my cells to differentiate??!!. Do you have got ideas? I want to perform some viability assays in presence of (synthetic) urine.

first of all, when they are growing in a confluent monolayer, this is their morphology (although under our inverted scope they look pale pink/yellow)
http://cellapplications.com/images/HEK.jpg

some good references for you can be found on www.cascadebiosciences.com, also www.cambrex.com

we feed ours (primary human keratinocytes) serum-free KGM, with supplements and antibiotics. note that they are very 'sticky' when you go to passage them. if you increase the calcium they will undergo terminal differentiation (the standard KGM is typically 0.15mM). if you let them become >90% confluent they will usually become senescent. if you seed them too thinly they will not establish a healthy monolayer, they are happiest when they can signal to each other and 'find' each other in their ECM.

we get all our stuff from Cambrex and it works well. they have some good information and technical support and I would recommend them to anyone who wants to work with this type of cell

I'm learning how to culture canine keratinocytes. I use using williams medium E supplemented with epidermal growth factor, cholera toxin, L-glutamine and antibiotics. I have been told that using media with a low calcium concentration reduces fibroblast contamination. So I am planning on using a decalcified media when I passage my cells.

Thanx for your answers. Is it a good or a bad thing that my keratinocytes will differentiate? I want to use them for the in vitro screening of the damaging potential of urine. Is the standard KGM medium ok?

after passaging into 24-, 6-, or 96-well dishes, depending on our experiment, we grow them
in KGM medium. after they hit ~80% confluence, we up the Ca in the medium to induce differentiation. we think a differentiated monolayer will act more like the 'real skin' keratinocytes in the epidermal layer

we up the Ca for about 3 to 5 days. you can test for differentiation using involucrin as a marker; there are other published methods (they still look the same

you can differentiate by other means, different chemicals and vitamins (D, for one; there are others) if you would like to scan the literature you should be able to find a protocol suited to your experimental design

if you just grow them up in regular KGM, they will not differentiate (the 0.15mM is not high enough. you can't grow them too long with 0 Ca or they will not be happy cells; Ca is required for all sorts of signalling events) they will undergo senesence, unless you passage them...and after a number of passages they will undergo senesence anyways as the cells go through their normal life cycles. this is, of course, for primary keratinocytes; for immortalized lines I believe this is different and you can get tons of passages? with the primary keratinocytes we use (HNEK) this is how it works; we typically do our experiments between 2 and 5 or 6 passages and toss them when they start to look a little old and flaky.

as far as your studies with urine, I'm not really sure if this is the best medium? I believe they will grow in other types. have you found any protocols or anything from other people doing similar experiments?

Thanx for your answers. I haven't found any literature about the same experiments. What I am going to do now: I let the keratinocytes grow in standard KGM medium untill they reach 70-80% confluence. Than I will subculture the cells and when they are 70-80% confluent again, I will do my actual experiment:

I let the cells grow (24hours) in KGM media with different adjusted pH levels. I will determine cellviability by MTT assay.

I hope my cells become less viable due to differences in pH.