Dear Nina,

The information that your fibroblast are from human aorta is a bit too sparse. From which histological region were they isolated, adventitia?

What type of activation are you looking for?

Would you like to know if they are in a quiescent or resting stadium after isolation, purification and re-culturing on TCPS (tissue-culture treated polystyrene) in what? Are you using a commonly used media supplemented with FCS and etc. or are you following a "better" or "more suitable" protocol for "specialized" human aortic adventitia-derived fibroblasts from a healthy young donor??

If the fibroblasts are normal activated fibroblasts (NAFs) they should or will show an increase in alpha-SMA and vimentin expression which goes along with a change in morphology: spindle-to-stellate-shape transformation;

If the cells were isolated from a fibrosis or ….diseased patient/donor you may/will have differently activated fibroblasts, e.g. fibrosis-associated fibroblasts (FAFs) or cancer-associated Fibros (CAFs).

The problem is that the different activated fibros as such are usually identified by investigating/analysing histological material. The whole process of isolation and culturing by applying common in vitro conditions leads/ may lead to stadium changes (or de-/transdifferentiation). That is the reason why we and many others (e.g. in the cancer research field, lung-fibrosis research field etc.) started longer ago to work on 3D culture methods.:-) Hope my questions, thoughts and notes will help you.

Kind regards,

Jochen

Hi Nina,

Using fibroblast-specific proteins as markers is probably the most viable and reliable option. There are several markers for fibroblast activity, as far as I know FSP-1 and vimentin are the main ones. You can find out more about them in the following article:

http://jcb.rupress.org/content/130/2/393

There is also another question on RG that might be interesting for your purposes: https://www.researchgate.net/post/IHC-fibroblast_staining-what_marker_to_use

Hope this helps!

Cheers,

LG

Dear Jochen and Lukas,

Thank you so much for your input! I was thinking my explanation was too little, I'm completely new to cell culture and am just in the initial planning phase of an experiment. We are planning on buying human aortic adventitial fibroblasts and essentially incubating them in this liquid we have in the freezer and see how/whether the fibroblasts react - whether they die, show abnormal growth etc.

Sorry, the explanation is probably very poor! But your ideas helped a lot, I will read that article and forum and take a closer look at vimentin, alpha-SMA and FSP-1!

Kind regards,

Nina

1. https://www.promocell.com/product/human-aortic-adventitial-fibroblasts-haoaf/ Promocell as a good and reliable source for cells. They are quite expensive, but we made good experiences.

2. Volume 56(4): 347–358, 2008

Journal of Histochemistry & Cytochemistry

http://www.jhc.org Nice article and a little bit about things I mentioned in my previous email.

3. Circ J. 2016 October 25; 80(11): 2269–2276. doi:10.1253/circj.CJ-16-1003. Defining a cardiac fibroblast / Good article.

"incubating them in this liquid we have in the freezer" Are you and/or your supervisor sure?? Hmmm, sounds very strange. Smile

I would like to recommend the following article

Chapter 16

Isolation and Culture of Cardiac Fibroblasts

Asish K. Ghosh Joseph W. Covington Douglas E. Vaughan Book Editor(s): Hossein Ardehali MD, PhD, FAHA Roberto Bolli MD Douglas W. Losordo MD First published: 20 December 2013 https://doi.org/10.1002/9781118495148.ch16 Cited by: 1

Finally, it is not a question of "philosophy" or "taste". And it is not really effective to say that many different ways lead to Rome. You should not take for your very specific question or approach an undefined "liquid", e.g. DMEM or RPMI.... Many colleagues/researchers already went this very hard way. So, best is to learn from their experience and not to waste time and money!!

This all is just for free!!

Good luck!

Kind regards, Jochen Salber

Sorry, I forgot s.th. very important to say particularly for beginners

You will read very often things like the following

"Fibroblast culture conditions,

This protocol can be used for any primary fibroblast culture. Examples of cell strains (all from Coriell) grown using this protocol include: Source Name Refered to as Tissue Source Gender AG20443 PF43 skin fibroblasts (71yo) male AG08395 PF95 skin fibroblasts (85yo) female AG08396 PF96 lung fibroblasts (85yo) female

Fibroblast Culture Medium: Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, cat. no. 11960) supplemented with 10% Fetal Bovine Serum (FBS; Invitrogen,), 2mM L-glutamine (Invitrogen, cat. no. 25030), and 0.1mM (0.7µl/100ml final media volume) 2-mercaptoethanol (Sigma, cat. no. M7522). Antibiotics can also be added at final concentrations of 50 units/ml penicillin and 50 g/ml streptomycin (Invitrogen, cat. no. 15070). Fibroblast Culture Medium is filter sterilized, stored at 4°C, and used for up to 2 weeks." It could work in terms of cells will survive and adapt somehow to the conditions and this works particularly in the case of fibroblasts of different origin and species very well, because of their enormous plasticity. But specific cell homeostasis including behaviour, metabolism, survival and stable proliferation under defined and microenvironment-adapted conditions needs to be taken into account.

Meanwhile we know much more from Proteomics and Transcriptomics and generalised statements are not good.

That is all you should keep in mind.

Dear Jochen,

Thank you again for all your help! We were indeed thinking about purchasing the HAoAF from Promocell, and the mysterious liquid that I mentioned is thrombus material from patients. We will plan this out carefully so your help is much appreciated, considering the jungle of information out there!

Best regards,

Nina