Hi Kelly,

In our lab we are using SH-SY5Y cells for PD research. N27 rat dopaminergic cell line is another choice of ours. However, I think, one of the best options you can use is the primary midbrain cultures isolated from rat embryos. We dissect midbrain from E17 rat embryos and culture the cells. It's, obviously, a mixed culture including dopaminergic and GABAergic neurons, as well as astrocytes and microglia. You can also inhibit the growth of glial cells in cultures dishes via different treatments if you wanna eliminate these. Since it's a mixed culture, you can see the effect of your treatment/chemical/compound on different neuronal or glial cells, which might be an advantages depending on the purpose of your neuroprotective assay.

I hope this helps a bit. Good luck!

Hi Kelly,

I have worked with SHSY5Y, N27, N2A and PC12 cells. SHSY5Y differentiation using RA always resulted in a mixed population with rather low efficiency, and I wonder whether that would affect the outcome of your experimental results. N2A cells and PC12s give rather uniform differentiation and both are Dopaminergic. PC12 are more troublesome to work with forming clusters in the plate when not differentiated. 

@ Zeynep Sena Agim, 

Thanks for the info. Any advice on doing primary cultures? And are these WT E17 strains? I'm entirely inexperienced in the murine model realm.

@Navneet Ammal Kaidery,

The mixed population could definitely be a problem. What line would you work with simply out of easy of use? which would you work with because of the highest level of application/reproducibility?

Thanks a ton for the help folks! it's greatly appreciated

Yes, we use wildtype rats. It requires training and practice to get the brains and dissect the midbrain. 

Check out this paper and cited articles in the Methods:

http://toxsci.oxfordjournals.org/content/140/1/179.long

It is not exactly how we do it, but I thought it might help you get a broad idea:

http://www.jove.com/video/3691/dissection-culture-mouse-dopaminergic-striatal-explants-three

Hi,

you can also try organotypic slices wherein all different cellty pes are organized and preserved.

See these references:

J Neurosci Methods. 2006 Oct 15;157(1):1-9. Epub 2006 May 15.

A method for a more complete in vitro Parkinson's model: slice culture bioassay for modeling maintenance and repair of the nigrostriatal circuit.

Kearns SM1, Scheffler B, Goetz AK, Lin DD, Baker HD, Roper SN, Mandel RJ, Steindler DA.

Neuroscience. 2014 Jan 3;256:10-22. doi: 10.1016/j.neuroscience.2013.10.021. Epub 2013 Oct 23.

Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease.

Daviaud N1, Garbayo E2, Lautram N1, Franconi F3, Lemaire L1, Perez-Pinzon M4, Montero-Menei CN5.

Hope it could help!

Obviously since my laboratory years ago did a lot of characterization of the RA/TPA differentiation of SH-SY5Y cells, I favor using those cells. We always got high yield and felt that there was a reaons to utilize human cells vs non-human cells. Any of the immortalized cell lines are going to be different from slice culture or primary cultures.

Fantastic! thanks to everyone that answered, i hope i can successfully apply your advice!

I can probably add a little in this thread and learn know something new.

I have recently started working with N27 cells and I have lots of characterization to do before I can say that they are dopaminergic. Papers from the Labs which developed this cell line have published several articles validating it as a very good model for DA neurons. However, I am always skeptic unless I analyze these cells myself and characterize them. If you do literature search on all these cell lines (SH-Sy5Y, MN9D, PC12, B63, N2A etc.) you'll find that there is a divided opinion on their validity as a DA cell line. In my opinion I think that one can be a little better than the other but it would not be fair to call one of the cell lines as the best in-vitro model for DA neurons. Also, if a cell line is expressing, DrD receptors, tyrosine hydroxylase or DAT does not mean its enough. There are many other factors that needs to be investigated like extracellular/intracellular DA levels, levels of homovanillic acid, extracellular/intracellular NA levels and also uptake of NA or DA. I am sure there are many more assays which can be done to further characerize the DA like properties of these cell lines. Another problem with these cell lines is their homogeneity. From my short experience of working with N27 cells I can see some heterogeneity in the cells (differentiated as well). I am saying these just from visual inspection but not from any analysis yet. Therefore, it becomes tricky to work with them depending on the nature of question you are asking. For toxicity studies, I am assuming the whole population of cells in the culture  will be affected by your treatment and results will be a a difficult interpretation.

Just like what others have recommended, using acutely dissociated culture is better than using a model cell line, provided you are able to make the primary culture survive for atleast three weeks without any contamination and cell death. It takes lot of practice to get there and even much more tricky if you are using postnatal animals. Embryonically derived cultures tend to survive easier than postnatal ones but postnatal ones are better models. Also pinpointing DA neurons in the culture is really tricky , therefore, using a transgenic line with GFP or some other label expressed in DA neurons will be the best way to go.

We sequentially treat SH-SY5Y cells with RA and BDNF, which induces cell differentiation accompanied by growth retardation and the robust neurite formation. 

https://www.ncbi.nlm.nih.gov/pubmed/15196967

Dr. Hasegawa,

SH-SY5Y differentiated with RA and a phorbol ester seems to be the current standard. My initail interest was in trying to find an assay that didnt involve differentiation, as a means to reduce variability in results and the potential for confounding factors.