I am trying to culture primary neuronal cells and am a bit confused by the many different media formulations. Does anyone have experience culturing neuronal cells and if so, what media do you use to keep them alive?
What you need is BrainPhys. It is a defined and serum-free neuronal culture medium for the culture of central nervous system (CNS)-derived neurons and human pluripotent stem cell (hPSC)-derived neurons. Based on the formulation published by Cedric Bardy and Fred H. Gage (C Bardy et al. Proc Natl Acad Sci USA, 2015).
I know that StemCell Technologies will be releasing it soon and because this medium is more representative of the brain's extracellular environment your primary cells will be physiologically more normal.
I've had the best results using the media that is used in the attached publication.
Article Brewer, G.J. & Torricelli, J.R. Isolation and culture of adu...
Have you tried Neurobasal Medium? Commercially available from Gibco and supplememnted with B27, N2, EGF, FGF.
What you need is BrainPhys. It is a defined and serum-free neuronal culture medium for the culture of central nervous system (CNS)-derived neurons and human pluripotent stem cell (hPSC)-derived neurons. Based on the formulation published by Cedric Bardy and Fred H. Gage (C Bardy et al. Proc Natl Acad Sci USA, 2015).
I know that StemCell Technologies will be releasing it soon and because this medium is more representative of the brain's extracellular environment your primary cells will be physiologically more normal.
Joel, why do neuronal cells require B27, N2, EGF, and FGF? Will this formulation keep down the growth of glial cells?
Hello! I always use Neurobasal with 1% B27 supplement and 0.5mM GlutamaX for culturing hippocampal mouse neurons, you can add Pen/Strep for initial culture to avoid contamination after dissection (for 1-2days). I make 100ml batches of media at the time to avoid keeping the complete media stored for too long (usually no longer than a month) and I also aliquot B27 supplement. Also you mentioned reducing the levels of glial cells, if you are keen in decreasing their growth some people use Ara-C but I don’t recommend it (depends what pathways you are looking at, I am interested in NF-кB and Ara-C is an NF-кB activator so I avoid it). I found the best way to decrease astroglial growth it by using embryos at earlier stages of gestation such as E16 (you will get a lot more glial cells at E19) and I plate the cells in the Neurobasal medium as described, but I supplement it with 10% horse serum and let the cells attach to coated surfaces for 2-3hrs and then I just add the serum-free media to the well. Glia love serum and they will grow better if you leave serum in the medium for longer, but some growth is necessary for healthy synapses and they will make your cultures more robust. Hope this helps!
Dear Charles,
I think the answer also lies in what you are aiming at or what you are trying to achive. For example, if you are trying to do electrophysiology or patch-clamping like myself, then the suggestion given by Sun Yung is ideal (Based on the formulation published by Cedric Bardy and Fred H. Gage (C Bardy et al. Proc Natl Acad Sci USA, 2015). If you are doing electrophysiology, to be able to intrepret your results you need to know exact ionic concentrations of your extracellular solutions which determines the membrane responses. With premade media, there are lots of additives that adds too many variables to the equation and makes it hard to calculate ionic conductances and interpret electrophysiological responses.
best wishes,
Refik
Thank you for your responses, I have another question. Is there a difference between using Penn/Strep vs. Gentamicin for neuronal cell culture media?
Another question, should glutamate be used for initial plating of neurons?
Hi Charles,
As Refik mentioned, the medium you use will depend slightly on what your application is. The medium that is traditionally used for this application is Neurobasal (Thermo) combined with a serum-substitute like SM1 or B-27 supplement. However, although this combination works well to keep the neurons alive, their functional activies are inhibited. BrainPhys is a new basal medium that improves neuronal function and increases physiological relevance (as well as supporting survival). We (at STEMCELL Technologies) worked with the Gage lab to make this medium available globally. Contact me or google it if you want to try!
Glutamate should be added for the initial plating, but not used beyond day 3 or 5 - the neurons become sensitive to toxic effects from it as they mature.
Best wishes,
Sam
Usually i use those products and i upload my protocol too..
Neurobasal A medium + 2 % B27 + 1 % Glutamax I + P/S(250ul/50ml)
B27 (cat #17504-044. CCF)_Gibco
Glutamax I (cat #35050-061)_Thermo
- Badges
- Science method