In the culture of neuron, the serum is avoided in the media. Does it just inhibit the growth of astrocytes? Or the serum in the neuron culture media would cause any other problems? What is the minimum concentration of serum to affect the neuron (0.1% or lower)?
Any suggestion will appreciate!
Serum is dense and thick biological, that contains many constituents mainly lipoproteins, enzymes, ferritin, albumin, growth factors and others, main problem with the astrocytes in culture is to keep its Na and K channels free of any obstruction because of their very high permeability and specific transport of chloride ions, because there are enzymes based receptor channels and need its purity and non adhesive nature for normal function, hence a minimum medium supplement with non obstruction in nature should apply. Hence, concentration below 0.1% is also considered safe and usable for astrocyte culture.
According to my experience, serum favors the growth of astrocytes, while causes neuronal death. When preparing cultures we dissect the cortex from E18 embryos, dissociate cells and plate them in serum containing medium (10% concentration) to allow cell adhesion to the substrate. If we want to culture neurons, after 4h we substitute the medium with a serum-free one. In this way, neurons survive while astrocytes grow slowly. If we want to culture astrocytes/glia, we keep on with serum-containing medium (again 10%), and they proliferate well while neurons die in few days. You may look at "Banker GA and Cowan WM (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res 126:397–442." or "Goslin, Banker. Rat hippocampal neurons in low-density cultures." for some details. I tried to culture neurons in the presence of 1% serum, and this is not toxic, but may favors unwanted proliferation of glia. It depends on your aims. Keep attention: neurons always need trophic support to grow. You can follow two strategies: either you culture them according to Banker, with a coverslip of neurons facing a feeder layer of astrocytes, or you use a medium (usually Neurobasal) supplemented with B27 or analogs.
If you need detailed protocols I can send them to you by e-mail.
Hello, Ravi Kant Upadhyae and Fabrizia Claudia Guarnieri
Thanks for your help, both of your answer is quite useful to me.
To Fabrizia Claudia Guarnieri, my email is [email protected]. If it's convenient, I'd like to read your protocols.
If I want to study the astrocyte' role in elinimating the neuron synapse, co-culture of the two cell types is required. As said by Fabrizia Claudia Guarnieri, 1% serum should be added to the medium? Or is there any other protocols to follow up?
Thanks.
Dear Fabrizia,
Do you have any advice that why the neuron is cultured in 25mM glucose concentration, which is 4 to 5 folds higher than physiological level no matter human or mice? It will be very appreciated if you could also send me a copy of your protocol. Thanks a lot.
email: [email protected]
Mengying
Dear Mengying Xia,
we usually use a glucose concentration of 3.3-4 mM.
Dear Xuebo Cheng,
Consider that in every neuronal culture (if not treated with AraC for example), a variable percentage of contaminating glial cells is always present. This include astrocytes, oligodendrocytes and microglial cells. If you want to study the exact role of glial cell populations in neuronal cell development, I would suggest to set up controlled co-cultures. Protocols for cultivating specifically astrocytes or microglial cells are available in the literature. You may envisage to prepare separate cultures, then trypsinize glial cells and incubate them over/together with neuronal cultures, in a known proportion.
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