I have done a lot of live cell imaging on hippocampal neuron-astrocyte cultures in Neurobasal medium and now I would like to look at astrocytes in pure culture. I normally culture the astrocytes in different medium with 10% horse serum, but I am changing the medium 1-3days to serum free neurobasal before the imaging experiment to keep it the same as during experiments with neuron-astrocyte culture. My question is: is serum necessary in maintaining appropriate astrocyte function or am I ok just to use serum free neurobasal? I am considering supplementing the neurobasal medium with 2% serum and grow my mixed neuron-astrocyte cultures as well as single astrocyte cultures in that medium, is this better idea? I would be grateful for some suggestions/discussion.
Dear Joanna,
Ben Barres at Stanford is very expert in culturing astrocytes, so you might want to look at the protocol in this paper (it's in the supplemental materials, not the body of the paper): Foo LC(1), Allen NJ, Bushong EA, Ventura PB, Chung WS, Zhou L, Cahoy JD, Daneman R, Zong H, Ellisman MH, Barres BA. Development of a method for the purification and culture of rodent astrocytes. Neuron. 2011 Sep 8;71(5):799-811. doi: 10.1016/j.neuron.2011.07.022.
In brief, they switched the astrocytes cultured with 10% serum (MD astrocytes) to a serum-free media that was 50% Neurobasal and 50% DMEM, along with some supplements. You might try the same supplements in pure Neurobasal and see what happens.
Otherwise, there is the serum-free supplements for glial cell lines that were developed by Bottenstein and Sato. I have tried the mixture they called G5, but I added it to DMEM. You might try that also.
Good Luck!
Jill
In general: chemically defined media supress astrocyte growth. Serum derived batches will enhance astrocytic growth.
2% serum should be fine. There are also some serum-free supplements that can support the growth. Based on my experience, it depends on how long you'd like to keep the cultures alive. If you keep the serum in for too long, the over proliferation of astrocytes might affect the viability of your neurons.
From my experience with cortical neuronal culture and serum free Neurobasal medium it will affect astrocyte proliferation rate and this rate also can be affected by seeding density and culture's age . and I Thinke the period of incubation with serum free can determine the functionality of your cells
Dear Joanna,
Ben Barres at Stanford is very expert in culturing astrocytes, so you might want to look at the protocol in this paper (it's in the supplemental materials, not the body of the paper): Foo LC(1), Allen NJ, Bushong EA, Ventura PB, Chung WS, Zhou L, Cahoy JD, Daneman R, Zong H, Ellisman MH, Barres BA. Development of a method for the purification and culture of rodent astrocytes. Neuron. 2011 Sep 8;71(5):799-811. doi: 10.1016/j.neuron.2011.07.022.
In brief, they switched the astrocytes cultured with 10% serum (MD astrocytes) to a serum-free media that was 50% Neurobasal and 50% DMEM, along with some supplements. You might try the same supplements in pure Neurobasal and see what happens.
Otherwise, there is the serum-free supplements for glial cell lines that were developed by Bottenstein and Sato. I have tried the mixture they called G5, but I added it to DMEM. You might try that also.
Good Luck!
Jill
I recently switched from culturing rat hippocampal astrocytes with neurons on PDL-coated coverglass (Neurobasal A, B-27, Glutamax), to pure astrocyte cultures on TCPS (DMEM, pyruvate, glutamine, 15% FBS). The astrocyte proliferation rate is markedly slower and morphology much flatter after switching culture conditions. Considering astrocyte monoculture in Neurobasal A + B-27 + Glutamax for comparison. If anyone has tried this, please share :)
- Badges
- Science method