Pozzi, D;
Ban, J;
Iseppon, F;
Torre, V;
(2017)
An improved method for growing neurons: Comparison with standard protocols.
Journal of Neuroscience Methods
, 280
pp. 1-10.
10.1016/j.jneumeth.2017.01.013.
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Abstract
BACKGROUND: Since different culturing parameters - such as media composition or cell density - lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons - both in vivo and in vitro - is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. NEW METHOD: Our culturing protocol is based on a novel serum-free preparation of astrocyte - conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7, 14 and 60days in vitro (DIV). RESULTS: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. COMPARISON WITH EXISTING METHOD(S): ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. CONCLUSIONS: We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.
| Type: | Article |
|---|---|
| Title: | An improved method for growing neurons: Comparison with standard protocols |
| Location: | Netherlands |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.jneumeth.2017.01.013 |
| Publisher version: | http://dx.doi.org/10.1016/j.jneumeth.2017.01.013 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Astrocyte, B27 Supplement, Calcium signaling, Fetal bovine serum, Neuronal branching, Neuronal culture |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Wolfson Inst for Biomedical Research |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1572932 |
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