Pozzi, 2017

2017 - J Neurosci Methods. 2017 Mar 15;280:1-10. doi: 10.1016/j.jneumeth.2017.01.013

An improved method for growing neurons: Comparison with standard protocols

Pozzi D, Ban J, Iseppon F, Torre V.

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.

 

Copyright © 2013 SISSA. Scuola Internazionale Superiore di Studi Avanzati

via Bonomea, 265 - 34136 Trieste ITALY