Highlightsï· An improved protocol for primary hippocampal cell cultures is proposed. ï· The method relies on serum-free astrocytes conditioned medium (ACM). ï· The ACM method is extensively compared with other two commonly used protocols. ï· ACM improved morphology and function of both short-and long-term cultures.
AbstractBackground: 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 astrocyteconditioned 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 60 days 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.