An improved method for growing neurons: Comparison with standard protocols

Abstract

Highlights An improved protocol for primary hippocampal cell cultures is proposed.  The method relies on serum-free astrocytes conditioned medium (ACM).  The ACM method is extensively compared with other two commonly used protocols.  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.

Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica

“…At DIV4, we counted on average 4.70 ± 0.13 neurites per neuron (the total number of TUJ1-positive neurites emerging from soma; 170 neurons analyzed). This result is similar to rat hippocampal neuronal cultures cultured in Neurobasal/B27-based medium (Pozzi et al, 2017), where also the enhanced branching was observed in the first 4 days in vitro.…”

“…The GCs had variable sizes with an average value of 39.50 ± 3.09 µm 2 (n = 40). To the best of our knowledge, these are the first experimental observations showing that neuronal GCs derived from the opossum cortex can be efficiently obtained in vitro, with a size comparable to rat hippocampal GCs (Pozzi et al, 2017).…”

“…The dissociation protocol was developed following the procedures previously described for rodents (Kaech and Banker, 2006;Beaudoin et al, 2012;Pozzi et al, 2017) and all efforts were made to minimize suffering and to reduce the number of animals used. Primary neurons were isolated from the cortex of neonatal opossums from two developmentally significant postnatal age groups: P3-5 and P16-18, respectively.…”

Considerations and Implications in the Purification of Extracellular Vesicles – A Cautionary Tale

“…Here, we have demonstrated the phenotypic changes that occur in stem cells after short term incubation in these different media types. Although serum-free alternatives for cell types such as neurons and astrocytes have been developed (Brewer, 1995; Pozzi et al, 2017), these remain poorly defined, protein rich alternatives that may induce unwanted molecular changes and lead to growth of selective populations (Balvers et al, 2013). Despite the growing number of studies comparing EV isolation methods, there is still only limited knowledge about the molecular profiles of EVs collected from protein rich media types and their implications on molecular contents of EVs.…”

Isolation and culture of porcine primary fetal progenitors and neurons from the developing dorsal telencephalon

“…Cell culture systems have added considerable value to research, in that cells can be grown for extended periods and used and treated accordingly. Fetal cell culture research has however been predominantly performed in the mouse, but also successfully performed in other small mammals including chicks and rats [12131415].…”