Diletta Pozzi1, Jelena Ban2, Federico Iseppon1, Vincent Torre3. 1. Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy. 2. Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy; Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, 51000 Rijeka, Croatia. 3. Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy. Electronic address: torre@sissa.it.
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.
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.
Authors: Aurore Tourville; David Akbar; Olga Corti; Jochen H M Prehn; Ronald Melki; Stéphane Hunot; Patrick P Michel Journal: Cells Date: 2022-05-13 Impact factor: 7.666
Authors: Antonela Petrović; Jelena Ban; Ivana Tomljanović; Marta Pongrac; Matea Ivaničić; Sanja Mikašinović; Miranda Mladinic Journal: Front Cell Neurosci Date: 2021-03-18 Impact factor: 5.505