title: An improved method for growing neurons: Comparison with standard protocols.
abstract: 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.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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.
  
  
  
  We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.
authors.affiliation: Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy.
authors.forename: Diletta
authors.initials: D
authors.lastname: Pozzi
authors.affiliation: 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.
authors.forename: Jelena
authors.initials: J
authors.lastname: Ban
authors.affiliation: Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Iseppon
authors.affiliation: Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy. Electronic address: torre@sissa.it.
authors.forename: Vincent
authors.initials: V
authors.lastname: Torre
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D006624:Hippocampus / Q000166:cytology / Q000502:physiology; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D000071446:Neuronal Outgrowth / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000502:physiology; D017208:Rats, Wistar; D013997:Time Factors; D056969:Voltage-Sensitive Dye Imaging
chemical_list: D017077:Culture Media, Conditioned; D002118:Calcium
keywords: Astrocyte; B27 Supplement; Calcium signaling; Fetal bovine serum; Neuronal branching; Neuronal culture