Pozzi Diletta, Ban Jelena, Iseppon Federico, Torre Vincent
Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste, Italy.
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.
J Neurosci Methods. 2017 Mar 15;280:1-10. doi: 10.1016/j.jneumeth.2017.01.013. Epub 2017 Jan 27.
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.
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.
We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.
由于不同的培养参数（如培养基成分或细胞密度）会导致不同的实验结果，因此定义用于神经元培养的方案很重要。星形胶质细胞在体内和体外维持神经元内环境稳态中的重要作用已得到充分证实：大多数用于原代解离神经元培养的改良培养条件都依赖于星形胶质细胞。
我们的培养方案基于一种新型的无血清星形胶质细胞条件培养基（ACM）制备方法。我们将提出的ACM培养方法与其他两种常用方法（基于Neurobasal/B27和胎牛血清的培养基）进行了比较。我们在体外培养1、7、14和60天（DIV）时，通过免疫细胞化学进行形态计量学表征，并通过钙成像对所有三种培养方法进行功能分析。
基于ACM的培养在所有测试标准下都取得了最佳结果，即生长锥的大小和形状、神经元的生长和分支、网络活动和同步性、成熟度和长期存活率。与基于胎牛血清的培养基相比，差异更为明显。对于年轻培养物（DIV1），基于Neurobasal/B27的培养与ACM相当，但对于培养时间超过DIV7的情况则不然。
基于ACM的培养在DIV1时显示出更强健的神经元生长。在DIV7和60时，在ACM中生长的神经元网络的活动具有更活跃的自发电活动和更高程度的同步性。
我们提出基于ACM的培养方案是一种改进的、更适合短期和长期神经元培养的方法。
