A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture
A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture
复制标题
DOI：
10.3791/53797
复制
发表时间：
2016-03-01
影响因子：
1.2
通讯作者：
Qiu, Shenfeng
中科院分区：
文献类型：
--
作者：
Lu, Zhongming;Piechowicz, Mariel;Qiu, Shenfeng
体外培养原代海马神经元有助于神经元发育的许多方面的机械询问。解离的胚胎海马神经元通常可以在无血清条件下以高密度在盖玻片上成功生长，但低密度培养物通常需要通过将它们与神经胶质饲养层共培养来供应营养因子，其制备可能是耗时且费力的。此外，神经胶质细胞的存在可能会混淆结果的解释，并排除对神经元特异性机制的研究。在此，提出了一种用于超低密度（类似于2，000个神经元/cm（2））、长期（>3个月）原代海马神经元培养的简化方法，该方法在无血清条件下并且没有胶质细胞支持。低密度神经元在聚D-赖氨酸包被的盖玻片上生长，并翻转在24孔板中生长的高密度神经元上。实验者可以简单地蚀刻高密度神经元所在的孔的塑料底部，而不是使用石蜡点在两个神经元层之间创建空间，以创建有利于低密度神经元生长的微空间。共培养可以容易地维持>3个月而没有明显的低密度神经元损失，从而有利于这些神经元的形态学和生理学研究。为了说明这种成功的培养条件，提供的数据显示在长时间培养后低密度细胞中大量的突触形成。这种共培养系统还促进了在多聚-D-赖氨酸底物的岛中生长的稀疏个体神经元的存活，从而促进了自适应连接的形成。
Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (similar to 2,000 neurons/cm(2)), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.