Shipley Mackenzie M, Mangold Colleen A, Szpara Moriah L
Department of Biochemistry and Molecular Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University.
Department of Biochemistry and Molecular Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University;
J Vis Exp. 2016 Feb 17(108):53193. doi: 10.3791/53193.
Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.
拥有合适的体内和体外系统以提供人类疾病的转化模型是神经生物学和神经科学研究不可或缺的一部分。神经生物学中使用的传统体外实验模型包括来自大鼠和小鼠的原代神经元培养物、神经母细胞瘤细胞系，包括大鼠B35和小鼠Neuro-2A细胞、大鼠PC12细胞以及短期切片培养物。虽然许多研究人员依赖这些模型，但它们缺乏人类成分，观察到的实验效果可能仅适用于各自的物种，在人类中可能不会以相同的方式出现。此外，尽管这些细胞是神经元，但它们的核型可能不稳定，这使得它们在基因表达研究和细胞信号转导的可重复研究中使用存在问题。因此，开发更一致的人类神经疾病模型非常重要。以下步骤描述了一种易于遵循、可重复的方法，通过分化染色体稳定的人类神经母细胞瘤细胞系SH-SY５Y来获得均匀且有活力的人类神经元培养物。该方法整合了几种先前描述的方法(1-4)，并基于从培养基中依次去除血清。时间线包括逐渐血清饥饿，同时引入细胞外基质蛋白和神经营养因子。这使得神经元能够分化，同时淘汰上皮细胞，从而得到均匀的神经元培养物。代表性结果表明，SH-SY５Y神经母细胞瘤细胞成功地从最初的上皮样细胞表型分化为更广泛且有分支的神经元表型。该方案提供了一种可靠的方法来生成均匀的神经元培养物群体，可用于后续的生化和分子分析，为研究人员提供了一个更准确的人类感染和疾病转化模型。
